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Selective Preconcentration of Gold from Ore Samples

A simple and selective method has been developed for preconcentration of gold in ore samples. The method is based on use of N, N-diethyl-N'-benzoylthiourea (DEBT) as selective chelating agent and Amberlite XAD-16 as solid sorbent. Sorption behavior of gold with DEBT impregnated resin under optimized conditions has been studied in batch process. The gold ion capacity of the impregnated resin is calculated as 33.48 mg g−1 resin (0.17 mmol g−1 resin). The selective preconcentration of metal was examined using gold chelates prepared in column process under optimized conditions: pH, flow rate, volume of sample solution, nature of eluent, flow rate, and volume of eluent. Under optimum conditions, gold ions at the concentration of 0.015 μg mL−1 with a preconcentration factor of 6.7 have been determined by flame atomic absorption spectrometry (FAAS). The accuracy of the proposed method was validated by the analysis of a Cu-ore (semi-certified) supplied by CMC (Cyprus Mining Company, North Cyprus) and a certified reference material, Gold Ore (MA-1b Canmet-MMSL). Satisfactory results were obtained with a RSD of 7.6%. The highly selective proposed method does not require any interference elimination process.

selective_preconcentration_of_gold_from_ore_samples

1. Introduction<!>2.1. Apparatus and Instrumentation<!>2.2. Chemicals<!>2.3. Synthesis of DEBT and Impregnation Process<!>2.4. Batch Method<!>2.4.1. pH Effect<!>2.4.2. Stirring Time<!>2.4.3. Gold Ion Capacity of Resin<!>2.5. Column Method<!>2.6. Proposed Method for Preconcentration of Gold<!>2.7. Preparation of Ore Samples<!>3.1. Characterization Studies<!>3.2.1. pH Effect<!>3.2.2. Effect of Stirring Time<!><!>3.3. Optimized Parameters in Column Method<!>3.3.1. Sample Flow Rate of Gold Chelates<!>3.3.2. Effect of Ligand Volume on Impregnation of Gold Chelates onto Resin<!>3.3.3. Effect of Optimized Ligand Concentration on Amount of Gold Chelates<!>3.3.4. Choice of Eluent: Its Nature, Concentration, Volume, and Flow Rate<!>3.4. Effect of Electrolytes and Competing Ions<!>3.5. Analytical Figures of Merit<!>3.6. Analysis of Real Samples<!>4. Conclusions

<p>Gold is one of the precious metals which occurs in very low natural contents such as 4 ng g−1 in basic rocks, 1 ng g−1 in soils, 0.05 μg L−1 in sea water, and 0.2 μg/L in river water. Due to its specific physical and chemical properties, gold is widely used in industry, agriculture, and medicine [1]. Low abundance and heterogeneous distribution of gold in geological samples and various interfering matrices requires the development of accurate and reliable analytical procedures for determination of gold in environmental samples. Therefore, a selective separation and preconcentration method is a critical need for sensitive, accurate, and interference free determination of gold [2].</p><p>In literature, various techniques have been recorded for separation and preconcentration of gold, such as liquid-liquid extraction [3], coprecipitation [4], solid-phase extraction [5], cloud point extraction [6], and electrodeposition [7]. Solid-phase extraction (SPE) is preferable over all these techniques due to its advantages like high enrichment factor, high recovery, rapid phase separation, low cost, minimum solvent waste generation, and sorption of the target species on the solid surface in a more stable chemical form [8, 9].</p><p>The use of solid sorbents for preconcentration and separation has received great attention from analytical chemists [10]. Among wide range of solid phases such as multiwalled carbon nanotubes [11], surfactant coated alumina [12], styrene-divinylbenzene matrix [5, 13, 14], and silica gel [10, 15] have gained much importance for the metal ion enrichment. Amberlite XAD series have been more preferably used as solid support due to their good physical properties such as high porosity, uniform pore size distribution, high surface area as chemical homogenous non-ionic structure, and good adsorbent properties for great amounts of uncharged compounds [5, 16, 17]. Compared to Amberlite XAD-2 and XAD-4 resins, Amberlite XAD-16 has larger surface area [9], which makes possible to increase the number chelating sites hence increasing the selectivity towards target metal ions. This can be achieved by selecting suitable chelating agents. The chelating groups that are widely used for preconcentration of precious metals are imidazole, thioguanidine, dithizone, mercapto groups, amino groups, and thioureas [18]. In several studies, N, N-diethyl-N'-benzoylthiourea (DEBT) has been recorded as a selective complexing agent for precious metals [19, 20]. Its selectivity is mainly controlled by pH. It has very high resistance to hydrolysis and oxidation. In addition to high pKs values, with resonance effects, DEBT can increase the electron density at sulfur donor atom when suitable acceptors are available. DEBT forms stable complexes only with Class b and border line acceptors. Noble metal ions, due to their specific Class b properties, form chelates with DEBT in low oxidation states in strongly acidic solutions [19, 20].</p><p>In the present study, DEBT as a chelating ligand and Amberlite XAD-16 as solid support have been used for selective separation and preconcentration of gold which is determined by flame atomic absorption spectrometry (FAAS). Optimum conditions for batch and column processes have been studied in detail. Then the proposed method has been applied to two real samples: Cu-ore supplied from CMC, North Cyprus, and a certified reference material Gold Ore (MA-1b) supplied by Canmet-MMSL, Ontario.</p><!><p>In order to prevent sorption of gold on silica surfaces, equipment made of polytetrafluoroethylene (PTFE) was used. 100 mL DuPont polyethylene containers for the storage and 5-50 and 100-1000 μL adjustable micropipettes (Transferpette, Treff Lab) with disposable polyethylene tips for preparation of solutions were used. In batch process, optimum conditions such as pH, stirring time, metal ion capacity, and agents suitable for desorption were studied by using 50-mL of Falcon tubes. NÜVE SL 350 horizontal shaker was used during sorption optimizations in batch process.</p><p>Columns were prepared from 12-mL syringe barrels (1.5 cm i.d., 7.8 cm height, PTFE, Supelco) where disposable porous frits were placed at the bottom of the barrels. 1.0 g resin (unless otherwise stated) slurred in 50 mL water was uniformly placed in column and was covered with cotton wool to prevent dispersion by the addition of sample solution. Tygon® tubing was used to connect the outlet tip of the syringe barrel to a Gilson Miniplus peristaltic pump. A calibration, flow rate mL min−1 versus rpm was carried out. This calibration was repeated for each column before the application. Each time, 15 mL blank solutions at a flow rate of 1 mL min−1 were passed before sorption and desorption studies.</p><p>Philips PU 9200 Atomic Absorption Spectrometer with Epson FX-850 printer was used for determination of gold ions.</p><!><p>All the reagents were of analytical reagent grade. Deionized water from a Milli-Q system was used throughout the study unless otherwise stated. Amberlite XAD-16 resin was supplied by Sigma. Gold standard solutions were prepared by diluting of 1000 μg mL−1 stock solution (Spectrosol) with 1 mol L−1 HCl (J.T. Baker, 36-38% w/w). During batch process, pH-adjustments were done using NaOH (Acros, 50% w/w). For desorption studies, Na2S2O3 (extra pure, Bilesik Kimya Mekanik) was used.</p><!><p>DEBT was synthesized according to the procedure modified in our laboratory [20] where potassium thiocyanate (Fischer, 0.1mol) was dissolved in anhydrous acetone (Riedel-deHaen, 100 mL) by stirring and heating in a reflux condenser. After cooling to room temperature, benzoyl chloride (Merck, 0.1 mol) was added dropwise and stirred for 30 minutes. Then potassium salt was filtered off. The filtrate in orange was reacted with 0.1 mol of diethylamine (Merck) dropwise. The reaction mixture was crystallized in 250 mL of 1 mol L−1 HCl solution. After filtering the mixture, the residue was recrystallized with ethanol.</p><p>Since the impregnation process deals with physical interactions between the chelating agent and solid support by either inclusion in the pores of the support material or adhesion process or electrostatic interaction, some parameters controlling the impregnation such as stirring time and chelating agent capacity have been optimized as mentioned elsewhere [21].</p><!><p>With batch studies, sorption behavior of high concentrations of gold on DEBT impregnated Amberlite XAD-16 was investigated. Some critical parameters such as pH, stirring time, and metal ion capacity of resin capacity have been studied to find out the optimum conditions for recovery of gold.</p><!><p>In order to investigate the pH effect on sorption of gold onto impregnated resin, different sets of 10 mL of 10 mg L−1 of Au3+ solution in the pH range of 1-5 were stirred with samples of 0.1 g impregnated resin for 50 minutes. After filtration under vacuum, metal ions in the filtrate were determined by FAAS.</p><!><p>Three different sets of 2 mg L−1, 10 mg L−1, and 100 mg−1L of 10 mL of Au3+ solutions in 1 mol L−1 HCl were stirred with samples of 0.1 g impregnated resin from the periods of 5 minutes to 1 hour. Then solutions were filtered and filtrates were aspirated into FAAS for metal ion determination.</p><!><p>In order to determine the resin capacity, samples of 0.1 g of impregnated resin (1 mmol DEBT g−1 resin) were stirred with 10 mL of gold ions solutions in the concentration range of 2 mg L−1 to 600 mg L−1 in 1 mol /L−1 HCl for 15 minutes. Then the solutions were filtered and metal ion concentrations were determined by FAAS.</p><!><p>Since the kinetic and equilibrium aspects of column process are different than batch process, optimization of column conditions is needed. Effect of flow rate and volume of ligand solution on impregnation and effect of ligand concentration on amount of metal chelate adsorption have been studied in column process.</p><!><p>100 mL of gold chelate solutions (0.15 μg mL−1 Au3+ and 3 mL of 2x10−3 mol L−1 DEBT) was percolated through the column (1.0 g pure resin) at a flow rate of 0.5 mL min−1. Then metal ions could be eluted with 15 mL of 0.2 mol L−1 Na2S2O3 in water with a recovery of 97.6 ± 2.3% (N=2).</p><!><p>An acid digestion procedure was applied to Cu-ore and Gold Ore (MA-1b) samples as suggested elsewhere [22]. Accordingly, two parallel 10.0 g of Cu-ore sample and 1.0 g of Gold Ore (MA-1b) were transferred into Teflon beakers. 20 mL of HCl was added to each where the beakers were covered and placed on a warm hot plate. After 15 minutes of digestion, 15 mL of concentrated nitric acid was added and the contents were digested for 20 minutes. Then 25 mL of concentrated HCl and 25 mL of deionized water were added. The contents were boiled to expel nitric acid digestion gases and to dissolve all soluble salts. After cooling they were filtered through Whatman white band filter paper into 100 ml PTFE flask. Once 3 mL of 2 X 10−3 mol L−1 DEBT solution was added, final volume was completed to 100 mL with deionized water. Later, the proposed procedure for preconcentration of gold was applied.</p><!><p>The structure of DEBT was characterized by FTIR and UV-VIS spectrophotometer. The synthesized DEBT exhibited 2 strong broad UV-absorption peaks at 237 and 278 nm which were consistent with those given in literature [20]. The characteristic absorption bands for N-H, C−H, and amide I (C=O), amide II, and amide III at 3276, 3066-2936, 1656, 1537, and 1306 cm−1, respectively, appeared in both of the spectra. FTIR studies for pure resin, DEBT, DEBT impregnated resin, and DEBT-metal chelates have been carried out [20]. The characteristic absorption bands for C-H and amide I (C=O), amide II, and amide III at 3276, 3066-2936, 1656, 1537, and 1306 cm−1, respectively, appeared only in the spectra of DEBT and impregnated resin. While the characteristic IR band of –N(CH2CH3)2 group in the ligand at 2875 cm−1 remained almost unchanged in the spectrum of the complex showing that this group is not involved in coordination, C-H vibration in the aromatic ring is blue shifted upon metal-ligand bond formation. The position of amide I, amide II, and III bands at 1656, 1537, and 1306 cm−1, respectively, arising from the carbonyl of the benzamide moiety and secondary amide of DEBT at 3276 cm−1 disappeared in the complex.</p><!><p>In literature, it is noted that DEBT forms stable and selective complexes with noble metals only in acidic or strongly acidic media [19]. Moreover, Shuster and coworker reported optimum pH range as 0-5 for liquid-liquid extraction of gold with DEBT was previously reported as 0-5 by Schuster and coworkers [31]. Therefore, the pH effect on chelation of gold ions with DEBT impregnated resin is investigated within the pH range from 1 to 5.</p><p>It was shown that the maximum percent sorption is obtained at pH ~ 1 (see Figure 1). Therefore, standard solutions were prepared by diluting AAS standard stock solutions with 1 mol L−1 HCl.</p><!><p>Referring to Figure 2, it can be concluded that 15 minutes of stirring is sufficiently good enough to achieve sorption equilibrium for three different concentrations of gold ion. Higher gold ion concentrations have no effect on the optimum time of sorption. Actually, fast kinetics can be expected in applications of macroporous resins. In addition, high ligand concentration, 1 mmol g−1 resin, used in impregnation may increase selectivity of the resin which can also be a reason for fast sorption rate.</p><p>As a result, 15 minutes of stirring can be accepted as a suitable stirring time during loading of resin with possible higher concentrations of gold ions to determine gold ion capacity of the resin.</p><!><p>Co is the initial concentration of metal ion (mg/L),</p><p>CA is the equilibrium concentration of metal ion (mg/L),</p><p>V is the volume of the solution (L),</p><p>W is the weight of the resin (g).</p><!><p>During the application of the proposed method in column process, it was noticed that excess volume of sample solutions during sorption leached the impregnated DEBT that lead to loss of selectivity and analyte. Moreover, the partial exhaustion of available chelating sites due to leaching of impregnated ligand caused irreproducible results of sorption percentages of metal ion [5]. Therefore, research was continued with preparation of metal chelates before transferring to column and certain limited volume of chelate solution would be percolated through the column containing pure resin under the optimized conditions.</p><!><p>During batch studies, it was recognized that DEBT showed similar kinetics during chelation with gold as that of silver which was reported in the previous study [5], as long as DEBT concentration is kept the same or close optimum pH for sorption is maintained [21]. In the previous study, considering application of larger volume of sample solutions for preconcentration, to be on safe 0.5 mL min−1 had been accepted as optimum sample flow rate [5]. The same was also found to be optimum sample flow rate for gold studies.</p><!><p>Maximum applicable ligand volume and concentration on analyte sorption are important. Therefore, maximum applicable ligand volume on retention is studied before further application of metal chelates for solid-phase extraction. For this reason, 3 mL of 3.75x10−4 mol L−1 DEBT solution was percolated through column for 4 times and DEBT concentration in the effluent determined by UV spectrometry. In Figure 4, it can be seen that maximum amount of DEBT retained on Amberlite XAD-16 was achieved with the first 3 mL of DEBT solution. Following additions of 3 mL of 3.75x10−4 mol L−1 DEBT solution showed a decrease in amount of DEBT retained on resin. This may be because of the leaching effect of ethanol on DEBT.</p><!><p>Ligand concentration is also important because if it is not excessively present, the chelate formation may not be complete so metal ions may not be selectively retained on resin. However, excess DEBT (in case of inadequate amount of resin) may prevent retention of metal chelates because of the competition for sorption on resin between excess DEBT and metal chelates.</p><p>Considering the further applications of the proposed method to a real sample and limitations related to ligand mentioned above, it was decided to use 1.0 g of resin and amount of DEBT as 3 mL of 2x10−3 mol L−1. This amount of DEBT is always in excess considering the amounts of analyte metal that is our concern (15 μg gold ions).</p><p>As indicated in Table 1, up to 100 μg gold ions in 10 mL sample solution can be safely retained on 1.0 g of resin as metal chelates. When 10 mL of 50 μg mL−1 metal chelate solution was passed through the column, a decrease in percent retention was observed.</p><p>During this study, we dealt with quite low amounts of metal ions and carried out the optimizations accordingly. Referring to results in Table 1, any researchers interested in higher amounts of gold up to 100 μg can study safely with the proposed method under the same conditions (such as amount of resin, sample flow rate, pH of sorption media, and ligand volume) as long as only the concentration of eluent and its volume are reoptimized according to the interested amount of metal ions.</p><!><p>In the previous study, we used sodium thiosulfate which was found as the most suitable eluent for desorbing silver ions from Amberlite XAD-16 [5]. The sorption of silver ions was governed by the chelation mechanism in that silver ions (belong to class of soft acids) have affinity for (S-O) chelating group of DEBT. During desorption, (S-S) chelating groups of Na2S2O3 provide a stronger complex formation as silver has a higher affinity for (S-S) than (S-O) chelating group [21]. Since gold metal also belongs to class of soft acid and same discussion could be valid as well, as a result, Na2S2O3 was selected as suitable eluent for desorbing gold ions.</p><p>Series of experiments were conducted to optimize the concentration and the volume of eluent [21]. When 0.1 mol L−1 Na2S2O3 in water was used as an eluent, at a flow rate of 0.3 mL min−1, only 65% desorption was obtained. Later, it was found that the highest recovery with 97.6 ± 2.3% desorption was achieved when 15 mL of 0.2 mol L−1 Na2S2O3 in water was percolated through the column at a flow rate of 0.3 mL min−1.</p><!><p>In geological samples like ores, some metals in higher concentrations such as Na+, K+, Cu2+, Ni2+, Pb2+, Mn2+, Fe3+, Zn2+, Al3+, and Cr3+ can coexist with gold. The anions Cl−, NO3−, SO42−, PO43−, and ClO4− are the anions that are capable of forming complexes with several metal ions.</p><p>Considering the real sample amount weighed according to gold digestion procedure (at least 10.0 g), 50 mL of 300 μg mL−1 of copper standard solution was prepared in 1 mol L−1 HCl. Initial metal ion concentration was determined by FAAS. Then the proposed method was applied. The metal ion concentration in the effluent was determined by FAAS. Initial metal ion concentration was found to be 292 μg mL−1 and the metal ion concentration in the effluent was found as 288 μg mL−1. As a result, only 1.37% of copper ions was adsorbed on resin. Although it is known that copper ion forms complex with DEBT in pH range of 0-7 [20], this result showed that the formation of Cu-DEBT complex in 1 mol L−1 HCl is quite lower to compete with gold ions. N-benzoylthioureas are bidentate chelating ligands with S and O as donor atoms. The possibility of increasing the electron density at the sulfur atom by means of resonance effect leads to selective complex behavior of DEBT which can be influenced by the adjustment of pH, where competing metal ions could be eliminated.</p><p>The effect of various electrolytes like NaNO3, Na2SO4, Na3PO4, and Na2CO3 on the sorption of gold (1 mg L−1) as Au-DEBT chelate on Amberlite XAD-16 resin was studied as well. Na2SO4 was tolerable up to 0.04 mol L−1, Na3PO4 up to 0.1 mol L−1, and NaNO3 and Na2CO3 up to 0.15 mol L−1.</p><!><p>The calibration graph for the determination of gold was plotted according to the proposed procedure under the optimum conditions. The equation of the line was derived as A = 0.0125C + 0.0003 with the regression coefficient 0.9998 where A is the absorbance and C is concentration of the metal ion (μg mL−1).</p><p>The limit of detection (LOD) and limit of quantitation (LOQ) for gold ions were determined employing the standard solutions giving absorbance signal slightly recognizable than blank. The LOD and LOQ were calculated based on 3s/slope and 10s/slope of 10 measurements of the blank, respectively, where s is the standard deviation of the sample solution. The results of the LOD, LOQ, and precision (RSD %) for gold and its concentration are shown in Table 2.</p><!><p>In order to demonstrate the accuracy of the proposed method, analyses of two real samples, one of which is the Cu-ore supplied by Cyprus Mining Company (CMC), North Cyprus, and the other one certified Gold Ore (MA-1b) by Canmet, Ontario, were carried out and the results were compared with the values reported. The results of spiked CMC samples and the results that were corrected for 97% desorption recovery value which was found for 100 mL sample solution were also tabulated in Table 3. Student's t-test was performed to statistically evaluate the found and certified values. The found values were in good agreement with the certified ones and the difference was found to be statistically insignificant (at 95% confidence interval level).</p><!><p>Highly selective, reliable, and low cost method has been proposed for preconcentration of gold ions from highly interfering matrices, namely ores. The validity of the proposed method was demonstrated by the analyses of two geological samples: Cu-ore (supplied by CMC) and Gold Ore (MA-1b) as a certified reference material. The results are in good agreement with the given values. Comparison of the proposed method with some similar studies in literature is summarized in Table 4. Although there are more sensitive methods applied to similar samples, such as ICP-MS and GFAAS, these are much more expensive and sophisticated. The gold ion at such a low concentration of 0.15 μg mL−1 could be preconcentrated selectively and determined by the proposed method without any matrix elimination processes.</p>

PubMed Open Access

Three-Dimensional Large-Pore Covalent Organic Framework with stp Topology

Three-dimensional (3D) covalent organic frameworks (COFs) are excellent porous crystalline polymers for numerous applications, but their building units and topological nets have been limited. Herein we report the first 3D large-pore COF with stp topology constructed with a 6-connected triptycene-based monomer. The new COF (termed JUC-564) has high surface area (up to 3300 m 2 g -1 ), the largest pore (43 Å) among 3D COFs, and record-breaking low density in crystalline materials (0.108 g cm -3 ). The large pore size of JUC-564 is confirmed by the incorporation of a large protein. This study expands the structural varieties of 3D COFs as well as their applications for adsorption and separation of large biological molecules.

three-dimensional_large-pore_covalent_organic_framework_with_stp_topology

<p>Covalent organic frameworks (COFs), [1][2][3][4][5] a remarkable class of organic porous crystalline materials with high surface areas and promising stabilities, have attracted wide interests in varied fields including gas adsorption and separation, [6][7][8][9] catalysis, [10][11][12][13][14][15] optoelectronics, [16][17][18][19][20] and some others. [21][22][23][24][25][26] Over the past decade, most researches have been focused on two-dimensional (2D) COFs with eclipsed AA stacking modes. [1][2][3][4] Three-dimensional (3D) COFs are considered as ideal platforms for abundant uses because of their interconnected channels, superior surface areas, and fully exposed active sites. [27][28][29][30][31][32][33][34][35][36] However, only few topologies are available for 3D COFs so far, such as ctn, bor, dia, and pts, and almost all of them are based on tetrahedral building blocks, which have extremely limited the structural diversities of 3D COFs. 5 Interestingly, Wang, Feng and co-workers synthesized the first 3D anionic COFs with rra topology, CD-COFs, in which each boron atom is joined to four γ-cyclodextrin struts, and each γ-cyclodextrin is connected to eight boron atoms. 37 Thomas, Roeser and coworkers have recently demonstrated a novel 3D anionic silicate COF adopting a two-fold interpenetrated srs-c topology by reticulating dianionic hexacoordinate [SiO6] 2nodes with 3connected triphenylene building blocks. 38 In principle, the employment of new building units, such as 6-connected monomer with D3h geometry, can establish novel architectures in 3D COFs; however, its realization has remained an enormous challenge.</p><p>Herein, we for the first time reported a 3D triptycene-based COF with large pores and stp topology. This novel COF, termed JUC-564 (JUC = Jilin University China), was constructed from a stereoscopic 6-connected triptycene-based building unit, 2,3,6,7,14,15-hexa(4′-formylphenyl)triptycene (HFPTP). As a result, JUC-564 showed high surface area (> 3300 m 2 g -1 ), the largest pore (43 Å) among 3D COFs, and the lowest density among all crystalline materials (0.108 g cm -3 ). Moreover, due to the presence of large pores, JUC-564 showed a favorable adsorption of a large protein with suitable dimensions.</p><p>Structural identification is one of major roadblocks for developing 3D frameworks with new topologies. Different from other crystalline porous materials, such as aluminosilicate molecular sieves 39 and metal-organic frameworks (MOFs), 40,41 single crystals are not common in COFs and their crystal structures are mostly obtained through powder X-ray diffraction (PXRD) patterns along with structural simulation. Usually, more than one possible topology is available for combinations of multiple building block geometries. After investigating RCSR database carefully, we fortunately found that only definite stp topology is available for [6 + 4] (3D-D3h + 2D-D2h) nets (Scheme 1), facilitating the structural determination of the target products. 42 To implement this strategy, we firstly designed a 6-connected 3D-D3h building block, HFPTP, based on a triptycene moiety with a link angle of 60º (Scheme 1a). Condensation of HFPTP and a synergistic 4-connected 2D-D2h monomer with a link angle of 120º (1,3,6,8-tetra(4-aminophenyl)pyrene, TAPPy, Scheme 1b) leads to an expanded [6 + 4] connected network (JUC-564, Scheme 1c and 1d). To the best of our knowledge, JUC-564 represents the first COF with a 6-connected 3D-D3h building block and a stp net.</p><p>The synthesis of JUC-564 was carried out through traditional solvothermal approach by suspending HFPTP and TAPPy in a mixed solvent of mesitylene and dioxane with the presence of 6 M acetic acid followed by heating at 120 ºC for 3 days. Complementary methods have been employed for detailed structural determination and characterization. Scanning electron microscopy (SEM, Figure S1) and transmission electron microscopy (TEM, Figure S2) images revealed isometric microcrystals. Fourier transform infrared (FT-IR) spectrum exhibited a new adsorption corresponding to the characteristic of the C=N bond at 1628 cm −1 . The concomitant reducing of the C=O stretching (1700 cm −1 for HFTPT) and N-H stretching (3312 cm −1 for TATPy) confirmed the transformation of aldehyde and amine groups (Figure S3). The solid-state 13 C cross-polarization magicangle-spinning (CP/MAS) NMR spectroscopy further verified the presence of imine groups by the peak at 157 ppm (Figure S4). High thermal stability (∼450 °C) was observed by thermogravimetric analysis (TGA, Figure S5). The crystal structure was resolved by PXRD measurements in conjunction with structural simulations (Figure 1). After a geometrical energy minimization of JUC-564 by the Materials Studio software package on the basis of stp net, 43 the simulated PXRD pattern was in good agreement with the experimental one. Furthermore, the full profile pattern matching (Pawley) refinement was conducted based on experimental peaks at 1.93, 3.34, 3.87, 5.47, 6.96, and 9.46° corresponding to (100), ( 110 2). Notably, benefiting from its highly void framework and light constitutional elements, JUC-564 has a calculated density of 0.108 g cm -3 , which is the lowest reported for any crystalline material known to date, such as MOFs (0.22 g cm -3 for MOF-200, 44 0.195 g cm -3 for IRMOF-74-XI, 45 and 0.124 g cm -3 for NU-1301 46 ) and COFs (0.19 g cm -3 for JUC-518, 20 0.17 g cm -3 for COF-108, 27 and 0.13 g cm -3 for DBA-3D-COF 1 47 ).</p><p>To investigate the porosity of JUC-564, gas sorption study of N2 was conducted at 77 K. As shown in Figure 3a, JUC-564 exhibited typical reversible type IV isotherms, which is one of the main characteristics of mesoporous materials. The surface area was calculated to be 3383 m 2 g -1 using the Brunauer-Emmett-Teller (BET) model (Figure S6). Pore size distribution calculated by nonlocal density functional theory (NLDFT) illustrated two kinds of pores with sizes of 15 Å and 41 Å (Figure 3b), which are in good agreement with those of the proposed structure (14 Å and 43 Å). Remarkably, the largest pore size of JUC-564 (43 Å) is far superior to that of other reported 3D COFs (Table S1), such as 13.5 Å for COF-102, 27 13.6 Å for DL-COF-1, 33 15.4 Å for JUC-518, 20 and 28 Å for DBA-3D-COF 1. 47 Furthermore, its BET surface area (3383 m 2 g −1 ) is much higher than that of other 3D imine-based COFs (Figure 4 and Table S2), such as 1360 m 2 g −1 for COF-300, 48 1513 m 2 g −1 for JUC-508, 15 2020 m 2 g −1 for LZU-111, 31 and 3023 m 2 g −1 for JUC-552. To further define the structure and large channels of JUC-564, incorporation of large biomolecules with suitable dimensions as probes was explored (Figures S7-12). The uptake ability of JUC-564 for myoglobin (Mb, about 21 Å × 35 Å × 44 Å) 49 was confirmed by UV-vis spectrum, which proves the existence of the wide channel (43 Å) in JUC-564. For comparison, no observable adsorption of Mb in the microporous COF-320 took place due to its smaller pore size (~12 Å). In summary, we have developed a large-pore 3D COF with novel stp topology utilizing a rare 6-connected D3h node based on triptycene. JUC-564 exhibited interconnected channel systems with high surface areas (3383 m 2 g -1 ), ultra-large channels (up to 43 Å), record-breaking low density (0.108 g cm -3 ), and positive uptake of a large protein molecule. This work not only opens a door to enrich 3D structures of COFs but also promotes new applications of 3D COFs in adsorption of large biological molecules.</p>

ChemRxiv

Rapid sampling of all-atom peptides using a library-based polymer-growth approach

We adapted existing polymer growth strategies for equilibrium sampling of peptides described by modern atomistic forcefields with a simple uniform dielectric solvent. The main novel feature of our approach is the use of pre-calculated statistical libraries of molecular fragments. A molecule is sampled by combining fragment configurations \xe2\x80\x93 of single residues in this study \xe2\x80\x93 which are stored in the libraries. Ensembles generated from the independent libraries are reweighted to conform with the Boltzmann-factor distribution of the forcefield describing the full molecule. In this way, high-quality equilibrium sampling of small peptides (4\xe2\x80\x938 residues) typically requires less than one hour of single-processor wallclock time and can be significantly faster than Langevin simulations. Furthermore, approximate, clash-free ensembles can be generated for larger peptides (up to 32 residues in this study) in less than a minute of single-processor computing. We discuss possible applications of our growth procedure to free energy calculation, fragment assembly protein-structure prediction protocols, and to \xe2\x80\x9cmulti-resolution\xe2\x80\x9d sampling.

rapid_sampling_of_all-atom_peptides_using_a_library-based_polymer-growth_approach

I. INTRODUCTION<!>II. FORMALISM<!>II.A. Forcefield, fragments and notation<!>II.B. Combination of fragments<!>II.C. Growth by reweighting<!>II.D. Resampling<!>II.E. Approximate ensembles<!>II.F. Assessment of sampling precision and efficiency<!>III. IMPLEMENTATION<!>III.A. Fragment libraries<!>III.B. Enrichment<!>III.C. Recycling of energy terms<!>III.D. Cartesian vs. internal coordinates<!>III.E. Software optimizations<!>III.F. Breadth vs. depth<!>IV. RESULTS<!>V.A. Limitations<!>V.B. Possible applications<!>V.C. Possible improvements<!>VI. CONCLUSIONS<!>

<p>This paper investigates whether decades-old polymer-growth algorithms 1–15 have promise for the study of biomolecules modeled by modern atomistic forcefields. Although polymer approaches have previously been applied to proteins 16–22, their application to atomistic forcefields at physiological temperatures has been problematic 23–25. Here we report a novel implementation of a growth algorithm based on pre-calculated statistical libraries of molecular fragment configurations and energies. The encouraging results from a limited set of small test peptides, reported below, suggest that further investigation is warranted.</p><p>The well-known problem of sampling biomolecules typically has been addressed by dynamical simulations and variants – molecular dynamics (MD), Langevin dynamics (LD), and Metropolis Monte Carlo with local moves. All these approaches suffer from the well-known problem of undersampling: dynamical simulations of proteins are far too short to probe timescales (and motions) thought to be of dominant biological importance. Even simulations of modest-sized peptides are slow to "converge" 26,27. Sophisticated variants of dynamical simulations, such as replica exchange 28–31, also have not convincingly solved the undersampling problem 32–34. While multi-resolution methods appear to have substantial promise 35–38, rigorous applications have been restricted to small systems thus far.</p><p>The importance of sampling biomolecules and the intrinsic limitations of dynamical simulation together suggest the value of exploring fully non-dynamical polymer growth algorithms. Such methods have a history dating back more than fifty years. Initial studies focused on straightforward build-up of lattice-polymer chains 1,3,4, but the early approaches were limited by the "attrition problem," in which the vast majority of chains encounter dead ends before reaching a significant size. Our own approach builds directly on methods developed to treat attrition, especially (i) the Rosenbluths' approach of re-weighting chains based on possible growth steps 5, and (ii) equally seminal work by Wall and Erpenbeck describing "enrichment" of successful partially grown chains by replication and appropriate weighting 7. Wall, Rubin and Isaacson noted that future increments of the growth of a lattice polymer were limited to a small set of configurations 6, partly anticipating the libraries we employ here. Many additional improvements have also been proposed 8–10. The basic theory behind polymer growth as we apply it, along with key practical insights, was fully set out by Garel and Orland in 1990 11. Important descriptions of growth algorithms are also provided by Grassberger 12,13 and by Liu 15.</p><p>Polymer growth algorithms have been applied previously to biomolecules. Highly simplified models of proteins were studied by Grassberger and coworkers 17, by Liu and coworkers 19,21,39, Liang and coworkers 20,22,40,41, and others 16,18. Garel, Orland, and coworkers applied polymer growth methods to all-atom peptide models — but their work employed extremely high-temperature sampling (T=1000 K) followed by energy minimization 11,23–25. Our use of pre-calculated fragment libraries emulates ideas from the ROSETTA software 42 as well as from work by Clementi and coworkers 43,44. However, none of these previous studies appears to have generated canonical sampling for a modern atomistic forcefield at T ~ 300 K.</p><p>In light of the significant body of historical work, the present contribution must be considered pragmatic rather than theoretical. In brief, our work shows that pre-generated libraries of statistically distributed monomer/fragment configurations can be used to sample all-atom molecular systems with a simple uniform dielectric solvent at temperatures of interest (T = 300 K). For high quality statistical sampling the present implementation is limited to small peptides – up to about eight residues and less than 100 atoms. However, besides equilibrium sampling, our growth procedure can be also used for rapid generation of approximate (i.e., steric-clash free) ensembles of larger peptides containing up to 32 amino acid residues in this study. Although the present work is formally similar to our previous use of fragments for free energy calculations 45, this study presents critical technique improvements which greatly improve efficiency.</p><p>Our study also employs recently developed statistical approaches 46 to quantify the degree to which efficiency has been gained. The library-based strategy is shown to be extremely efficient in some cases — decreasing the required wallclock time by over one order of magnitude. However, we believe that several improvements are possible, as described in the Discussion section.</p><p>In our approach the choice of fragments is flexible and they can correspond to different groups of atoms in the molecule. For proteins natural choices of fragments are the amino acid residues because proteins consist of only 20 building blocks. However, other choices are possible. When the fragments correspond to the backbone and side chains, the procedure is essentially a multi-resolution method. The backbone can be sampled using other methods such as our previously developed library-based Monte Carlo 47, followed by the gradual addition of more atomistic detail embodied in side chains.</p><!><p>As noted in the Introduction, polymer growth algorithms have been developed and used over decades 1–13,15. Our approach follows earlier work in many regards, but is specifically tailored to the use of modern atomistic forcefields with implicit solvent. Our presentation of the algorithms relies solely on straightforward re-weighting concepts 15,48. We describe a simple and apparently novel approach to using libraries of molecular fragments which can save significant computational cost.</p><!><p>In this study we generate equilibrium configurations according to the OPLS-AA forcefield 49 with a simple solvent model (with uniform dielectric constant of 60) at 298 K. This dielectric constant has been chosen to give reasonable agreement for Ramachandran propensities as compared to GBSA solvent model 50.</p><p>The potential energy of the forcefield including any implicit solvent terms will be denoted by U (x), where the full set of 3N-6 internal coordinates x = (x1, x2, …, x3N−6), consists of N-1 bond, N-2 bond angles and N-3 dihedrals. The full set of coordinates corresponding to a single molecular fragment y will be denoted by xy with y = A, B, C, …. The collection of forcefield terms for fragment y, denoted by Uy will contain all terms internal to the particular subset of atoms included in the fragment. That is, it will include all bonded and non-bonded terms for those atoms. Dummy atoms may be added to a fragment, as in the present study, to include the six degrees of freedom that specify the orientation of fragments relative to each other. However, dummy atoms will have no effect on the final distribution.</p><p>We assume that fragments are non-overlapping and exactly divide all coordinates, so that for the whole molecule the full set of coordinates may be written as</p><p>It is important to realize that the full forcefield U can never be written as a sum of fragment forcefields Uy (xy ). The reason is that, regardless of which intermediate coordinates are included via dummy atoms, no coordinate set xy includes distances between atoms from different fragments. Needless to say, such inter-atomic distances are fundamental to the full molecular forcefield. Inter-fragment interactions are fully accounted for in our growth procedure, as described below.</p><!><p>In our approach, a molecule is sampled by growing it from scratch using pre-calculated molecular fragments. Here we describe the process of joining fragments which may be repeated inductively by adding additional "monomers" onto the growing chain. Configurations for each fragment are calculated in advance so that they are distributed according to the Boltzmann factor of the forcefield describing the fragment. The set of Boltzmann-distributed configurations for each fragment is called a "library".</p><p>The basic procedure for joining fragments is simple. A new fragment configuration is drawn with uniform probability from its library and added to the partially grown chain. The interaction energy between the new fragment and other previously added fragments is evaluated. The generated configurations are reweighted to the Boltzmann factor distribution describing the partially grown molecule to correct for the new interactions.</p><p>Consistent with free energy calculations using our growth process 45, we will define a set of intermediate models {Uj} which correspond to different stages of the growth process. We note that these intermediates are a little different than those employed (before) in ref 45.</p><p>For a molecule consisting of k fragments, we will employ k intermediate models with interactions between fragments gradually "turned on". The first intermediate U1, sampled at the library generation stage, includes interactions internal to each fragment, while subsequent intermediates add the indicated interactions among fragments A, B, C, …. These intermediate models can be written as</p><p> (2)U1(x)=UA(xA)+UB(xA)+UC(xC)+…U2(x)=U1(x)+UAB(xA,xB)U3(x)=U2(x)+UAC(xA,xC)+UBC(xB,xC),⋯U(x)=Uk−1(x)+∑y=A,B,…Uyz(xyz) where Uyz denotes all forcefield interaction terms between fragments y and z. The last intermediate U (x) is simply the full molecule and the sum ∑y=A,B,…Uyz(xyz) represents interactions between the last fragment z and all other fragments in the molecule.</p><!><p>Our polymer-growth approach heavily relies on the re-weighting concept 15,48 because interactions between fragments are not included in the libraries of individual fragments. In essence we generate configurations with non-interacting fragments and gradually reweight them into an ensemble with all interactions. In other words the purpose of reweighting is to effectively put back all the interactions and correlations between fragments into the molecule.</p><p>At each stage, we want to generate a suitably distributed ensemble – called the target ensemble Pjtarg∝exp[−βUj(x)] for stage j with the set {Uj} defined in Eq. (2). When j<k, this target ensemble based on Uj includes interactions only for the partially "grown" molecule. Yet configurations for stage j, as will be seen, are generated according to a different distribution, denoted Pjgen. Hence, configurations must be reweighted according to</p><p> (3)uj(x)=Pjtarg(x)Pjgen(x), where uj (x) is the weight of a configuration at stage j. (In fact, as explained below, uj (x) is an intermediate weight.) In Eq. (3) and subsequent equations, the symbol x does indeed represent the full set of coordinates. In intermediate stages j < k, however, some interactions are omitted: see Eq. (2).</p><p>To perform the reweighing procedure, we need to define the Pgen and Ptarg for each intermediate stage. Let us consider each stage in detail. The first stage U1 includes interactions within each fragment which are sampled according to exp[−βU1 (x)] at the library generation stage. We note that in general it is not necessary to employ fragment libraries distributed according to the Boltzmann factor distribution and other distributions can be employed 20,40.</p><p>The second stage U2 corresponds to turning on interactions between fragments A and B, starting from configurations already distributed according to U1. Thus the generating distribution P2gen is simply proportional to the Boltzmann factor describing the first intermediate with non-interacting fragments: (4)P2gen(x)∝exp[−βU1(x)].</p><p>The distribution targeted at the second stage P2targ proportional to the Boltzmann factor describing the second intermediate: (5)P2targ(x)∝exp[−βU2(x)].</p><p>At the third stage, similarly, interactions are turned on between fragment C and previously combined fragments A and B. As before P3gen is nothing but P2targ</p><p>Likewise, P3targ distribution is proportional to the Boltzmann factor describing the third intermediate: (7)P3targ(x)∝exp[−βU3(x)].</p><p>It is not difficult to generalize this combination process for any other intermediate. For the kth intermediate (corresponding to the full molecule) Pjgen and Pjtarg can be written as</p><p>It is important to note that in our procedure Pgen is built sequentially based on Ptarg from the previous stages. This is the essence of "sequential importance sampling" 15 i.e., the probability distribution of the full molecule is built sequentially step by step. The advantage of sequential importance sampling is that the probability distribution is changed in small increments to give the better overlap between Pgen and Ptarg at each stage.</p><p>The required partial weights wj can be calculated based on the incremental weights of Eq. (3). Specifically, the weight of a configuration at stage j can be written recursively based on the weights from previous stages: (10)wj=wj−1uj</p><p>Substituting the corresponding Pgen and Ptarg from Eqs. (4)–(9) into Eq. (10) the partial weights can be written as</p><p> (11)w1(x)=1w2(x)∝w1(x)exp[−βU2(x)]exp[−βU1(x)]=w1(x)exp[−βUAB(xA,xB)]w3(x)∝w2(x)exp[−βU3(x)]exp[−βU2(x)]=w2(x)exp[−β(UAC(xA,xC)+UBC(xB,xC))],⋯w(x)∝wk−1(x)exp[−βU(x)]exp[−βUk−1(xk−1)]=wk−1(x)exp[−β∑y=A,B,…Uyz(xy,xz)] where w(x) is the total weight for the full molecule i.e., with interactions "turned on" between all fragments. Note that w1 (x) is equal to one by construction because fragment configurations in the libraries are distributed according to the corresponding Ptarg – i.e., the Boltzmann factor describing the individual fragments.</p><p>Our "resampling" protocol, described later, will use the partial weights {wj}. However, it is instructive to note that the total weight w(x) in Eq. (11) can be re-written by expanding the weights and rearranging terms, resulting in</p><p>Eq. (12) shows that the total weight takes into account all the interactions missing in the non-interacting fragments described by the first intermediate U1.</p><p>Note that the weights in Eqs. (11) and (12) are proportional to the ratio of the Boltzmann factors up to the constant which is the ratio of the corresponding partition functions. However, this constant is not needed for re-weighting because only the relative weights are important.</p><!><p>In general, configurations with low weights have low importance in the ensemble and therefore it is desirable to save computer time by eliminating such configurations from future consideration. However, such elimination must be performed statistically to preserve the correct distribution 15. Such a "resampling" process refers to eliminating, duplicating, and/or adjusting weights of configurations in the original ensemble resulting into an alternative ensemble 15. Both ensembles are formally equivalent in representing the desired distribution.</p><p>A number of resampling algorithms have been suggested in statistics and data processing 15,51. We implemented several resampling schemes in our growth algorithm and found a scheme termed "optimal resampling" 51 to be the most efficient. The main advantage of optimal resampling compared to other schemes is that it guarantees distinct configurations 22.</p><p>Optimal resampling guarantees drawing the desired number of distinct configurations, denoted by M, from an original ensemble containing N configurations and corresponding weights. This is achieved by employing a threshold weight c which uniquely defines M. The configurations are accepted with probability min{1,wj(x)c}, where wj (x) are the partial weights at stage j. The resampling of only distinct configurations is guaranteed by employing a special numerical cumulative distribution function (cdf) 51.</p><p>We implemented the optimal resampling in our growth algorithm at the end of each combination stage. After the fragments are joined and the weights are calculated, the configurations are resampled into a smaller ensemble containing 10% of the original configurations. The 10-fold reduction factor was found to be the most efficient based on trials of different N and M values. The typical ensemble size employed in our simulations is N=105 configurations, which is resampled into an ensemble of size M=104. As we describe in Sec. III.B, an "enrichment" procedure is employed to compensate for configurations eliminated by resampling and to maintain a constant ensemble size at different combination stages.</p><p>It is worth noting that after the last combination stage, configurations with weights may be resampled into an ensemble without weights. We implemented several different resampling algorithms to eliminate weights in the final ensemble. However, we consistently found that such resampling considerably reduces information contained in the weights. Therefore, after the last combination stage we use the same optimal resampling scheme as at other stages and save configurations with weights for further analysis. This is similar to keeping a larger number of correlated "snapshots" from a dynamics trajectory 52.</p><!><p>Besides equilibrium sampling, our growth procedure can be adapted for rapid generation of approximate ensembles. This may be useful for larger systems for which precise ensembles are not required – for instance, in schemes which assemble protein configurations from multi-residue segments 42,53–55. The only new feature of our approximate procedure is that after the last combination stage configurations are used without weights. This way, weights are used only to identify configurations at intermediate stages without steric clashes. In other words, resampling works as a "bump check".</p><!><p>In the present work efficiency of the growth algorithm is defined as the savings in wallclock time to achieve the same level of statistical precision in sampling of configuration space distribution relative to standard Langevin dynamics. This precision can be quantified by the number of statistically independent configurations contained in the trajectory (i.e., effective sample size (ESS)). To assess efficiency, the time to generate a single statistically independent configuration can be compared between two methods. Thus, we define efficiency as</p><p> (13)γ=tLangevintGrowthESSGrowthESSLangevin where ESSGrowth and ESSLangevin are the effective sample sizes of the growth and Langevin simulations respectively. The symbols tGrowth and tLangevin denote wallclock times of growth and Langevin simulations respectively.</p><p>To calculate the ESS for both growth and Langevin simulations we used a recently developed statistical analysis 46. Qualitatively, the idea is to divide configuration space into approximate physical states and calculate population variance in each state. The variance is inversely proportional to the effective sample size. The approximate physical states can be constructed using Voronoi bins in configuration space 45. The reference structures for the Voronoi procedure 56 are derived from the protocol described in Ref. 27.</p><p>To check the results of the previous method we also used a second method to calculate the ESS for Langevin simulations. This method employs our previously developed "de-correlation time" analysis and can be used only for dynamic simulations 27. Briefly, the idea is to determine how much simulation time must elapse between configurations in the trajectory in order for them to exhibit the statistics of fully independent samples. Using the de-correlation time and the total simulation length the number of statistically independent configurations in the trajectory can be calculated.</p><!><p>The growth formalism described in Sec. II does not lead to a unique algorithm, but can be implemented in many different ways. Implementation details are particularly important because modern forcefields are much more complicated than the early simple polymer models. Indeed, in our study we found that the efficiency of the growth algorithms depends significantly on the implementation. Here, we describe the technical approaches that helped to significantly improve the efficiency of our growth algorithm.</p><!><p>The advantage of using libraries is that some interactions and, therefore correlations within a molecule, can be calculated in advance and then used in multiple simulations saving CPU time. Instead of generating new fragment configurations on the fly, they can be cheaply retrieved from the memory. This approach is well suited for proteins which consist of only 20 different building blocks. We can build up libraries for different amino acids and then combine them according to the sequence to sample any peptide or protein. The idea to use molecular fragments in molecular simulations is well established in the literature 57,58 and has been successfully implemented in the protein structure prediction software Rosetta 42. Earlier we have used libraries in a Monte Carlo approach 47. Although our libraries are Boltzmann-factor distributed by design, other choices are possible 20,40.</p><p>Fragment libraries can be generated using any canonical method such as Langevin dynamics or Metropolis Monte Carlo. The only requirement for the libraries is that they should represent the true equilibrium distributions. In practice we used internal coordinate MC because it allows fixing some degrees of freedom such as some bond angles and dihedrals introduced with the dummy atoms. The dummy atoms were employed for two reasons. First they provide the six degree of freedom that specify the orientation of fragments relative to each. Second, the dummy atoms were chosen to interact with the real fragment atoms to provide better overlap with the full molecule distributions. We used libraries containing 105 configurations.</p><p>We note that our fragments contain the same degrees of freedom and are sampled according to the same forcefield as employed in our previous study 45. The only difference is that in our previous work the fragment libraries were generated by sampling the internal coordinates independently with subsequent reweighting into the full fragment distributions.</p><!><p>Enrichment entails making multiple copies of configurations at different stages of growth without introducing statistical bias, in order to increase the chances of partially grown chains to survive 6. We implemented enrichment in our growth algorithm and found that it significantly increased the efficiency. One drawback of enrichment is that when chains are replicated, they are not longer statistically independent, limiting the degree to which enrichment can ameliorate attrition. If chains are replicated too much, the configurations become too statistically correlated, and ultimately limit efficiency. We found that the most efficient implementation of enrichment in our growth algorithm is when it is applied after each combination stage and chains are replicated 10–100 times.</p><!><p>In addition to coordinates, the potential energy of each fragment configuration can be calculated in advance, stored with the libraries and then used during a simulation to save CPU time. For example, when two fragment configurations are combined, their internal potential energies need to be evaluated. However, instead of calculating these energy terms over and over again (when the same fragment configurations are combined), they can be calculated only once at the library generation stage, saved with the libraries and then cheaply retrieved from the computer memory during a simulation. This way the potential energy is recycled rather than calculated each time from scratch.</p><p>We note that these CPU savings will only be moderate for long molecules containing many fragments because interactions between fragments will dominate. We implemented recycling of energy terms in our growth algorithm and found that it helped to increase the efficiency for all the systems studied.</p><!><p>To implement the growth formalism of Sec. II., it could seem natural to use internal coordinates, particularly for connecting fragments. However, each configuration ultimately must be converted to Cartesian coordinates for potential energy evaluation. In our original implementation fragment configurations were combined in internal coordinates and then converted to Cartesian for energy calculation. But we found that a large fraction of CPU time was actually spent on coordinate conversion.</p><p>The efficiency of our growth procedure was significantly improved when fragments were combined in Cartesian coordinates. This was implemented by storing "connector coordinates" – i.e. the six relative degrees of freedom – along with transformation matrices for each fragment configuration. First, the six degrees of freedom that specify the orientation of fragments relative to each other were used to set up the local coordinate systems. Given the local coordinate systems for each fragment, the appropriate transformation matrices were applied to generate the full Cartesian coordinates. In practice, configurations in the libraries were pre-oriented in the local coordinate system at the N-terminus of our residue based fragments and only one transformation matrix (at the C-terminus) was saved for each configuration in the library.</p><p>All transformation matrices were calculated using quaternion operations which allow fast and accurate transformations 59.</p><!><p>The cost analysis of our growth algorithm revealed that it is "memory bound" – i.e., the bottleneck is not the CPU operations but rather the transfer of data from memory to CPU 60. It is memory bound because it heavily relies on pre-calculating and storing configurations and energies in the memory. The transfer rate of data between the main memory and CPU is limited and becomes the bottleneck. To alleviate the memory latency problem, modern CPUs utilize "cache" memory which allows much faster communication with CPU. However, the size of cache is much smaller than the main memory size so the data can be cached only in relatively small chunks. The memory bound algorithms can be improved by reusing the data and "neighbor use" 60. Reuse helps to reduce the transfer of data from main memory to CPU by reusing as much as possible the data stored in cache and CPU registers. Neighbor use helps to perform computation on data (physically) close in memory reducing the transfer of data from memory to cache.</p><p>We implemented several standard optimization techniques in our C code including array linearization and blocking 60,61 both aimed at improving the reuse and neighbor use of fragment configurations and energies stored in the libraries.</p><!><p>The growth algorithm can be implemented in two different ways: "breadth first" and "depth first". In breadth first a whole ensemble of configurations is obtained at each intermediate stage before proceeding to the next one. In depth first only one full configuration is grown at a time. Both implementations have their own advantages and can be better suited for a particular resampling scheme etc.</p><p>Our implementation of the growth algorithm is a hybrid between breadth and depth. It is a hybrid because we grow a whole ensemble at once (typically 105 configurations). However, to achieve a good statistical precision we repeat the whole growth process many times and simply combine configurations, energies and weights from different simulations into one large ensemble. Specifically, we used 10 repeats for Ace-(Ala)4-Nme, 100 for Ace-(Ala)6-Nme, and 1000 for Ace-(Ala)8-Nme and Met-enkephalin. The approximate ensembles for Ace-(Ala)12-Nme, Ace-(Ala)16-Nme, Ace-(Ala)24-Nme, and Ace-(Ala)32-Nme were generated using only one repeat.</p><!><p>We applied our polymer-growth algorithm to equilibrium sampling of several peptides including Ace-(Ala)4-Nme, and Ace-(Ala)6-Nme, Ace-(Ala)8-Nme and Met-enkephalin. The equilibrium ensembles were sampled according to OPLS-AA forcefield 49 and for this initial study we used a simple solvent model with uniform dielectric of 60 at 298 K. The dielectric constant was chosen based on several trial simulations to give reasonable agreement for Ramachandran propensities with GBSA simulations 50. As discussed in Sec. III.F. Ace-(Ala)4-Nme was run for 10 repeated simulations resulting in 105 saved structures, Ace-(Ala)6-Nme was run for 100 repeats leading to 106 configurations. Ace-(Ala)8-Nme and Met-enkephalin were run for 1000 repeats also resulting in 106 saved configurations.</p><p>To compare the growth results we ran standard Langevin dynamics simulations for the same four peptides described by the same forcefield and solvent model. We also ran Langevin dynamics simulations and generated approximate ensembles (see Sec II.E) for four larger peptides Ace-(Ala)12-Nme, Ace-(Ala)16-Nme, Ace-(Ala)24-Nme, and Ace-(Ala)32-Nme. The Langevin dynamics was used as implemented in Tinker software package 49. All systems were sampled at the temperature of 298 K with a friction constant of 5 ps−1. Ace-(Ala)4-Nme, and Ace-(Ala)6-Nme, Ace-(Ala)8-Nme and Met-enkephalin were run for 200 ns, and all other peptides were sampled for 100 ns. All growth and Langevin dynamics simulations were performed on a single Xeon 3.6 GHz CPU and 2 GB of system memory.</p><p>We first checked that our algorithm can correctly sample the equilibrium distributions by comparing it with Langevin dynamics for the smaller systems. The equilibrium distributions were compared using structural histograms constructed using Voronoi procedure as described in previous work 45. The results are shown in</p><p>Figure 1 and indicate mostly good agreement between the two methods – although there appears to be slight bias in the Met-enkephalin results: see Discussion section.</p><p>To assess the efficiency of growth simulations we calculated the effective sample size (ESS) of Langevin simulations using two different statistical tools described in Sec. II.F. The first method is based on calculating the variance in the approximate physical states 46. The second method employs our previously developed de-correlation time analysis 27 and was used to check the results of the first method which we recently developed 46. The results are reported in Table 1 and indicate a reasonable agreement between two statistical methods. We note that the de-correlation time analysis can be used only for dynamic trajectories and, therefore, was not used for growth simulations.</p><p>The ESS of growth simulations was calculated using the first statistical tool i.e., by computing the variance in the populations of approximate physical states 46. The results of this analysis are reported in Table 2 and indicate that a large efficiency gain of over one order of magnitude was achieved for most peptides.</p><p>We emphasize that the efficiency of polymer growth algorithms applied to atomistic forcefields at 298 K depends significantly on implementation. In fact our original, naive implementation was not efficient at all – it was several times slower than Langevin simulations. However, in a series of implementation improvements described in Sec. III, we achieved good efficiency.</p><p>To aid future research in the field, we report how different improvements contributed to the efficiency of growing the peptide Ace-(Ala)4-Nme. The largest improvement, of about two orders of magnitude, can be attributed to using Cartesian coordinates and recycling energy terms. Software optimizations improved the efficiency by about three times. Implementation of the optimal resampling algorithm increased the efficiency by almost another order of magnitude.</p><p>Besides equilibrium sampling of small peptides, our growth procedure can be also used for rapid generation of approximate ensembles (i.e., steric-clash free) of larger peptides. As described in Sec. II.E, we generated approximate ensembles for Ace-(Ala)12-Nme, Ace-(Ala)16-Nme peptides, Ace-(Ala)24-Nme, and Ace-(Ala)32-Nme. Each required less than a minute of a single-processor wallclock time. To check whether these approximate ensembles provide reasonable distributions in configuration space, we compared them with equilibrium ensembles from Langevin simulations. The distributions were compared based on structural histograms constructed using a Voronoi procedure 45. A larger number of bins (20) were used to investigate whether reasonable coverage of configuration space was obtained; the issue of coverage could be important in fragment-assembly applications. The results for both peptides are shown in Figure 2 and indicate that all bins are reasonably populated by approximate ensemble configurations; for many bins the error bars overlap between the equilibrium and approximate ensemble distributions. Based on these observations we can conclude that our approximate ensembles yield reasonable coverage of configuration space.</p><!><p>The key limitation of the present implementation of the growth algorithm is that it can be applied for precise equilibrium sampling only of relatively small peptides. The limit is about eight amino acid residues and less than 100 atoms, although we showed that significantly larger peptides can be treated approximately. The size limitation for precise sampling appears to result from the small overlap between non-interacting and fully interacting fragment distributions. In the present implementation, the overlap significantly decreases with system size because configurations which predominate in the partially grown ensemble may be less important in the full molecule. For example, if one is growing a largely helical peptide, partially grown configurations will not "know" about hydrogen bonds which will be formed later in the growth process. In Sec. V.C we describe possible solutions to the problem of small overlap.</p><p>The present procedure is also limited to implicit solvent models. It is not clear whether explicit solvent could be treated practically, but as discussed below, it should be possible in principle to grow explicit solvent.</p><!><p>There are numerous applications for any fast peptide sampling scheme, and some that are specific to the growth scheme. First, it is important to recognize that relatively small peptides have important functions as hormones, neurotransmitters, antigens, and drugs 62. Examples include enkephalins and vasopressin. Pharmacologically, peptides are actively being investigated as potential drugs 63. Fast growth procedures can permit the investigation of large numbers of sequences.</p><p>Unlike dynamic methods, the polymer growth approach can be used to calculate the absolute free energy without any additional cost 45. This is possible because all the required generating probabilities and weights are known 64. In our previous study 45, we calculated absolute free energies for several peptides based on pre-calculated molecular fragments; however, that study did not employ the critical efficiency improvements described here. In principle, different peptides (or other molecules) can be grown in the presence and absence of a protein to yield relative binding affinities via a standard thermodynamic cycle.</p><p>The approximate growth procedure could be of particular use in conjunction with fragment assembly protocols for protein structure prediction 53–55. Presently, these protocols rely on expensive dynamic sampling of fragment configurations for subsequent assembly into native-like structures. Our growth procedure can rapidly generate approximate ensembles of peptides suitable for such assembly or perhaps with other fragment-based programs like Rosetta 42,65.</p><p>Interestingly, our growth procedure can be adapted to multi-resolution sampling because of flexibility in how a molecule is divided into fragments. For example, fragments can correspond to the backbone and side chains of different types. In such a version of the growth algorithm – that we will call "decorating" 36 – given a backbone ensemble, side chains can be added one at a time. Decorating is a true multi-resolution technique because the backbone can be sampled using other canonical methods, for example, our previously developed library-based Monte Carlo 47. Initial data obtained from decorating (not shown) suggest it can be a useful approach. We note that polymer growth algorithm has been applied before to modeling of side chains 41.</p><!><p>There are several possible solutions to the problem of small overlap between early and late growth stages. One possibility is to bias the growth based on some prior knowledge of the full molecule's configuration-space distribution. This knowledge may be obtained from previous dynamics or growth simulations even if these simulations are not fully sampled, provided there is some information on correlations among all fragments. For example, the biasing can be implemented as a "self-guided" algorithm: a regular growth simulation can be performed first and then subsequent growth simulations can be biased toward important parts of configuration space based on the information obtained in the first simulation. Libraries could also be biased based on simulations and/or databases like the protein data bank.</p><p>Efficiency for larger systems might be improved by expanding the ensemble at every intermediate stage j. For instance, ensemble expansion could be effected using local "relaxation" of the growing configurations with a canonical sampling method, such as library-based Monte Carlo 47. This idea is based on "annealed importance sampling" 66,67. An enlarged canonical ensemble at stage j should have more configurations pertinent to stage j+1. In general, growth and dynamic approaches have features that can help each other to better sample configuration space. Growth can instantaneously cross potential energy barriers but is not good at exploring local configuration space. On the other hand, relaxation of partially grown configurations may help to remove strains and better explore local configuration space. Canonical relaxation preserves the correct distribution 66–68.</p><p>It is natural to consider fragments larger than those used here, which were single amino acids. For instance, fragments can correspond to amino-acid dimers or even larger peptides. There are two practical limitations on fragment size, however, both of which will become less severe with increasing memory. One restriction stems from sequence variations within a fragment. For example, for (ordered) dimer fragments it will be necessary to generate and store at least 2 0×2 0 = 4 0 0 different libraries. Another practical limitation is the number of configurations required to adequately describe each library. Our present procedure employs 105 configurations per library but larger fragments may require significantly larger libraries. On the other hand, biasing libraries toward the most pertinent parts of configuration space will decrease the storage requirements. Again, as computer memory increases and becomes more affordable, these limitations may become less important.</p><p>Despite these limitations we tested the potential of using larger fragments in our growth procedure. We employed (Ala)2 and (Ala)3 fragment libraries each containing 105 configurations to sample Ace-(Ala)8-Nme but found that the efficiency was inferior compared to using a single Ala fragment. One reason why larger fragments did not help may be that they require much larger libraries (compared to single-residue fragments) to represent their large configuration space.</p><p>In this initial study we employed a simple solvent model with uniform dielectric although more accurate models such as GBSA 50 can be implemented. When using a new solvent model, fragment libraries will have to be regenerated although it requires only one time cost. Additional energy terms for the solvent model will have to be implemented in the algorithm.</p><p>In principle, polymer growth algorithms are not limited to implicit solvent models. Similar to growing peptides, water molecules can be added one at a time to solvate the system. In fact, our group has already "grown" a simple Lennard-Jones fluid 69.</p><p>The polymer growth algorithms are well suited for modern graphics processing units (GPUs) because multiple configurations can be grown at once in contrast to dynamic simulations where only one configuration can be processed at a time. The advantage of GPUs is that they have hundreds of arithmetic units where multiple interactions and/or configurations can be simultaneously processed.</p><!><p>We report the use of a polymer-growth algorithm that employs pre-calculated molecular fragment libraries for equilibrium sampling of peptides using an atomistic forcefield (OPLS-AA) at 298 K. To authors' knowledge this is the first application of the polymer-growth technique for equilibrium sampling of atomistic protein models at a semi-physiological temperature. The results show that the approach is correct and can be considerably more efficient than standard Langevin dynamics for several peptides. Approximate ensembles for larger peptides (up to Ace-(Ala)32-Nme in the present study) can be generated in less than a minute of single-processor computing time.</p><p>The considerable speed of the calculations can be attributed to the implementation of several optimization techniques, some of which are not applicable to standard dynamics methods. Future improvements such as biased libraries, self-biasing, and relaxation may help to further improve speed and efficiency, especially for large systems. Our results seem to warrant further studies of the polymer growth strategy for equilibrium sampling of polypeptides.</p><!><p>Fractional population of Voronoi bins constructed from growth and Langevin simulations for four peptides: (A) Ace-(Ala)4-Nme, (B) Ace-(Ala)6-Nme, (C) Ace-(Ala)8-Nme, and (D) Met-enkephalin. The bins were constructed based on a Voronoi classification of configuration space as described in Ref. 45. Error bars represent one standard deviation for each bin, based on 12 independent simulations for both Langevin and growth.</p><p>Fractional populations of Voronoi bins constructed from approximate growth procedure and Langevin simulations for four peptides: (A) Ace-(Ala)12-Nme, (B) Ace-(Ala)16-Nme, (C) Ace-(Ala)24-Nme, and (D) Ace-(Ala)32-Nme. The bins were constructed based on a Voronoi classification of configuration space as described in Ref. 45. Error bars represent one standard deviation for each bin, based on 12 independent simulations for growth and 10 for Langevin.</p><p>The results of statistical analysis of Langevin dynamics simulations are reported for four peptides. The effective sample size (ESSLangevin) was calculated using two different statistical tools as described in Sec. II.F.</p><p>The results of the statistical analysis of growth simulations are reported for four peptides. The effective sample size (ESSGrowth) was obtained based on calculating the variance in the approximate physical states as described in Sec. II.F. The efficiency gain γ relative to Langevin dynamics was calculated using Eq. (13). Note that γ was obtained using ESSLangevin calculated from the variance in the physical states.</p>

PubMed Author Manuscript

Heterocycle-derived β-S-enals as bifunctional linchpins for the catalytic synthesis of saturated heterocycles

We demonstrate how heterocycle-derived β-S-enals can be employed as bifunctional substrates in a cascade of two rhodium-catalysed C-C bond forming reactions to deliver substituted heterocyclic products. A single rhodium-catalyst, generated in situ from a commercial salt and ligand combination, is used to promote both an initial alkene or alkyne hydroacylation reaction, and then a Suzuki-type cross-coupling, resulting in a three-component assembly of the targeted heterocycles. Substrates based on N-, Oand S-heterocycles are included, as are a range of alkenes, alkynes and boronic acid derivatives. Scheme 1 Saturated and partially saturated heterocycles in pharmaceuticals and natural products. Scheme 2 Heterocycle-derived β-S-enals as building blocks towards saturated heterocycles. † Electronic supplementary information (ESI) available: Experimental details and supporting characterisation data. See

heterocycle-derived_β-s-enals_as_bifunctional_linchpins_for_the_catalytic_synthesis_of_saturated_het

<p>Due to the favourable physiochemical properties often associated with their incorporation into candidate structures, saturated, or partially saturated, heterocycles are becoming increasingly targeted in drug discovery programs. 1 Their presence in biologically active molecules has significant precedent, and Scheme 1 shows several examples of N-, O-and S-heterocycles embedded in pharmaceuticals and natural products used in a variety of applications. 2 The N-based congeners such as pyrrolidines, piperidines, and tropanes are the most commonly encountered structures. 3 In order to access saturated and partially saturated heterocycles decorated with a variety of substituents, we conceived an approach based on a common class of bifunctional building blocks that could be elaborated using cascade catalytic reactions. The key building blocks that we settled on were heterocycle-derived β-S-enals (1, Scheme 2). Variants of 1 featuring N, O, and S-atoms are all accessible from the parent ketones using established methods. 4 With the key building blocks available we speculated that a single rhodium-catalyst could mediate an initial alkene or alkyne hydroacylation reaction, 5 and then a Suzuki-type cross-coupling, to convert enals 1 into difunctionalised products 2 in a single step, joining together three separate components. A variety of methods could then be used to convert enones 2 into the fully saturated heterocycles. The design of the β-S-enals allows the S-atom to function as the directing atom for the initial chelation-controlled hydroacylation reaction, and then for the O-atom of the resul-tant enone to direct the Suzuki-type coupling. The use of aldehydes with β-S-directing groups in hydroacylation reactions is well established. [6][7][8][9] In addition, our laboratory, 10 and others, 11 has recently reported on the required aryl methyl sulfide Suzuki chemistry, including a cascade reaction on a benzenederived substrate.</p><p>We began by evaluating the basic reaction sequence using pyran-derived enal 1a, tert-butyl acetylene and p-tolyl boronic acid as the reaction partners (Scheme 3). Earlier precedent, 10,12 and initial investigations, 13 suggested that a Rh(I) catalyst incorporating the small-bite-angle bis-phosphine ligand dcpm should be able to mediate both of the key C-C bond forming reactions. When a catalyst of this type was generated in situ and applied to the targeted transformation, good yields of enone 2a were obtained. Achieving a short reaction time for the initial hydroacylation reaction was key to obtaining a high yield for the final product. Accordingly, warming to 55 °C resulted in the hydroacylation reaction being complete in only 5 minutes, with the following Suzuki-type coupling then requiring 5 hours. It was pleasing to note that the β-S-enal substrate appeared to allow significantly faster reactions than the previously explored fully aromatic system.</p><p>With a catalyst and appropriate reaction conditions for the cascade process in hand, we next explored the scope of the alkynes and alkenes that could be employed in the hydroacylation step. Given the importance of N-heterocycles in medicinal chemistry, we chose the tetrahydropyridine substrate, 1b, in combination with tolyl boronic acid, as a suitable platform to evaluate the chemistry (Scheme 4). As can be seen the use of tert-butyl acetylene was successful, delivering the three-component product, enone 2b, in high yield. A terminal alkyne substituted with a benzyl ether (2c), and an internal alkyne (2d) were both successful substrates. Less sterically demanding alkyne substrates resulted in mixtures of linear and branched regioisomers in the hydroacylation step, resulting in lower overall yields. For example, phenyl acetylene (2e) and hex-5ynenitrile (2f ) delivered 4 : 1 and 5 : 1 mixtures of linear : branched isomers, respectively. Terminal alkenes could be employed in the desired cascade process, however, their lower reactivity in the hydroacylation step, relative to alkynes, necessitated the use of five equivalents to achieve suitably fast reactions. Using these conditions with octene delivered the desired three-component coupled product (2g) in 84% yield. Terminal alkenes substituted with bromo (2h), phenyl (2i) and free hydroxyl groups (2j) were also successfully employed. Disubstituted alkenes were unreactive in the described system. 14 Tetrahydropyridine enal 1b was then used to evaluate the scope of the boronic acid coupling partner (Scheme 5). Initially employing tert-butyl acetylene as the hydroacylation coupling partner, a range of electronically varied aryl boronic acids could be readily employed (2k-2p), including halogen substituents. Although substitution at the meta-position was possible (2n), attempts to employ ortho-substituted boronic acids were unsuccessful. A heterocyclic boronic acid, in the form of 3-thienyl, and also an alkenyl boronic acid performed well (2q and 2r). Using octene as the hydroacylation coupling partner allowed a similar range of boronic acids to be successfully incorporated into the cascade process (2s-2x).</p><p>One of the goals of the present chemistry was to show that a variety of different heterocycles could be accessed using a single strategy. Accordingly, in Scheme 6 we demonstrate the successful use of N-, O-, and S-based heterocyclic building Scheme 3 Establishing reaction conditions for the hydroacylation-Suzuki cascade sequence to prepare dihydropyran 2a.</p><p>Scheme 4 Substrate scope of the alkyne and alkene component in the cascade synthesis of tetrahydropyridines 2. Reaction conditions: 1b (1.0 equiv.), alkyne (1.5 equiv.) or alkene (5.0 equiv.), [Rh(nbd) 2 ]BF 4 (5 mol%), dcpm (5 mol%), acetone, 55 °C, 5-10 min; then p-tolyl boronic acid (1.5 equiv.), Ag 2 CO 3 (1.0 equiv.), 55 °C, 5 h. Isolated yields of the major isomer. a Measured by 1 H NMR spectrometry on the crude reaction mixture.</p><p>blocks. For O-based heterocycles, a dihydropyran and a 2Hchromene-based substrate were both combined successfully with alkene and alkyne coupling partners and aryl boronic acids (2a, 3a-3d). S-heteocycles were represented by a 2H-thiochromene-based substrate, which was employed without incident (3e, 3f ). Unfortunately, it was not possible to prepare the dihydrothiopyran substrate due to stability issues. Finally, in addition to tetrahydropyridine substrate 1b already described, we were able to prepare and exploit a tropane-based substrate (1e), allowing access to alkyne and alkene coupled products in good yields (3g and 3h).</p><p>All of the scoping experiments described in Schemes 4-6, were performed on a relatively small scale (0.2 mmol of enal), and as such 5 mol% of catalyst was employed due to ease of use. However, for larger preparative scale reactions, it was possible to lower the catalyst loading. Scheme 7 shows the use of tetrahydropyridine substrate 1b, and tropane-derived sub-strate 1e, used in alkene hydroacylation initiated cascades, employing just 3 mol% of catalyst, to deliver gram scale quantities of coupled products (2g and 3i) in excellent yields.</p><p>Finally, as an illustration of synthetic potential of the enone products obtained from the developed cascade processes, we have shown that tetrahydropyridine-derived product 2g undergoes high-yielding detosylation and alkene reduction in a single-step, providing piperidine 4 (Scheme 8). This one-step Scheme 5 Substrate scope of the boronic acid component in the cascade synthesis of tetrahydropyridines 2. Reaction conditions: 1b (1.0 equiv.), alkyne (1.5 equiv.) or alkene (5.0 equiv.), [Rh(nbd) 2 ]BF 4 (5 mol%), dcpm (5 mol%), acetone, 55 °C, 5-10 min; then boronic acid (1.5 equiv.), Ag 2 CO 3 (1.0 equiv.), 55 °C, 5 h. Isolated yields.</p><p>Scheme 6 Variation of the heterocyclic enal component in the preparation of di-coupled products 3. Reaction conditions: 1 (1.0 equiv.), alkyne (1.5 equiv.) or alkene (5.0 equiv.), [Rh(nbd) 2 ]BF 4 (5 mol%), dcpm (5 mol%), acetone, 55 °C, 5-10 min; then boronic acid (1.5 equiv.), Ag 2 CO 3 (1.0 equiv.), 55 °C, 5 h. Isolated yields.</p><p>Scheme 7 Preparative scale synthesis of products 2g and 3i. transformation was achieved using magnesium metal in methanol under sonication conditons. 15 In conclusion, we have shown that heterocycle-derived β-Senals are efficient substrates for rhodium-catalysed hydroacylation-Suzuki type coupling cascade processes. Both alkyne and alkene hydroacylation reactions can be used as the initial C-C bond-forming event, and a variety of boronic acids can be employed as substrates in the Suzuki-type coupling. The products are obtained in good to excellent yields, and show potential as precursors to access biologically relevant compounds.</p>

Royal Society of Chemistry (RSC)

The extraordinary richness of the reaction between diazomethane and tetracyanoethylene: can computational calculations shed light on old papers?

In the quest of the structure of the intermediate between D 1 -and D 2 -pyrazolines, the reactivity of these molecules tetrasubstituted by cyano groups in adjacent positions (3,3,4,4 or 4,4,5,5) has been explored in their neutral and protonated forms. Many reactions reported in the literature for pyrazolines have been studied and quantified (energies and transition states). Thirty-three structures of pyrazolines, their open-ring counterparts and their complexes are described. Acid-base equilibria, rotations, electrocyclic reactions and sigmatropic transpositions are reported.

the_extraordinary_richness_of_the_reaction_between_diazomethane_and_tetracyanoethylene:_can_computat

Introduction<!>Computational details<!>Results and discussion<!>3.4.<!>Acid-base equilibria, rotations, electrocyclic reactions and sigmatropic transpositions<!>Conclusions

<p>The reaction of diazomethane 2 with tetracyanoethylene 1, both very common compounds, has only been studied two times. In 1962, Bastu ´s and Castells reported the reactions of Fig. 1 (their numbering of formulae is different). 1 They indicated that 3 can be explosive and this probably prevented other authors from repeating its preparation.</p><p>D 1 -pyrazoline 3 (4,5-dihydro-5H-pyrazole-3,3,4,4-tetracarbonitrile) was isolated and it spontaneously evolved nitrogen to yield 1,1,2,2-tetracyanocyclopropane 4 that was already known having been prepared by other methods. 2 Compound 3 was washed with benzene to eliminate all traces of 1 and was slowly dissolved in dry ether to yield a compound to which structure 5 (2,2,3,3-tetracyano-1,5-diaza-bicyclo[2.1.0]pentane) was assigned. Compound 5 when treated for about 90 min with a 5% solution of 1 in dry ether afforded D 2 -pyrazoline 6. The isomerization of 6 to 5 was performed using wet ether or dry ether containing traces of hydrogen chloride. Both substances can be kept for several weeks without alteration. According to Bastu ´s and Castells, the 6 to 5 isomerization involves the pyrazolinium cation 6bH + . 1 The role of TCNE (1) in the 5 -6 isomerization was assigned to a 1 : 5 complex.</p><p>Huisgen et al. repeated the reaction. 3,4 They cited Banu ´s and Castells but they isolated only 4 and 6. Leaving aside 4 (also 6bH + was not characterized), we have summarized in Table 1 all the available information on the compounds in Fig. 1.</p><p>Calatroni and Gandolfi reported a series of reactions that are related to the work of Banu ´s and Castells (Fig. 2). 5 D 1 -pyrazoline I reacted in two ways with TCNE (1) to afford a charge-transfer complex II and adduct III that was not isolated nor identified. They assumed that the reaction I " III is fast and reversible. Besides, TCNE promotes the isomerization I -IV in agreement with Castells' results. A reaction product V was postulated corresponding to the reaction of IV with TCNE.</p><p>Calatroni and Gandolfi indicated in their paper that they wanted to determine the crystal structure of II (yellow crystals) but they probably failed because no structure like II was reported in the CSD. 6 The only one that bears resemblance to II is FEJDUT (VI), see Fig. We decided to study theoretically structures 3, 5 and 6 and their corresponding protonated cations (Fig. 4) as well as some concerted reactions related to Woodward-Hoffmann rules. [8][9][10]</p><!><p>The geometries of pyrazolines (Pz) and pyrazolinium cations (PzH + ) have been fully optimized using the functional B3LYP 11 and the 6-311++G(d,p) basis set 12 in the spin restricted formalism as implemented in the Gaussian 16 package (the coordinates of all the optimized geometries are gathered in the ESI †). 13 The minimum energy and transition state structures of all compounds were characterized using frequency analysis. The solvent effects have been evaluated by re-optimizing the structures at the B3LYP/6-311++G(d,p) level and using the self-consistency reaction field (SCRF) method 14 based on the polarized continuum model (PCM) of Tomasi and co-workers 15 in ethanol, diethylether and benzene as solvents using the standard parameters provided by the Gaussian-09 program. For the infrared spectra, the frequencies have been scaled by a factor of 0.9679. 16 Absolute chemical shieldings have been calculated with the GIAO approximation 17 and then transformed into chemical shifts using empirical equations. 18 The static intrinsic reaction coordinates (IRCs) 19 were analyzed in two cases. pyrazoles. 20 For the reaction 1 + 2 -3, D 1 -pyrazoline 3 lies 17.1 kJ mol À1 higher than the potential surface minimum, the D 2 -pyrazoline 6 (Table 2), and the barrier with regard to 3 is 73.8 kJ mol À1 . For the reaction between diazomethane and different olefins, Ess and Houk calculated barriers between 57 and 70 kJ mol À1 . 21 Calculated IR, the NMR properties of compound 5 are reported in Table 3.</p><!><p>This compound has two conformations depending on the position of the NH bond, towards or out of the ring (Fig. 5).</p><p>Compounds 5 lie 165.8 (5a) and 151.0 (5b) kJ mol À1 over the potential surface minimum and D 2 -pyrazoline 6 (Table 2). Calculated IR and NMR properties of compound 3 are reported in Table 3. D 2 -pyrazoline 6 is the most stable of the three isomers (Table 2). The 3 -6 tautomerization involves a 1,3 CH to NH prototropy. A direct proton transfer has a high barrier that in similar fivemembered rings prevents this mechanism, while the following one involves assistance by solvent molecules (one or two) such as water or alcohols with a low barrier. 22 The calculate TS between 3 and 7 is 289.7 kJ mol À1 and between 7 and 8, it is 277.8 kJ mol À1 . The experimental IR spectrum (KBr) and the calculated bands (gas phase) agree exceptionally well assuming that there is an intercept. The corresponding linear regression line is: Exp. = (126 AE 37) + (0.93 AE 0.1) Calc., n = 7, R 2 = 0.999, RMS residual = 32 cm À1</p><p>(1)</p><p>We have removed Castells' bands at 1592 and 1595 cm À1 assigned to NH bending because according to eqn (1) they should appear at 1269 and 1417 cm À1 . On the other hand, if the 1592 cm À1 band is assigned to NQNQC of 7, the agreement is good (fitted value 1544 cm À1 ). In conclusion, on IR grounds, structure 5b should be rejected while structure 7 is acceptable.</p><p>The available 13 C NMR data of compound 6 3 agree with the calculated values according to the linear regression, Exp. = (173.5 AE 3.4) À (0.91 AE 0.04) Calc., n = 7, R 2 = 0.991, RMS residual = 2.9 ppm</p><p>(2)</p><p>The most different signals of the three compounds are the 15 N chemical shifts of the ring nitrogen atoms: +91.3 and +105.4 ppm for 3; À236.8 and À29.5 for 6; À285.8 and À297.0 for 5b.</p><!><p>A first attempt to find a more stable structure for intermediate 5</p><p>We have considered that the intermediate instead of being diaziridine 5b could be zwitterion 7 (Fig. 6).</p><p>Compound 7 is much more stable than 5b, 88.3 vs. 151.0 kJ mol À1 , but still too high for allowing the 6 -5 backward reaction. Solvent effects (amongst them those used by Castells 1 ) calculated using the PCM model decrease the difference by a small amount: benzene, 79.7; diethylether, 75,4; and ethanol, 70.4 kJ mol À1 .</p><p>Calculation of the IR spectrum of compound 7 leads to scaled bands at 3419 cm À1 (nNH) and 1523 cm À1 [(NQNQC) AE ]. Using eqn (1), these values in KBr should be 3319 and 1520 cm À1 . Castells et al. did not report a CQN band but 3319 cm À1 is consistent with the experimental value of 3295 cm À1 .</p><!><p>Two reviews provide an overview of the electrocyclic reactions and sigmatropic reactions involving pyrazolines. 23,24 We have already reported the 1 + 2 -3 reaction with a TS of 73.8 kJ mol À1 with regard to 3 (IRC, Fig. 7). Other reactions that we have studied theoretically are reported in Fig. 8 and 11 together with acidbase equilibria (protonation) and with rotations about single bonds. Note that the loss of dinitrogen to afford 4 is a very exergonic reaction. Regarding Fig. 8, as we have already commented, the proposed structure of intermediate 7 lies 88.3 kJ mol À1 higher than 6 (71.2 kJ mol À1 above 3). A 1,2 (C to N) transfer of hydrogen has a barrier of 289.7 kJ mol À1 ; the subsequent 1,2 (C to N) transfer of hydrogen to afford 6 has a barrier of 289.7 kJ mol À1 . These very high barriers do not correspond to real pathways because proton transfers assisted by solvent molecules have much lower barriers (see previous discussion).</p><p>Ring opening of 7 to 8a should occur thermally in a disrotatory way. 23,24 However, the geometry of 8a (Fig. 9) allows only a conrotatory mechanism with a barrier of 71.9 kJ mol À1 with regard to 7. A rotation about the single NC bond affords the more stable 8b while prototropy results in the less stable azine 8c. Note that according to the Woodward-Hoffmann rules, 8-10 a conrotatory mechanism is thermally forbidden.</p><p>We then considered that the complex 1 : 5 (Fig. 1) could be the cycloaddition product of 7 (an azomethine imine) with 1 but the resulting 9 is a bicyclic compound very destabilized by the eight cyano groups (Fig. 8).</p><p>D 2 -Pyrazoline (4,5-dihydro-1H-pyrazole-4,4,5,5-tetracarbonitrile) 6 can result from the reaction of tetracyanoethylene 1 with diazen-1-ium-1-ylidenemethanamide (10), a 1,3-dipole with octet stabilization (a nitrile imine). [23][24][25][26] The cycloreversion barrier of 166 kJ mol À1 is much higher than that of diazomethane.</p><p>Isomerization 6 -12a [(2,2,3,3)-tetracyanocyclopropyldiazene, 67.8 kJ mol À1 ] corresponds to a [1,3]-sigmatropic transposition that has been reported in other compounds. 23 In particular, Rosenkranz and Schmid described the photochemical transformation of 5-phenyl-D 2 -pyrazolines into compounds similar to 12a (1-methylazo-2-phenyl-cyclopropanes); on thermal treatment, these compounds are reconverted into the corresponding pyrazolines. Compound 12b, an isomer of 12a, also possible from a similar mechanism, lies much higher in energy.</p><p>Loss of HCN from 6 affords 15. According to Rodrı ´guez Mora ´n, when they carried out the reaction of 1 + 2 to afford 6, 3 release of hydrogen cyanide was observed. 27 The last reaction we have studied is the cycloaddition of diazomethane 2 on D 2 -pyrazoline 6. It is known that D 2 -pyrazolines react with 2 to afford 1,2-diazabicyclo[3.1.0]hexanes, related to 14. 28 These compounds should result from the loss of dinitrogen of either 13a or 13b. According to the literature, the cycloaddition of diazomethane on imines (or Schiff bases) results in 1,2,3-triazolines related to 13a. 29 In our case, the reverse addition leads to a more stable compound, 1,3,4-triazoline 13b instead of 1,2,3-triazoline 13a; this is probably related to the fourth N atom, i.e. our compounds are hydrazones, not imines. The loss of dinitrogen to form 14 is strongly favored (À151.4 kJ mol À1 ).</p><p>We then decided to carry out calculations parallel to those of Calatroni and Gandolfi. 5 They are reported in Fig. 10.</p><p>Compound 16 is the complex of 1 and 3 (compared with the 1 : 5 complex of Fig. 1 and with complex II of Fig. 3); it is located 7.1 kJ mol À1 above 6 and at À10.0 kJ mol À1 from 3. The zwitterion 17 (compare with III of Fig. 3) is not stable and reverts to 16 both in the gas phase and in ethanol (PCM); this sheds doubt on the hypothetical III structure. 5 Structure 19 lies 66.0 kJ mol À1 above 6, also an indication that structure V in Fig. 3 was probably never formed. Charge-transfer complex 18a is not stable and evolves to the hydrogen-bonded complex 18b; it is located at À20.1 kJ mol À1 from 6. The stabilization results from the hydrogen-bond and from a CN/CN stacking between both molecules. It is also interesting to study the reactivity of the conjugated acids of structures of Fig. 8 because Castells et al. indicated that 5/6 equilibration was acid catalyzed (Fig. 11). 1 Usually, D 2 -pyrazolines protonate on N1 (type a) 30 but probably due to the presence of the four cyano groups, in this case, the protonation takes place preferably on N2 (6bH + ), which corresponds to the hypothesis of Castells et al.. However, the most important finding is that 5H + (even the most stable 5bH + , Table 2, 154.3 kJ mol À1 ) has an energy that makes it impossible to isomerize 6H + into 5bH + . The cations have relative energies similar to those of the neutral molecules, 6 o 3 { 5. Although the synthesis of D 2 -pyrazolines protonated on N1, 6aH + , from 1 and 10H + , is unknown, both the stability and the barrier made it a feasible possibility.</p><p>One of the reactions of D 2 -pyrazolines protonated on N1 is to open into compounds related to 20b; this has been proven experimentally. 30b However, in the present case, the optimization led to a quaternary azetinium (2,3-dihydroazetium) cation 20a, still too high in energy.</p><p>The loss of HCN from 6bH + generates a protonated isopyrazole 15H + (1H-isopyrazolium) that is slightly less stable. Protonation of the cyano groups leads to cations 6cH + (CN at position 4) and 6dH + (CN at position 5) of similar energies to 6aH + , i.e. the CN groups are similar to the amino group of a pyrazoline. This is a surprising result because the PA of CH 3 CN is 787 kJ mol À1 and that of CH 3 NH 2 is 899 kJ mol À1 , 31 but the role of the four CN groups and the D 2 -pyrazoline structure may modify the proton affinity; it is known that cyano derivatives can be superbases. 32 From the most stable of these cations, loss of protonated hydrogen cyanide results in the formation of 15 with an energy of 59.9 kJ mol À1 .</p><!><p>Castells intermediate 5 (Fig. 1) 1 is characterized by IR in KBr by a stretching NH at 3295 cm À1 and a bending NH at 1592 cm À1 as well as by the absence of a CQN band although these last bands appear in D 2 -pyrazolines at 1555-1570, 33 1557, 34 1560, 35 1580-1592, 36 1600 37 and 1618-1622 cm À1 . 38 Therefore, the compound has a NH group, i.e., it is not a D 1 -pyrazoline, but the presence or absence of a CQN band is dubious.</p><p>The reactivity of intermediate assumed to be 5 is that it never affords 3 but it is in equilibrium with 6. Light and TCNE isomerize 5 to 6; water, HCl and time isomerize 6 to 5. 1 If pure 6 can be transformed into 5, this latter must be an isomer not containing TCNE.</p><p>Of all the compounds that we have studied, which is the best candidate? Our calculations of IR spectra and its high energy show that 5 can be definitely excluded. That the intermediate could be a salt was possible when HCl was used but not with wet ether. If, by an experimental error, TCNE still remained in the reaction medium, 16 is a good candidate due to its energy and absence of barrier.</p><p>Finally, the best candidates because they are consistent with the IR data are 7, as already discussed, and 8b. The linear equation relating both variables is:</p><p>The problem is that the available information is too scarce, there is not even the complete IR spectrum of 5 let alone the 1 H NMR data. This added to the non-reproducibility of the preparation of 5 3,27 made it impossible to ascertain its structure. Charge transfer complexes are colored (yellow/red) but no indication of the color is reported. 1 On the other hand, a plethora of structures and reactions surrounding the chemistry of tetracyanoethylene (1) has been explored covering different aspects of the chemistry of pyrazolines 39 that would prove useful in related studies because several of them were not experimentally known.</p>

Royal Society of Chemistry (RSC)

Stereoselective synthesis of chromane derivatives via a domino reaction catalyzed by modularly designed organocatalysts

A highly enantio- and diastereoselective method for the synthesis of functionalized chroman-2-ones and chromanes was achieved by using an organocatalytic domino Michael/hemiacetalization reaction of aliphatic aldehydes and (E)-2-(2-nitrovinyl)phenols followed by a PCC oxidation and dehydroxylation, respectively. Using the modularly designed organocatalysts (MDOs) self-assembled from cinchona alkaloid derivatives and amino acids in the reaction media, the title products were obtained in good to high yields (up to 97%) and diastereoselectivities (up to 99:1 dr) and excellent enantioselectivities (up to 99% ee).

stereoselective_synthesis_of_chromane_derivatives_via_a_domino_reaction_catalyzed_by_modularly_desig

Introduction<!>Results and discussion<!>Representative procedure for the synthesis of chroman-2-ones via the domino Michael/hemiacetalization followed by an oxidation reaction:<!>General procedure of the dehydroxylation reaction:<!>Conclusions

<p>Michael addition to nitroalkenes is a powerful tool in organic synthesis that enables the synthesis of complex organic molecules bearing the synthetically useful nitro group. Not surprisingly, organocatalytic nitro-Michael reactions have been extensive investigated in the past decades.1</p><p>Chroman-2-one and chromane are important classes of benzopyran derivatives.1 The dihydrocoumarin and chromane scaffolds are found in many natural products and synthetic molecules that frequently exhibits unique biological and pharmacological activities,2 such as antineoplastic activity,3 antiherpetic activity,4 and the inhibitive activities against protein kinases,5 aldose reductase,6 and HIV-1 reverse transcriptase.7 Owing to the importance of the chromane scaffold, its stereoselective synthesis has attracted considerable attention.8 Indeed, several organocatalytic methods have been developed to access this core structure in an asymmetric manner.9–13 For examples, Ramachary,9 Enders,10 Gong,11 and Hong12 have independently developed organocatalytic domino14 Michael/hemiacetalization reactions followed by an oxidation reaction for the efficient synthesis of chroman-2-one derivatives in a highly stereoselective manner.</p><p>Our group is interested in developing novel catalytic methods15 using the modularly designed organocatalysts (MDOs),16,17 which are self-assembled in the reaction media from cinchona alkaloid derivatives and amino acids. Herein, we wish to report that, using MDOs as the catalysts, the reaction between aliphatic aldehydes and (E)-2-(2-nitrovinyl)phenols gives the expected domino Michael/hemiacetalization products, which may be converted to functionalized chroman-2-ones and chromanes by PCC oxidation and dehydroxylation, respectively (Scheme 1). The desired chroman-2-ones and chromanes were both obtained in good yields and high stereoselectivities.</p><!><p>Hydrocinnamaldehyde (1a) and (E)-2-(2-nitrovinyl)phenol (2a) was adopted as the model substrates. Several cinchona alkaloid derivatives and amino acids (Figure 1) were adopted as the precatalyst modules. These two modules have complementary basic and acidic functional groups that can help them self-assemble in situ in the reaction media. The most interesting results of the catalyst screening are collected in Table 1. As the results in Table 1 show, when quinidine thiourea 6a and L-proline (7a) were adopted as the stereocontrolling module and the reaction-center module, respectively, the reaction of 1a and 2a gave product 4a (after oxidation with PCC) in a high yield (94%) and excellent diastereoselectivity (96:4 dr) and ee value (99%, entry 1). Control experiments conducted with either 6a or 7a alone as the catalyst did not yield any product under otherwise identical conditions (entries 2 and 3). These results confirm that the observed catalytic activity is indeed due to the in-situ generated MDO.</p><p>Similar results were obtained when the MDO self-assembled from cinchonine thiourea 6b and 7a was applied, except that the obtained product yield (80%) and diastereoselectivity (87:13 dr) were slightly lower (entry 4). Much lower product ee value (78% ee) was obtained when the MDO 6c/7a was employed as the catalyst (entry 5). The MDO 6d/7a yielded very similar stereoselectivities as 6c/7a did, but the product yield (97%) was much better (entry 6). Similar results were also obtained for the MDOs 6e/7a and 6f/7a (entries 7–8). In contrast, a poor product ee value (32% ee) was obtained when the MDO 6g/7a was applied (entry 9). These screening identified the stereocontrolling module 6a is the best one for this reaction in terms both the product yield and stereoselectivities (entry 1). Using 6a as the stereocontrolling module, we next screened several amino acids as the reaction-center module. The pseudo-diastereomeric MDO formed from 6a and D-proline (7b) led to the formation of the enantiomer of 4a in a high yield, but only moderate stereoselectivities (84:16 dr, 75% ee) (entry 10). Very good results were also obtained from the MDO 6a/7c (entry 11), which was only slightly inferior to that of 6a/7a (entry 1). However, almost no product could be isolated from the reaction catalyzed by the MDO self-assembled from 6a and L-thioproline (7d) (entry 12). Thus, the above screening identified MDO 6a/7a (entry 1) as the best catalyst for this domino Michael/hemiacetalization reaction. Next the solvent was screened for this best MDO. Common organic solvents, such as xylenes (entry 13), benzene (entry 14), and CH2Cl2 (entry 15) all yielded inferior diastereoselectivities. Slightly inferior results in terms of both yield and stereoselectivities were also obtained from the environmentally benign solvent cyclopentyl methyl ether (entry 16). On the other hand, much poorer product ee value was obtained (14% ee) in MeOH (entry 17). THF (entry 18), 1,4-dioxane (entry 19), and CH3CN (entry 20) also turned out to be poor solvents for this reaction since either only trace amount product or no product could be obtained from these solvents. When the catalyst loading was reduced to 5 mol%, the yield and stereoselectivities obtained for 4a were only slightly lower (entry 21).</p><p>Once the reaction conditions were optimized, the scope of this reaction was studied and the results are collected in Table 2. As the results in Table 2 show, besides hydrocinnamaldehyde (1a, entry 1), other linear aldehydes, such as propanal (entry 2), butanal (entry 3), pentanal (entry 4), heptanal (entry 5), also react with (E)-2-(2-nitrovinyl)phenol (2a) to give the desired chroman-2-ones 4b-e after oxidation in high yields (83–97%), good to excellent diastereoselectivities (81:19 to 98:2 dr), and excellent ee values (97–99% ee). In general, higher diastereoselectivities were obtained with longer chain aldehyde substrates. With the branched 3-methylbutanal high diastereoselectivity of 99:1 dr and enantioselectivity of 93% ee were obtained for the corresponding chroman-2-one 4f (entry 6). Similarly, 2-methylpropanal also yielded the expected 4g after oxidation in 96% ee, although in a lower yield (69%, entry 7). Using pentanal as the aldehyde component, various substituted (E)-2-(2-nitrovinyl)phenols were then screened. It was found that these substituted (E)-2-(2-nitrovinyl)phenols usually led to slightly lower yields (65–87%) and diastereoselectivities (80:20 to 95:5 dr) of the corresponding chroman-2-ones (4h-n, entries 8–14) as compared to those obtained from the unsubstituted (E)-2-(2-nitrovinyl)phenol (entry 4). However, the product ee values remained high (entries 8–14). On the other hand, the electronic nature and the position of the substituent on the phenyl ring of (E)-2-(2-nitrovinyl)phenol had no significant effects on the diastereoselectivities or the product ee values (entries 8–14), except that a slightly lower ee value was obtained for the chroman-2-one product of the 4-nitro-substituted phenol (entry 10). Using the branched 3-methylbutanal as the aldehyde component yielded comparable results with those of pentanal (entries 15–16 vs. 8–9).</p><p>To demonstrate the synthetic utility of this method, the same reaction was also carried out at 0.5-mmol scale of 1a and 2a. As the results in Table 2 show, product 4a was obtained in comparable yield, diastereoselectivity, and ee value as those of the small-scale reaction (entry 17 vs. entry 1).</p><p>To obtain the 3,4-substituted chromanes 5, the primary domino Michael/hemiacetalization products 3 were dehydroxylated by treating with triethylsilane and boron trifluoride diethyl etherate in dichloromethane (Table 3). As shown in Table 3, the dehydroxylation reaction provided the desired products 5a-c in good to excellent yields (72–94%) with preservation of the diastereoselectivities (85:15 to 95:5 dr:) and enantioselectivities of the domino reaction (92 to 98% ee).</p><p>The absolute stereochemistry of the major enantiomeric products of compounds 4 and 5 was determined as shown in the Tables by comparing the measured optical rotation of compounds 4d and 5a with those reported in the literature.12 Based on the product stereochemistry and a recent computational study our MDO catalytic system,18 a plausible transition state is proposed to account for the formation of the major stereoisomer of the domino Michael/hemiacetalization reaction (Scheme 2). As shown in Scheme 2, the Si-Si attack of the preferred syn-(E)-enamine18 of hydrocinnamaldehyde onto the (E)-2-(2-nitrovinyl)phenol (2a) yields the Michael addition intermediate 6 with the expected stereochemistry of the two stereogenic centers, which, after an intramolecular hemiacetalization reaction, gives product 3a. Product 3a yields the expected 4a upon oxidation.</p><!><p>To a vial were added sequentially the precatalyst modules 6a (5.9 mg, 0.010 mmol, 10.0 mol %) and 7a (1.1 mg, 0.010 mmol, 10.0 mol %) and dry toluene (1.0 mL). The resulting mixture was stirred at room temperature for 15 min. Compound 1a (16.0 mg, 0.12 mmol, 1.2 equiv.) was then added and the mixture was further stirred for 5 min. before the addition of compound 2a (16.5 mg, 0.1 mmol, 1.0 equiv.). The resulting solution was stirred at room temperature for 16 h until the reaction was complete (monitored by TLC). Then the reaction mixture was concentrated under reduced pressure and the residue was purified by flash column chromatography to give the chroman-2-ol 3a as a colorless oil (29.9 mg). A solution of the chroman-2-ol 3a (29.9 mg, 0.10 mmol) in CH2Cl2 (3.0 mL) and PCC (64.5 mg, 0.30 mmol, 3.0 equiv.) was stirred at room temperature for 24 h until the completion of reaction (monitored by TLC). The suspension was filtered through a short pad of silica gel and washed with ethyl acetate. Removing the solvents under reduced pressure afforded the crude product 4a, which was then purified by flash chromatography (30:70 EtOAc/hexane as the eluent) to afford product 4a (28.0 mg, 94%) as a colorless oil.</p><!><p>10,12 To a solution of chroman-2-ol 3 (0.10 mmol, 1.0 equiv.) in CH2Cl2 (3.0 mL) at 0 °C were added triethylsilane (34.9 mg, 0.30 mmol, 3.0 equiv.) and boron trifluoride etherate (42.6 mg, 0.30 mmol, 3.0 equiv.) with stirring. The ice bath was removed after 15 min and the mixture was further stirred for 2 h. Then the reaction was quenched with saturated aqueous NaHCO3 solution (3 mL) and the mixture was extracted with CH2Cl2 (3 × 20 mL). The combined organic phases were dried over MgSO4 and the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel to afford the corresponding chromane 5.</p><!><p>In summary, we have developed a highly stereoselective synthesis of cis-3,4-disubstituted chroman-2-ones and chromanes using a domino Michael/hemiacetalization-reaction of aliphatic aldehydes and (E)-2-(2-nitrovinyl)phenols catalyzed by modularly designed organocatalysts (MDOs) followed by a PCC oxidation or dehydroxylation. The corresponding chroman-2-ones and chromanes were obtained in good to excellent yields and diastereomeric ratios and high ee values.</p>

PubMed Author Manuscript

Extraction of heavy metal complexes from a biofilm colony for biomonitoring the pollution

An extraction method was tested for biomonitoring the biofilm samples containing heavy metals. The fractionation of metal complexes was performed via C-18-HPLC-ICP-MS and MALDI-MS, respectively. The extraction power of some reagents was determined for the heavy metal extraction from biofilm samples collected in Erdemli coast in the Mediterranean Sea. The ammonium acetate solution giving the highest extraction results was found as a suitable extraction reagent. The concentration and pH of the ammonium acetate solution were optimized and found as 1 M and 5, respectively. The chromatograms of metal complexes with the C-18-HPLC-ICP-MS system were taken to determine the effect of the pH of the metal complexes. After performing the extraction, metal bounded biomolecules were characterized by MALDI-MS for the fractions in the C18-HPLC system. It was seen that ammonium acetate extraction (1M, pH 5) might be used in biomonitoring studies due to relatively simple procedure, short analysis period, and low cost. The evaluation of the applicability of the method in biomonitoring studies might be supported by further studies with biofilms having similar characteristics.

extraction_of_heavy_metal_complexes_from_a_biofilm_colony_for_biomonitoring_the_pollution

1. Introduction<!>2.1. Preparation of biofilm samples<!><!>2.2. Digestion of biofilm samples<!>2.3. Selection the extraction reagent and optimization of parameters<!>2.4. Separation of extracted metals with HPLC<!>2.5. Characterization of separated metal complexes in MALDI-MS<!>3.1. Extraction of heavy metals from biofilm colony<!>3.2. Selection of extraction agent for metal complexes<!><!>3.2. Selection of extraction agent for metal complexes<!>3.3. Optimization of the concentration of ammonium acetate solution<!><!>3.3. Optimization of the concentration of ammonium acetate solution<!>3.4. Optimization of pH of ammonium acetate solution<!><!>3.4. Optimization of pH of ammonium acetate solution<!>3.5. Extraction efficiency<!><!>3.5. Extraction efficiency<!>3.6. Separation of extracted heavy metal complexes<!><!>3.6. Separation of extracted heavy metal complexes<!>3.7. Characterization of separated heavy metal complexes by MALDI-MS<!><!>3.7. Characterization of separated heavy metal complexes by MALDI-MS<!><!>3.7. Characterization of separated heavy metal complexes by MALDI-MS<!>4. Conclusions

<p>Heavy metal pollution has been growing, and its release into the environment on a large scale has become an alarming issue. The mean contaminant concentration of seawater cannot be regarded as a reliable measure of related location [1]. Trace metal concentrations were determined in water, biofilms, and sediment matrices of the Bílina River in the Czech Republic [2]. In a recent study, heavy metal levels were investigated by digesting surface sediments on the Mediterranean coast of Morocco [3]. Sediment analysis provides total contaminant load instead of direct ecotoxicological relevance. Concentrations of pollutants may not be correlated with data from sediments in the overlying water column [4]. The relative growth, metal accumulation, and tolerance of different plant parts were examined [5]. The relation between the elemental composition of biofilms (algal biomass) and the water column was tested in a study [6]. Variations in physical, chemical, and biological properties are directly and sensitively observed with a microalgal response. They are excellent indicators sensitive to several environmental variables, including heavy metals [7,8]. Microalgae are capable of accumulating a significant amount of metals [9], and they have some other important characteristics such as rapid growth, wide habit, etc., to be used as bioindicators [10].</p><p>Biofilms are composed of algae, bacteria, etc., their extracellular polymeric substances (EPS) and nonliving materials [11–13]. Metals are included almost in all parts of the biofilm samples. Especially, EPS of algae in mature biofilms [14] is very effective in metal complexation, so reduces the bioavailability of metals and their toxicity to algal cells.</p><p>The capacity of algae both in living and nonliving form on a metal-binding is very high and effective. The first step in metal uptake starts with the adsorption of ions or complexes on a cell wall. Active transport of metal (either essential or toxic) into phytoplankton cells is realized with a chelator. Responsibility for algal survival in metal-polluted environments has been described with several mechanisms; increases in the extracellular metal chelators production [15–17], metal immobilization with binding at the cell surface [18,19] or tolerance in internal mechanisms of storage and detoxification [20].</p><p>MT (metallothioneins) induction was carried out to a single-metal exposure in most experiments [21–25]. However, assessments of MTs should include metal mixtures if used as a biomarker. In that case, it might reflect the actual hazard of contamination when compared to a single toxicant. Studies with MTs response to metal mixtures are scarce. Lecoeur et al. [26], studied the influence of metal mixtures, as Cu+Cd, on MT response, and compared the results with single-metal exposure for the bivalve D. polymorpha.</p><p>All the higher and lower plants were analyzed in terms of the induction of PCs (phytochelatins) with the presence of intracellular heavy metals [27–31]. Recently, the online coupling of HPLC with ICP-MS has been tried for characterizations of heavy metal-binding peptides. Due to its high resolution, the most commonly used HPLC technique is the reversed-phase, as far as the PC separation is concerned [32,33]. Krauss et al. [34] extracted and measured the thiol-containing peptides according to the lines of Grill et al. [35], and reversedphase Cl8-HPLC-ICP-MS was used for the detection. Speciation of 4 metals has been studied for sediments in Egypt using sequential extraction with a 4-step procedure [36].</p><p>In this study, the contents of 8 different metals in biofilms were determined in the extracted form after obtaining a suitable extraction reagent and optimizing its concentration and pH of its solution. Moreover, the usability of the biofilm for the extraction of heavy metals from biofilm samples was also checked. Fractionation and determination of metal complexes without destroying the structure in the biofilms were also studied with C-18-HPLC separation and Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) characterization.</p><!><p>Plexiglass slides (artificial substrate) were used to accumulate biofilm. They were supported by polyvinyl chloride (PVC) holder with plastic clips. PVC was prepared in a rectangular frame (Figure 1). For each station, substrates within PVC frame support were dipped in water with a depth of 40–50 cm for 60 days. Then, plates were taken all at once.</p><!><p>PVC frames with plexiglass slides.</p><!><p>Dry algal sample (0.10 g) was accurately weighed and decomposed with a mixture of 65 % HNO3 (Merck), 35 % H2O2 (Merck), and 37 % HCl (Merck) with the volume percent ratios of 60/20/20, respectively. Digest was quantitatively transferred into volumetric flasks after cooling. It was completed to a final volume of 25 mL with deionized water. After decomposition, the supernatant was centrifuged for 15 min. It was waited to a constant room temperature and then again diluted 10 times with DW when measured in the instrument. Blank and standard solutions of the metals were also prepared in the same conditions. Signal of blank, standards, certified standard material and samples were observed and the total heavy metal contents of sample were determined in ICP-MS instrument. The instrument was calibrated using multi-element standards solutions prepared by mixing HNO3, HCl, and H2O2 and diluting ICP-MS stock solutions of individual elements with the proper ratio of sample content. Aliquot of digest (5 mL) was spiked with internal standard (indium) with a final concentration of 5 μg/L and completed to 10 mL with deionized water. Standard solutions of in 0.01, 0.1, 1.00, 10.00, and 100.00 ng/mLconcentrations were prepared from their stock solutions. Indium was also added to these calibration standard solutions as an internal standard to monitor and compensate for the possible instrumental drift. Various isotopes were selected and used for the determination of the related elements.</p><!><p>Three kinds of reagents (ammonium acetate, ammonium nitrate, potassium sulphate) were selected for the desorption of metals from algal biofilm samples. The concentration of each reagent was 0.1 M in 10 mL. For each treatment, 100 mg of algal samples (weighing is performed after stripping algae from the plexiglass slide surfaces) were added to each solution. Mixtures were kept at 4 °C in a final volume of 10 mL during the extraction step. Samples were put into a shaker, and the tube was shaken for 30 min with an ultrasonic shaker and centrifuged at 5000 rpm for 15 min at 4 °C. Afterward, the supernatants were filtrated using a 10 KDa ultrafiltration membrane (Filter Code: YM10 Dia: 63.5 mm) and Millipore Stirred ultrafiltration cell (8400 Model) to obtain a clear solution. The metal contents of the filtered solutions were determined in ICP-MS after the instrument was calibrated using multi-element standards solutions prepared in different concentrations of ammonium acetate solutions. Aliquots of extracts (8 mL) were spiked with internal standard (indium) and completed to 10 mL with deionized water.</p><!><p>The column chosen for the separation of the metal complexes was ion-pairing C18 (Dionex C18); heptafluorobutyric acid (HFBA) was selected as the ion-pairing reagent. In the C18-HPLC-ICP-MS system, 10.0% of CH3OH in 0.12% HFBA was used as a mobile phase at natural pH (pH = 6.0). The flow rate of mobile phase was adjusted to 1.2 mL/min.</p><!><p>MALDI matrix sinapinic acid (SA, 3,5-dimethoxy-4-hydroxycinnamic acid) was prepared in an acetonitrile:water: trifluoroacetic acid mixture (1:1:0.001, v/v) at a concentration of 10 mg/mL. MALDI samples were prepared by mixing HPLC fractions with the matrix solution (1:10 v/v) in a 1.5 mL Eppendorf microtube. Finally, 1.0 μL of this mixture was deposited on the sample plate, dried at room temperature (RT), and then analyzed for heavy metal complexes. Mass spectra were acquired on a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems, USA) equipped with a nitrogen UV-Laser operating at 337 nm. Spectra were recorded in linear mode with an average of 100 shots.</p><!><p>Instead of quantifying metals simply as adsorbed or absorbed in algal cells, we preferred to determine the metals in both forms at the cell surface and within the cell. The extraction procedure for total metal concentration was given with the optimized parameters in this study.</p><p>A few chemicals to desorb the metals bound to molecules' physical or chemical either in the ion-pair or complex form were examined. Optimizations of concentration and the pH of the selected desorption reagent were also performed in the experiments.</p><p>Three kinds of reagents were selected for the desorption of metals from algal biofilm samples. Ammonium acetate, ammonium nitrate, and potassium sulphate were the reagents, pH of which are near neutral and are thought as unaffected while the treatment.</p><!><p>The suitability of the selected chemicals was examined to be used as an extraction agent for the metal complexes. The concentrations of the reagents were prepared as 0.1 M in 10 mL for each metal complex. The extraction power of 3 reagents is selected as seen in Table 1. It was concluded that the ammonium acetate solution was the extractant, which provided the highest extraction recovery among the solutions studied.</p><!><p>Amounts of metals extracted with different reagent solutions from the biofilm samples (for standard deviations: n = 3. Extracted samples were measured without further dilution except for solid-liquid extraction procedure).</p><!><p>The most remarkable difference has appeared in the iron, cadmium, and copper extractions. It was about 50, 80, and 150% higher with the ammonium acetate reagent than with ammonium nitrate with the significant uncertainty in Cd concentration, respectively. The concentrations of all metals with the potassium sulphate reagent had the lowest values in the extraction. We observed that the extraction recoveries were too low to evaluate the metal complexes in the instruments worked. The reason for the low recoveries was probably due to the low concentration values of the extraction reagents and high pH. Determination of the total content of each metal in the extraction procedure was not possible since the extraction recoveries of metals were much lower than 100%. Nevertheless, the extraction of metals without destroying their complex structures gives an idea about both absorbed and adsorbed metals, separately. Additionally, it was possible to see each metal of the original structure in biofilm.</p><!><p>In this part of the extraction study, the concentration of the selected extraction solution was optimized to obtain the best extraction efficiency. Better extraction results based on ammonium acetate concentration were investigated to reach a reasonable extraction value. The concentration range was limited to appreciable values, which were 0.1, 0.5, and 1.0 M, while the pHs of the solutions were kept constant near to the neutral value of pH 7.0. Values below 0.1 M and above 1.0 M were not studied since the lower values approach to the behaviour of water, and higher values may block the instrument.</p><p>A suitable condition value for ammonium acetate concentration was evaluated to increase the extraction of metals. In the previous part, the concentration of extraction reagent (ammonium acetate) was prepared as 0.1 M and it was seen as too low for the extraction of the metals from the results. Two other concentration values were also tried to increase the extraction efficiencies of the metals. Results of 3 values such as 0.1, 0.5, and 1.0 M are given in Table 2.</p><!><p>Amounts of metals extracted with ammonium acetate solutions (pH = 7) with different concentrations from the biofilm samples (for standard deviations: n = 3. Extracted samples were measured without further dilution except for solid-liquid extraction procedure).</p><!><p>It showed that the concentration of ammonium acetate solution had a serious effect on the extraction of metals. The higher concentration of ammonium acetate was recorded as having the highest recoveries. Nevertheless, concentration values above 1.0 mol.L-1 for ammonium acetate were not tried since high levels of salt concentrations may plug the way to plasma and stop it in off position. Therefore, ammonium acetate solution in 1.0 mol.L-1 concentration was decided to be used for the further extraction studies.</p><!><p>Ammonium acetate solutions in 1.0 M concentration were prepared at different pH values to find out the effect of the pH on the extraction of metals as seen in Table 3. When compared with the extraction results of ammonium acetate solutions prepared at different pH values, it was obvious that there was a reverse relation between the pH and the extraction values as expected. Although each change in pH would not result in a change in the extraction values for some of the conditions, a continuous increase in the extraction of the metals was detected for many of the gradual decreases in pH.</p><!><p>Amounts of metals extracted from biofilms with ammonium acetate solution (1M) with different pH values (for standard deviations: n = 3. Extracted samples were measured without further dilution except for solid-liquid extraction procedure).</p><!><p>The Cell wall matrix of algae contains complex hetero-polysaccharides, which offer carboxyl and sulphate groups. Since deprotonated free carboxyl or sulphate groups were negatively charged, they would electrostatically attract any cation [37]. The magnitude of the overall negative charge increases as the pH increases. Therefore, more sites were deprotonated for the adsorption of metals. Due to these properties of algal cell walls, algal biomass may accumulate metals in the cationic form in the aqueous media in the sea. The accumulated metals in the algal cell walls in the sea may be desorbed when the biosamples were contacted with a solution having a high hydronium ion or salt concentration after they were dried and homogenized in the laboratory.</p><p>Considering the effect of the pH and concentration of ammonium acetate reagent together, a continuous increase in the extraction of metal complexes becomes not surprising due to the high concentrations of salt and hydronium ion. As expected, the ammonium acetate solution to desorb the metal complexes which were not only adsorbed in the cell walls of the algal biosamples extracellularly but also absorbed in the cells intracellularly as the phytochelatin complexes.</p><p>When the pH was changed from 7.0 to 6.0, the desorption values of Cr, Cu, and Cd were almost constant. An interesting result was seen in the Mn-extraction. It was lower in the pH 5.0 than 6.0, unexceptionally. Lower values of pH of the solution were recorded as having the highest recoveries. Also, pH values below 5.0 were not analysed since decreasing pH may create the deformation of the metal complexes. Therefore, ammonium acetate solutions with 1 M concentration were decided to be used at a pH of 5.0 for further extraction studies. To determine whether metal complexes deform or not, extracted metals and their complexes should be separated and characterized.</p><!><p>The total digested content of selected 8-heavy metals was also determined to compare the results with the extracted metal contents of biofilm samples as seen in Table 4. The sequence of the contents of 8-heavy metals in biofilm has appeared as Fe>Mn>Zn>Cu>Cr>Ni>Pb>Cd.</p><!><p>Comparison of the ammonium acetate (1 M, pH 5.0) extracted metals with the acid digested ones from the biofilm samples (for standard deviations: n = 3. Digested samples were measured after 100-fold dilution. Extracted samples were measured without further dilution except for solid-liquid extraction procedure).</p><!><p>The extraction values of the metals were given in the extraction part of the study after the parameters affecting the extraction recovery were optimized. As the concentrations of the metals were thought as taken totally after dissolved in the digestion, the percent extracted values of the metals would be given in terms of total values. Percent extraction values were calculated to form maximum recovery values per total digested values. Extraction efficiencies range from about 5% to 60% for different metals as seen in Table 4. The highest recovery value of 60% belonged to Ni. The lowest recovery was found for Fe concentration with 5%, and the nearest one was Cd recovery with about 18%. The percent recovery values of Cu and Mn were very close to each other at around 36%. Pb, Cr, and Zn recovery values were somehow close to each other, and they were 41%, 46%, and 53%, respectively. As a summary, the sequence for the recovery values was in the order of Ni>Zn>Cr>Pb>Mn>Cu>Cd>Fe.</p><p>There would be a few reasons to have different recovery values for each metal for the same sample. Indeed, the amount of metal sorbed in total components of biofilms may probably vary with metal and their concentrations distributed as EPS, cell wall, and intracellular metal complexes in biofilm. Percent recovery value for each metal depends on its chemical and/or physical form in which the bound structure is mostly affected with the extraction reagent. Although our suggestion for extraction values instead of digestion ones may also be used in biomonitoring studies due to relatively simple procedure, short analysis period, and low cost, more studies with biofilms having similar characteristics should be applied for the evaluation of the applicability of extraction procedure in biomonitoring studies.</p><!><p>After the determination of extraction agent and optimization of its concentration, pH value was not defined for extraction since there was a continuous increase in the desorbed metal content of the sample, while the pH of the agent was decreased step by step. Not knowing the effect of pH of ammonium acetate in the structure of desorbed metal complexes, whether it decomposes the structure or not, all the supernatants of solutions at different pH values were observed in HPLC-ICP-MS system.</p><p>The effect of pH on the extractions of metals was discussed in the previous sections, and there was a gradual increase in the extraction of most metals with decreasing the pH of the ammonium acetate solution. The chromatograms of metal complexes from 3 solutions (pH: 7.0, 6.0, 5.0) in the C-18-HPLC-ICP-MS online system were taken and compared in this part of the work to determine the effect of pH on the metal complexes.</p><p>As chromatograms of metals at different pH values were compared (Figures 2–9), some of them show no change in peak areas, whereas others have different chromatograms like variations in the extraction results. While the peaks in the chromatograms of Cu and Cd were observed as not much changed, those of others were recorded as more or less changed. One peak was observed for Cr, Fe, Ni, Cu, Cd, and Pb metals separately, whereas Mn and Zn have 2 peaks in their chromatograms. Whether there are unresolved peaks or not, we wanted to observe the peak areas whether or not they match the extraction results at the same pH values. Although the areas of the extracted metals in HPLC-ICPMS do not match the results of extracted metals without HPLC, tendencies of results are parallel to each other. The difference in nebulization efficiencies with different reagents (water in extraction and methanol-water-HFBA mixture for HPLC) is the main reason for reaching different results. Since organic solvents mostly have high nebulization efficiencies due to the low surface tension, metal peak areas in an organic solvent are recorded as higher than those in water.</p><!><p>Chromatograms of Cr extracted at 3 different pH values.</p><p>Chromatograms of Fe extracted at 3 different pH values.</p><p>Chromatograms of Mn extracted at 3 different pH values.</p><p>Chromatograms of Ni extracted at 3 different pH values.</p><p>Chromatograms of Cu extracted at 3 different pH values.</p><p>Chromatograms of Zn extracted at 3 different pH values.</p><p>Chromatograms of Cd extracted at 3 different pH values.</p><p>Chromatograms of Pb extracted at 3 different pH values.</p><!><p>Orange, red and green colors were related to pH values of 5.0, 6.0, and 7.0, respectively in all chromatograms.</p><p>As the chromatograms of metals were observed, fascinating results were recorded. A peak was seen at a retention time of 400 s in which no metal had a certain signal except lead chromatogram of extraction sample, which is hardly detected due to the low signal to noise ratio to evaluate. Therefore, selective isolation, determination, and characterization of lead complexes may be possible if the signals are enhanced with increasing the sample amount and/or decreasing pH.</p><p>Observing the peaks near 700 s for Mn and Zn chromatograms is another interesting result due to the signals just belonging to these metals. Although a considerably high increase was observed from pH 7.0 to 6.0 in the first peak, it seems similar in the second one in the Mn chromatogram. Another attractive result was marked in the change from pH 6.0 to 5.0. While they look similar in the first peak, the peak was lower in pH 5.0 in the second retention time for Mn. Peaks are similar at pH 6.0 and pH 7.0 for chromatograms of Zn; however, a specific increase was observed at pH 5.0 at a retention time of about 200 s. The signals were recorded as similar at pH 5.0 and pH 6.0 chromatograms of Zn, however, a certain decrease was observed at 700 s signal of pH 5.0.</p><p>Even though a little peak with a big noise is seen below 200 s for Cd, it is very obvious that Cd levels in the biofilm and extracted solution are too low to evaluate with the conditions performed. The peak was though not meaningful with a signal to noise ratio of about 3. It was clear that observing the change in signals in different chromatograms was unsuitable. Moreover, it seems unchanging while the conditions change.</p><!><p>Selection of the proper matrix is important in MALDI-MS, because different matrices allow analysis of varying types of biomolecules [38]. SA is routinely used in profiling experiments of biomolecules, although other matrices (CHCA, and ferulic acid) can be used. SA, CHCA, and ferulic acid have been shown to be useful for the detection of proteins [38,39]. SA matrix was selected for the characterization of the metal bound MTIII metallothioneins having molecular weights ranging from 2 to 10 kDa and recognized as peptides induced most strongly by metals in the algae. Mixures of MALDI matrix SA (in an acetonitrile:water:trifluoroacetic acid mixture) and HPLC fractions (1:10 v/v) were prepared in 1.5 mL Eppendorf microtubes. 1.0 μL of each mixture was deposited on the sample plate, dried at room temperature (RT), and then analyzed for heavy metal metallothionein complexes.</p><p>Ammonium acetate solution with 1.0 M concentration at pH 5.0 used in the extraction step was used to characterize the metal bounded biomolecule forms by MALDI-MS after the fractions were taken from the C18-HPLC system. The chromatograms can be investigated as dividing into 3 fractions since peaks appeared in 3-regions. The first fraction was taken from the start to 300 s and the second one started from 300 to 500 s. The last fraction was collected from 500 up to 1000 s. These 3 fractions were analyzed to characterize the metal complexes. Methanol, HFBA, and water mixture (5%, 10mM, 95%, respectively) were used in the HPLC separation step.</p><p>Almost all the metals have signals in the HPLC-ICP-MS system in the first fraction, and so the signals in the MALDI- MS system may be related to these metals (see Figure 10).</p><!><p>MALDI-MS spectrum of the first fraction from the chromatograms of extracted metals from the biofilm sample.</p><!><p>The second fraction was very interesting since the only peak appeared in the lead chromatogram. However, certain signal(s) was/were not observed in the MALDI-MS instrument due to very low concentrations of lead in the fraction. Only Zn and Mn peaks were recorded in the last fraction of the chromatograms. Therefore, the peaks in the MALDI-MS may be related to these metal and their complexes (see Figure 11).</p><!><p>MALDI-MS spectrum of the third fraction from the chromatograms of extracted metals from the biofilm sample.</p><!><p>In each fraction (fraction I and fraction III), MALDI-MS spectra showed an almost similar spectrum, which had some mass shifts on the similar biomolecule-metal complexes that appeared at around 4296, 5779, and 8611 Da masses with + 5 Da mass deviations onto the first 2 peaks. These mass deviations might be the different metal complexes of the same biomolecule complexes, such as from manganese to zinc.</p><!><p>Percent recovery value for each metal depends on its chemical and/or physical form in which the bound structure is mostly affected with the extraction reagent. It was found that extraction values could be used in biomonitoring studies due to their advantages. Nevertheless, more studies with biofilms should be applied for the evaluation of the applicability of the extraction procedure in biomonitoring studies.</p><p>Metal chromatograms of the C-18-HPLC-ICP-MS system can be used for each metal determination. It is very interesting that no metal had a certain signal except lead chromatogram of the extraction sample. This relates to the separation of Pb complex from other metal complexes. Therefore, selective isolation, determination, and characterization of Pb complexes may be possible. Observing the peaks near 700 s for Mn and Zn chromatograms is another interesting result due to the signals just belonging to these metals. Even though a little peak with a big noise is seen below 200 s for Cd, it is very clear that Cd levels in the biofilm and extracted solution are too low to evaluate with the conditions performed.</p><p>The second fraction form HPLC was very interesting since the only lead peak appeared in the chromatogram. However, the signal was not observed in MALDI-MS due to very low concentrations of lead in the fraction. However, it can be used in the work where the Pb concentration is higher than this work. Only Mn and Zn peaks were recorded in the last fraction of the chromatograms. Therefore, the peaks in the MALDI-MS may be related to these metals and their complexes. When the metal-biomolecule complex peaks were examined in detail, it could be noticed that each complex peak did not occur. It also had some isotopic mass distribution, such as metal isotopic peak distribution. This showed that the type of metal ion in each MALDI-MS peak could be identified when the high resolved mass spectra were obtained.</p>

PubMed Open Access

Langmuir-Blodgett Films of Supported Polyester Dendrimers

Amphiphiles with a dendritic structure are attractive materials as they combine the features of dendrimers with the self-assembling properties and interfacial behavior of water-air affinities. We have synthesized three generations of polyester dendrimers and studied their interfacial properties on the Langmuir films. The behavior obtained was, as a rule, the lowest generation dendrimers behaving like traditional amphiphiles and the larger molecules presenting complicated isotherms. The Langmuir films of these compounds have been characterized by their surface pressure versus molecular area (π/A) and Brewster angle microscopy (BAM) observations.

langmuir-blodgett_films_of_supported_polyester_dendrimers

1. Introduction<!>2. Experimental<!>3.1. Synthesis<!>3.2. Isotherms Surface/Atmospheric Pressure<!>3.3. Brewster Angle Microscopy (BAM)<!>4. Conclusions

<p>Various synthetic routes and structural modifications of dendrimers have been proposed, and several of their properties are now understood [1–4]. Therefore, potential applications of dendrimers at surfaces are clearly growing, for example, in surface-based catalysts [5, 6]; in the synthesis and stabilization of metal nanoparticles or as drug delivery supports [7]. Nevertheless, the general principles determining molecular conformation at the air-water interface are still under study [8–10], with unsolved questions such as the differences in the molecular shape near the interface and their relationship to the three-dimensional; the importance of the chemical functionalities of the different parts of the molecule; the shape of the formed monolayers, and so forth. So far, the knowledge of dendrimer structure in Langmuir films (at the air-water interface) comes largely from pressure-area (Π-A) measurements, which are however open to multiple interpretations [11–16].</p><p>During the past few years there has been a considerable interest in the use of dendrimers as surface and interface active materials. The preparation of Langmuir films from amphiphilic dendrimers or monodendrons has been investigated by several research groups [17–21], since the Langmuir technique helps to access valuable information about the behavior of dendrimers at the air-water interface. Previous publications showed possible approaches to achieve surface activity for dendritic molecules, but few of them have described Langmuir films obtained from dendritic molecules [22–25] and their application.</p><p>In this paper, we report the incorporation in Langmuir films of three generations of polyester dendrimers, in order to contribute to the understanding of the supported dendrimer properties and their behavior.</p><!><p>3,5-Dihydroxybenzoic acid (DHBA) (97+%), 1,3,5-tris(2-hydroxyethyl) cyanuric acid (THECA) (97+%), and p-toluenesulfonic acid (PTSA) (98.5+%) were purchased from Sigma-Aldrich and used without further purification. Tetrahydrofuran (99+%) was purchased from Sigma-Aldrich, degassed, and dried using standard procedures.</p><p>Thin-layer chromatography (TLC) was performed with TLC plates, silica gel on aluminum (Sigma-Aldrich), and Hexane : THF (mix v/v 3 : 2) as an eluent. Column chromatography was performed with Sigma-Aldrich silica gel 60 Å with a pore diameter range of 63–200 μm. 1H (TMS at 0.0 ppm internal reference) and 13C NMR (DMSO-d6 at 39.5 ppm internal reference) experiments were carried out in a 400 MHz NMR spectrometer Varian Unity Inova. Elemental analyses were made on a Fisons EA1108 elemental analyzer. UV-visible absorption spectra, in colloidal dispersion, were taken with an Ocean Optics USB200 miniature fibreglass optic spectrometer and fluorescence spectra were taken with a Jobin Yvon Horiba Fluoromax spectrometer.</p><p>The hyperbranched polyester dendrimers were produced by the polycondensation of stoichiometric amounts of the monomer DHBA corresponding to each generation with 1 mol of THECA as a trifunctional central core. According to the literature methods [26], the coupling reaction preceded without protection of the hydroxyl groups. The monomer to core ratio was varied between 3 and 12 equivalents. p-Toluenesulfonic acid (PTSA) (2 wt% based on DHBA monomer) was used as an acid catalyst in all reactions. The polymerization reaction was carried out in bulk at 220°C. After 10 min, the temperature was reduced to 180°C, the reaction mixture was kept at this temperature for 5 min to obtain several generations of growth, and the procedure was repeated, with a stepwise addition of stoichiometric amounts of monomer to a core molecule in a synthesis designed to mimic the generation of a dendrimer (Figure 1). The yield of all obtained polymers was found to be above 90% for generation 1, and 50% for generations 2 and 3.</p><p>G1 was considered to be the first generation after coupling 3 mol of the monomer with 1 mol of the central core. THECA (0.01 mol, 2.61 g), DHBA (0.03 mol, 4.624 g), and PTSA (0.092 g) were placed in a three-necked condensation flask equipped with a nitrogen inlet, a drying tube, and a stirrer. The reaction vessel was evacuated for 10 min and flushed with nitrogen three times and then placed in an oil bath that was preheated to 220°C. The reaction mixture was left to react for 10 min under a stream of alternated nitrogen and vacuum to remove the water formed during the reaction. After the solvent was evaporated, the crude product was purified with column chromatography, resulting in a purified compound in the form of yellow crystals, yield (90%). 1H and 13C NMR spectroscopic results are consistent with the formulation proposed. Elemental analysis results also agree with the proposed formulation.</p><p>For the Langmuir Films, data were collected with a KSV 5000 system 3 using a Teflon trough and barriers in a dust-free environment. The temperature was controlled to ±0.1°C. All the isotherms presented here were taken at 20°C. Ultrapure water (ρ = 18.2 MΩ·cm) obtained from a Milli-DIPAK/Milli-Q185 ultrapurification system from Millipore was used for the subphase. Surface pressure was measured by means of a platinum Wilhelmy plate. The spreading solutions were prepared by dissolving the dendrimers in chloroform at 1 mg/mL concentrations. The solutions were spread on the water surface with a microsyringe, and the film was then left for 15–20 min to equilibrate before the compression started. The monolayers were compressed at 4 mm/min. The phase transitions of the spread monolayer were observed using a Nanofilm Technologie Brewster angle microscopy (BAM) fitted with a Teflon trough such that images of the air-solution interface could be taken by the CCD camera. The images were corrected by subtracting a reference image to account for the nonuniform illumination by the laser beam across the image. The image contrast consisted of 256 gray levels.</p><!><p>The dendrimers were synthesized according to common acid-catalyzed esterification procedures [26]. All prepared dendrimers are phenolic-terminated polyesters and they are soluble in methanol, acetone, tetrahydrofuran and DMSO, and partial soluble in chloroform.</p><p>The 1H NMR (DMSO-d6) spectrum shows two sets of signals at 4.4 ppm (2H) and at 3.8 ppm (2H) assigned to the protons on the CH2–O groups with another two sets of signals at 4.1 and 3.4 corresponding to the protons on the CH2–N groups. Finally three sets of signals at 7.9, 7.8 and 7.4 ppm (9H), were assigned to phenyl protons of benzilic rings. 13C NMR (DMSO-d6) exhibits a set of various singlets at 160 ppm (2C), at 168 ppm (2C), and 149 ppm (3C) assigned to the carbonyl carbon atoms and to the carbon atoms on the N–C=O, CH2 groups, respectively; a second set of signals at 158 ppm (6C), 132 ppm (6C), and 107 ppm (6C) assigned to the aromatic carbon atoms; a third set of signals at 40.9 ppm (3C) and 40 ppm (3C) assigned to the aliphatic carbons. The FAB mass spectrum shows a peak at 669 m/z correspondent to the fragment [M +-Cl]. Anal. Calc. for CHN: C, 55.4; H, 4.65; N, 7.18%. Found C, 53.8; H, 4.56; N, 6.96%.</p><p>The IRs of the different generations show slight differences; in the region around 3200 cm−1, these differences are attributed to interactions between terminal OH groups, which increases with the generation; thus the band becomes wider. Another area that can be identified as being changed is around 1595 cm−1, which corresponds to carboxylate groups; 1200 cm−1 which is assigned to DHBA aromatic rings; 900 cm−1 corresponding to amide symmetry and anti-symmetry vibrations; 700–650 cm−1 to ester groups, which actually are the more affected groups when the generation is growing up. All of these regions are marked in Figure 2.</p><p>The photophysical properties of the obtained dendrimers were studied by UV-vis absorption and fluorescent spectrometry. The UV-vis absorption spectra of the three dendrimers (G1, G2, and G3) in DMSO are shown in Figure 3. The spectrum displays differences between the three dendrimers, with two intense bands at 261 and 330 for G1; a broad band at 330 nm (which includes both the bands of G1) for G2; two intense bands at 330 (again this one include both bands from G1) and at 424 nm for G3. Upon excitation at 260, 330, and 420 nm, correlated with the absorption maxima, the polymers were found to be mainly blue emitters which solutions exhibited intense fluorescence, with two maxima at 330 and 720 nm for G1; at 330, 455 (shoulder) and 720 nm for G2; at 330 and 594 nm, for G3 (Figure 3). The 720 nm emission is less intense and is associated to far-red emitters. As in previous publications [26], both the peak position and the emission profiles remained independent of the excitation wavelength. According to the literature [27], the intense peaks could be attributed to the emission from the intramolecular interaction between excimers. As already suggested, these polymers could be considered fluorescent materials promising for applications in molecular photonics or in fluorescent markers.</p><!><p>The LB technique allows measuring changes in surface pressure according to area at constant temperature, in this case 25°C, and with a defined value of absorbed molecules. The isotherms (Figure 4) showed that the polyester dendrimers are dispersed spontaneously over the water-air interphase and that, when compression begins, the film does not collapse, forming Langmuir monolayers even when generation increases; that is, the isotherms showed that it is possible for Langmuir monolayers to be formed with the three polyester dendrimers even though they have a large proportion of hydrophobic aromatic groups, which explains the fact that high surface pressures are not reached.</p><p>The collapse pressures that were reached (π c) are summarized in Table 1. The G1 polyester dendrimer reached the highest collapse pressure. The area held by each molecule in the Langmuir monolayer is determined by drawing a tangent over the liquid region of the surface pressure isotherms. The previous graph shows that the G1 polyester dendrimer is the one taking up the smallest area, with 133 Å2/molecule, followed by the G2 polyester dendrimer with a molecular area of 291 Å2/molecule, whereas the G3 polyester dendrimer held a larger area of 806 Å2/molecule. The previous indicates that, when dendrimer generation increases, the molecular area doubles, because molecule size also increases, therefore taking up a greater molecular area. The found areas are close to the expected area for a chain containing phenyl rings [11] and agree perfectly with the molecular modeling calculations (Table 1). G1 has a final molecular area of A 0 = 133 Å2, G2, A 0 = 291 Å2, and G3, A 0 = 806 Å2; for this series, the final molecular area varies almost linearly with the number of OH groups, as can be seen in Figure 5. The final packing must therefore be very dense for G1 molecules and decreases as the dendrimer generation increases.</p><p>The films for the polyester dendrimer G1 show excellent reversibility in successive compression-expansion cycles, as long as the π is kept below the collapse pressure π c ≈ 16 mN/m, and BAM observations revealed the quality of the films (Figure 6). The film obtained for G1 is noncontinuous at large area values, with holes through it, where water can be seen. These domains smoothly weld together, when the molecular area goes below A ≈ 133 Å2.</p><p>For the polyester dendrimers G2 and G3 the behavior is different, that is, showed no reversibility in successive compression-expansion cycles as long as the π is kept below the collapse. At the same time, defects can be seen in the BAM pictures confirming that the films are of poor quality, perhaps due to steric hindrance and the imbalance between the hydrophilic and hydrophobic groups with increasing generation of dendrimers [28].</p><p>Hysteresis curves of the G1–G3 polyester dendrimers were drawn, making it possible to observe the methods' reproducibility and stability. These were obtained with the same experimental conditions as the isotherms (concentration, barrier speed, injected volume, and temperature), compressing down to a surface pressure lower than that of the collapse, so as to immediately relax the barrier completely and repeat the cycle.</p><p>The first graph depicted in Figure 7 shows the hysteresis curve of dendrimer G1. It is possible to observe how the compound continues to have the same behavior after 6 cycles of compression and decompression. A very slight and continuous change is observed in the consecutive isotherms toward low molecular area, which is due to the organization that was reached during previous cycles, which is not completely lost. The previous implies that, for G1 polyester dendrimer, as long as compression is made under the same conditions, the same molecule organization will be reached over the water/air interphase of the Langmuir trough.</p><p>Comparison of the hysteresis curves (Figure 7) shows that only the G1 polyester dendrimer showed irreversibility, that is, it formed more stable films than dendrimers of higher generation, which confirms that this family of dendrimers is not apt for the transfer of solid supports since it is not possible to maintain equilibrium conditions. Apparently, larger surrounding polyester chains decrease the attraction to the SIO2 molecules from the support. The explanation used by Pao et al. [11], for similar results, can be employed here; thus, the hysteresis curves results indicate that the molecular area on larger generations of the studied dendrimers must be largely, but not entirely, determined by the sum of loosely packed individual alkyl chains, with the chains extending radially away from the surface. G1 polyester dendrimer has a somewhat smaller area, but not unphysically so. The limiting molecular area for G1 and G2 is much larger, but the area after compression to the plateau is again in the same range. A provisional explanation for this effect could be that the molecule lies essentially in a "flat" way on the surface in the gas phase, but lifts off and extends under compression.</p><!><p>Figure 8 shows a series of images that are representative of the interfacial behavior of the G3 polyester dendrimer, in a wide range of areas between 806 and 133 Å2/molecule. The first image shows the interface during the injection process, when the solvent is still present. It is possible to observe two shades, the clearer one being the solvent, which in this case is water. The second image shows the film aspect during the compression process, where it is possible to observe that the film is homogeneous. The third image, which corresponds to a surface pressure of 6 mN/m, shows the emergence of a liquid phase. In the following images, it is possible to observe the emergence of domains that extend over the surface as pressure increases, until reaching the collapse pressure, at which monolayer rupture occurs and structural systematic defects appear.</p><p>In a comparison of BAM images for the polyester dendrimers (Figure 9), the effect of generation on molecular orientation in the water/air interface can be seen. Considering the variations in contrast between experiments, a similar molecular arrangement is observed in the gas phase for all three dendrimers. Subsequently, it is possible to detect how the domains coalesce into a homogeneous liquid phase in the following series of images. Finally, it is shown that the structural defects are more important, proportional to the generation.</p><!><p>In conclusion, we have shown that the polyester dendrimers are suitable amphiphilic derivatives for the preparation of Langmuir films; however, the quality of the films decreases with increasing dendrimer generation, which suggests that larger dendrimers are not apt for the transfer of solid supports since it is not possible to maintain equilibrium conditions. The amphiphilic character of G2 and G3 polyester dendrimers might be reduced due to the relatively low hydrophilicity of the central triazine-2,4,6-trione group. This may result in films that are relatively unstable to multilayer collapse compared with the small generation dendrimer studied. Thus we are currently working on the synthesis of composites with the polyester dendrimers and Pd(0) NPs, to study their modification on the solid support transference. As expected, it can be observed that the surrounding polyester chains' length of the dendritic branches has a remarkable influence on the isotherm phases as well as on the packing of highest generation dendrimers in the monolayers. The studies on the composites, polyester-dendrimers-Pd(0) NPs will conduce on the transference of the Langmuir monolayers onto solid substrates, using the LB technique to study their catalytic reactions on heterogeneous catalysis.</p>

PubMed Open Access

Expanding the Substrate Scope of Nitrating Cytochrome P450 TxtE by Active Site Engineering of a Reductase Fusion

AbstractAromatic nitration reactions are a cornerstone of organic chemistry, but are challenging to scale due to corrosive reagents and elevated temperatures. The cytochrome P450 TxtE nitrates the indole 4‐position of l‐tryptophan at room temperature using NO, O2 and NADPH, and has potential to be developed into a useful aromatic nitration biocatalyst. However, its narrow substrate scope (requiring both the α‐amino acid and indole functionalities) have hindered this. Screening of an R59 mutant library of a TxtE‐reductase fusion protein identified a variant (R59C) that nitrates tryptamine, which is not accepted by native TxtE. This variant exhibits a broader substrate scope than the wild type enzyme and is able to nitrate a range of tryptamine analogues, with significant alterations to the aromatic and aminoethyl moieties.

expanding_the_substrate_scope_of_nitrating_cytochrome_p450_txte_by_active_site_engineering_of_a_redu

<!>Conflict of interest<!>

<p>R. Saroay, G.-D. Roiban, L. M. Alkhalaf, G. L. Challis, ChemBioChem 2021, 22, 2262.</p><p>Aromatic nitration is an important industrial process used in the preparation of dyes, pesticides, food additives and pharmaceuticals.[1] Large scale aromatic nitration is currently achieved using nitric acid and sulfuric acid. However, this method has several drawbacks, including a lack of selectivity and a high environmental impact.[2] Alternative, more environmentally benign methods for aromatic nitration thus need to be developed.</p><p>In 2012, the cytochrome P450 (CYP) TxtE was shown to catalyse regiospecific nitration of l‐tryptophan at the indole 4‐position during the biosynthesis of thaxtomin A, a phytotoxin produced by Streptomyces scabies and related plant pathogens.[3] TxtE employs a combination of O2, NO and an electron from NADPH (transferred via ferredoxin (Fd) and ferredoxin reductase (Fr) redox partner proteins) to effect the nitration reaction. In S. scabies TxtD, a nitric oxide synthase, supplies NO to TxtE by converting l‐arginine to l‐citrulline using O2 and NADPH.[3, 4] In vitro TxtD can be replaced with the sodium salt of 2‐(N,N‐diethylamino)‐diazenolate‐2‐oxide (DEANO), which functions as an NO donor in aqueous solution.</p><p>TxtE has been shown to accept several tryptophan analogues with additional substituents appended to the carbocycle or α‐carbon as substrates, although product yields were generally low (Figure 1).[5] However, analogues containing other heterocycles are not tolerated, and both the carboxyl and amino groups in tryptophan and the majority of accepted analogues appear to be required for productive binding to the active site (Figure 1).[5] Indeed, tryptophan analogues that are not accepted as substrates, such as tryptamine, have been shown by UV/Vis spectroscopy to ligate the heme iron, preventing dioxygen binding.[6] The requirement for an l‐configured α‐amino acid is a significant impediment to developing TxtE into a useful biocatalyst, due to the complexities associated with enantioselective substrate synthesis. Moreover, the carboxyl and amino groups are both reactive and awkward to manipulate, necessitating multistep protection and functional group interconversion strategies to utilize products of wild type TxtE‐catalysed nitration in complex molecule synthesis. Whilst a TxtE variant that nitrates l‐Trp at the 5‐position has been identified, alteration of the enzyme's substrate specificity has yet to be reported.[7]</p><p>(A) Reaction catalysed by TxtE in thaxtomin biosynthesis. (B) Tryptophan analogues accepted by wildtype TxtE (C) X‐ray crystal structure of TxtE with l‐tryptophan bound (PDB accession: 4TPO), highlighting hydrogen bonds between the amino/carboxyl groups of the substrate and the side chains of residues lining the active site including R59 which was mutated in this study. Red spheres represent ordered water molecules.</p><p>Here we describe the development of high‐throughput methodology for rapid screening of TxtE variants, resulting in the identification of an active site mutant (R59C) that is able to nitrate tryptamine and several analogues with a range of modifications to the indole and/or aminoethyl groups. The discovery of TxtE variants able to accept structurally‐simplified and more diverse substrate analogues is an important first step towards the development of useful nitration biocatalysts.</p><p>To identify TxtE variants capable of accepting a wider range of substrates, it was necessary to develop a high‐throughput screen for enzymatic activity. Thus, we initially sought to eliminate the dependence of TxtE on exogenous Fd and Fr (which are expensive and unstable) for electron transfer from NADPH by fusing it with a suitable reductase. The reductase domains from naturally occurring self‐sufficient CYPs, such as P450RhF and P450BM3, which are [2Fe‐2S]/FMN and FAD/FMN‐dependent, respectively, have previously been fused to several CYPs resulting in self‐sufficient fusion proteins.[8, 9] We therefore initially created a TxtE‐RhF reductase fusion, which was found to be capable of self‐sufficient nitration. However, turnover was low, prompting us to pursue a TxtE‐BM3 reductase (BM3R) fusion instead. A gene encoding a codon optimised His6‐TxtE‐BM3R fusion (to promote high level expression in E. coli) that included the full length heme‐reductase linker (27 aa) from CYP102A1 was designed, synthesised and cloned into a suitable expression vector (Figures S1 and S2). The resulting protein was overproduced in E. coli and purified to homogeneity (Figure S2). Incubation of the purified protein with l‐tryptophan, NADPH and DEANO resulted in production of 4‐nitrotryptophan (Figure S3). The total turnover number (TTN) for the fusion protein was 450, which is similar to that for our previously reported His6‐TxtE construct,[3] when spinach Fd and Fr are employed as redox partners (TTN=580). We then developed and optimised a cell lysate‐based assay system that employs glucose and glucose dehydrogenase to regenerate NADPH in situ from NADP+.</p><p>With this cell lysate‐based assay system in hand, we turned our attention to the creation of TxtE variants with altered substrate specificity. The X‐ray crystal structure of TxtE with l‐tryptophan bound shows an extensive network of hydrogen bonds and electrostatic interactions between the amino and carboxyl groups of the substrate and the side chains of the R59, Y89, N293, T296 and E394 active site residues, involving two ordered water molecules (Figure 1C).[5] Among these, a direct electrostatic interaction between the guanidinium group of R59 and the carboxyl group of the substrate was hypothesized to be a key selectivity determinant for substrates containing a carboxylic acid. Using saturation mutagenesis, we created a library of His6‐TxtE‐BM3R mutants in which R59 is substituted by other proteinogenic amino acids. Randomisation was achieved in a single step using the Q5‐mutagenesis protocol in conjunction with the 22c‐trick.[10] Colonies were arrayed into a 96‐well plate and grown, and expression of the variant genes was induced using IPTG. The ability of cell lysates to nitrate tryptamine 1 was analysed using LC‐MS. The plasmids from wells displaying activity were isolated and their inserts were sequenced, identifying mutants in which R59 has been replaced with S or C (Figure S4).</p><p>The R59C mutant appeared to be slightly more active than the R59S mutant and was thus selected for further analysis. Purified His6‐TxtE‐BMR3(R59C) was incubated with tryptamine 1, NADPH and DEANO. LC‐MS analysis of the reaction mixture revealed a major and a minor product with m/z values corresponding to [M+H]+ for nitrated tryptamine (Figure 2). The molecular formulae of these products were confirmed using UHPLC‐ESI‐Q‐TOF‐MS (Table S5). However, purification of the major product in sufficient quantities for NMR spectroscopic analysis proved challenging. While our work was in progress, Zuo et al. reported that TxtE‐BM3R fusions in which the heme‐reductase linker is shortened from 27aa to 14aa have increased activity.[11] We therefore created a His6‐TxtE‐BMR3(R59C)Δ construct with a similar shortened linker (Figure S1), which enabled purification of the major product using semi‐preparative HPLC and subsequent 1H NMR spectroscopic analysis. The pattern of signals due to the aromatic protons was consistent with nitration at the indole 4 or 7‐position and comparison with a synthetic standard of 4‐nitrotryptamine confirmed this is the major product of the reaction (Figure S5). The minor product could not be purified in sufficient quantity to characterize by 1H NMR spectroscopy, but is presumably an indole nitration regioisomer.</p><p>Nitration of tryptamine catalysed by TxtE‐BMR3(R59C). Extracted ion chromatograms at m/z=206.924±0.005 (corresponding to [M+H]+ for nitrotryptamine) from LC‐MS analyses of tryptamine incubated with DEANO, NADPH, and His6‐TxtE‐BM3R(R59C) (black) or heat denatured enzyme (red).</p><p>Consistent with our hypothesis that R59 plays an important role in the recognition of substrates containing carboxylic acids, the R59C mutant was much less active towards l‐tryptophan than the unmodified reductase fusion (Figure S6). UV‐Vis difference spectra indicated that the mutant protein has a lower affinity for l‐tryptophan (Figure S6) and a concentration of 5 mM was insufficient to saturate its active site (K d=84±2 μM for binding of l‐tryptophan to His6‐TxtE‐BMR3 (Figure S3)).</p><p>A type II difference spectrum is observed for binding of tryptamine to wild type TxtE,[6] indicative of heme iron coordination, which explains why it is not a substrate. Intriguingly, a similar spectrum is observed for binding of tryptamine to His6‐TxtE‐BMR3(R59C) (Figure S7). Despite this, it appears that the R59C mutation permits some tryptamine binding in an alternative mode, allowing nitration to occur.</p><p>To further explore the substrate scope of His6‐TxtE‐BM3R(R59C)Δ, we initially tested three analogues of tryptamine (2, 3, and 4) with additional functional groups on the indole (Fig. 3), for which the corresponding l‐tryptophan analogues have previously been reported to be substrates of wild type TxtE (Figure 1).[5] LC‐MS analyses showed all of these are converted to nitrated products (Figure S8 and Table S5). While 3 and 4 yielded one main product, the nitration of 2 was less selective, resulting in a ∼1 : 1 mixture of two regioisomers (Figure S8).</p><p>Encouraged by these results, we examined more diverse tryptamine analogues with structures that do not correspond to substrate analogues accepted by wild type TxtE (Figure 3). Tryptophanol 5 yielded a single nitrated product (Figure S8 and Table S5). On other hand, indole‐3‐propionic acid 6, tryptophol 7, acetamide 8 and hydrazide 9 could not be nitrated by the engineered enzyme, indicating that the nature and location of the heteroatom(s) in the substituent attached to the indole 3‐position plays an important role in substrate recognition. We also investigated the tricyclic analogues tryptoline 10 and 2‐hydroxycarbazole 11. While the former gave a ∼1 : 1 mixture of two regioisomeric nitration products (Figure S8 and Table S5), the latter was not turned over.</p><p>Analogues of tryptamine investigated as substrates of His6‐TxtE‐BMR3(R59C)Δ. Compounds in green yielded nitrated product(s), whereas those in black did not.</p><p>We recently proposed a catalytic mechanism for TxtE based on a combination of stopped‐flow kinetics experiments and density functional theory calculations (Figure 4).[12] This involves formation of a ferric superoxide complex that couples with NO to form a ferric peroxynitrite intermediate. Homolytic cleavage of the O−O bond in this intermediate affords an Fe(IV)=O complex and NO2, which adds to the indole 4‐position of l‐tryptophan. Abstraction of the ipso‐hydrogen atom by the Fe(IV)=O complex yields the nitrated product. However, N‐methyl‐l‐tryptophan has been shown to bind to the active site of TxtE, but does not undergo nitration,[5] suggesting that abstraction of hydrogen from the indole N−H may precede the addition of NO2 to the aromatic ring. This would be in accord with the mechanism for nitration of 3‐substitued indoles by NO2 in free solution, which involves hydrogen abstraction from the indole nitrogen atom.[13]</p><p>Proposed catalytic mechanism of TxtE. Substrate binding, heme reduction, loss of the water ligand and dioxygen binding results in a ferric‐superoxide complex, as in hydroxylating CYPs. Reaction with NO forms a ferric‐peroxynitrite intermediate that homolyses to form an Fe(IV)=O complex and nitrogen dioxide, which adds to the π system of the substrate. Abstraction of the ipso‐hydrogen atom by the Fe(IV)=O complex affords the product and a ferric hydroxide complex, which undergoes protonation to complete the catalytic cycle.</p><p>To further probe the catalytic mechanism of TxtE, we tested tryptamine analogues with modifications to the heteroatom of the indole (Figure 3). Neither N‐methyltryptamine 12, nor the benzothiophene analogue 13 gave nitrated products, whereas the benzofuran analogue 14 afforded a single nitrated derivative (Figure S9). The observation that N‐methyltryptamine 12 is not nitrated by His6‐TxtE‐BM3R(R59C)Δ is consistent with the inability of wild type TxtE to nitrate l‐tryptophan.[5] However, the fact that benzofuran 14 undergoes nitration provides further evidence that the enzyme is able to catalyse the direct attack of NO2 on the HOMO of moderately electron rich aromatic species. The failure of benzothiophene 13 to undergo nitration probably reflects the relative inability of the sulphur lone pair, compared with the nitrogen and oxygen lone pairs, to stabilise the radical resulting from addition of NO2 to the aromatic ring, which is a consequence of poorer orbital overlap.[14]</p><p>In conclusion, we have created an expression construct for a TxtE‐BM3R fusion that enables direct high throughput screening of E. coli cell lysates for enzyme variants with altered properties. This construct was exploited to create and screen a saturation mutagenesis library targeting R59, which is hypothesized to be a key determinant of substrate specificity. R59C and R59S mutants were identified that catalyse efficient nitration of tryptamine, a substrate that is not accepted by the native enzyme. The R59C mutant was also able to nitrate structurally diverse tryptamine analogues with modifications to both aromatic rings and the aminoethyl substituent. This demonstrates the applicability of protein engineering approaches to the creation of TxtE variants with altered and expanded substrate tolerance, highlighting the potential for CYPs to be developed into useful nitration biocatalysts.</p><!><p>G.L.C. is a non‐executive director of Erebagen Ltd.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Pathobiochemical effect of acylated steryl-\xce\xb2-glucoside on aggregation and cytotoxicity of \xce\xb1-synuclein

Objective Cycad seed consumption by the native islanders of Guam is frequently associated with high rates of Amyotrophic Lateral Sclerosis-Parkinsonism Dementia Complex (ALS-PDC); furthermore, accompanying pathological examination often exhibits \xce\xb1-synuclein inclusions in the neurons of the affected brain. Acylated steryl-\xce\xb2-glucoside (ASG) contained in cycad seeds is considered as causative environmental risk factor. We aimed to investigate whether ASG influences aggregation and cell toxicity of \xce\xb1-synuclein. Methods To understand whether ASG is a causative factor in the development of ALS-PDC, soybean-derived ASG was tested for its effect on in vitro aggregation of \xce\xb1-synuclein using Thioflavin-T. ASG was also tested to determine whether it modulates \xce\xb1-synuclein cytotoxicity in yeast cells. In addition, we determined whether an interaction between ASG and \xce\xb1-synuclein occurs in the plasma membrane or cytoplasm using three factors: GM1 ganglioside, small unilamellar vesicles, and ATP. Results In the present study, we found that ASG-mediated acceleration of \xce\xb1-synuclein aggregation is influenced by the presence of ATP, but not by the presence of GM1. ASG accelerated the \xce\xb1-synuclein aggregation in the cytoplasm. ASG also enhanced \xce\xb1-synuclein-induced cytotoxicity in yeast cells. Conclusions This study demonstrated that ASG directly enhances aggregation and cytotoxicity of \xce\xb1-synuclein, which are often observed in patients with ALS-PDC. These results, using assays that replicate cytoplasmic conditions, are consistent with the molecular mechanism that cell toxicity is caused by intracellular \xce\xb1-synuclein fibril formation in neuronal cells.

pathobiochemical_effect_of_acylated_steryl-\xce\xb2-glucoside_on_aggregation_and_cytotoxicity_of_\xc

Background<!>Materials<!>Preparation of small unilamellar vesicles (SUVs) by sonication<!>Analysis of \xce\xb1-synuclein fibril formation<!>Effect of ASGs on \xce\xb1-synuclein cytotoxicity<!>Statistical analysis<!>Time course for \xce\xb1-synuclein fiber formation<!>ASG-induced \xce\xb1-synuclein\'s cytotoxicity<!>Discussion

<p>ALS-Parkinsonism Dementia Complex (ALS-PDC) is a unique neurodegenerative disease. It is remarkable that ALS and PDC, either in isolation or in combination occur with a high frequency in the Chamorro people of Guam, Kii Peninsula of Japan, and west New Guinea (1-4). The etiology of ALS-PDC has led to a growing awareness of the involvement of purely genetic and/or environmental factors. A potential environmental factor that has been considered to be involved in the etiology of ALS-PDC is the consumption of traditional foods made from flour of the cycad seed (Cycas micronesica), which has often been used as a primary foodstuff, especially during periods of famine (3-5). Strong epidemiological data links disease development to cycad seed consumption. Cycad seeds are harvested, cut open to expose the starchy endosperm, sliced into 'chips', and then washed for periods of up to 10 days. Components of cycad seeds, particularly, steryl-β-glucosides including acylated steryl-β-glucoside (ASG in Fig. 1) and beta-N-methylamino-L-alanine (BMAA) have been focused upon as to their neurotoxicity (6, 7). ASG and BMAA have been considered the principal components of cycad neurotoxin, a conclusion that is supported by a feeding experiment in CD1 mice (8, 9).</p><p>However, BMAA induces acute toxicity in mice, making it difficult to explain the overall symptoms of ALS-PDC. Moreover, it has been shown that cycads were not used for food in two other high-incidence epicenters: Kii Peninsula and west New Guinea. In addition, ASG is generally found in many other edible plants in addition to cycads, and is consumed as a primary ordinary foodstuff all over the world; hence a particular genetic background of patients cannot be ignored in an etiological study of ALS-PDC(8). Thus, we hypothesize that ASG might be an environmental risk factor contributing to the pathogenesis of ALS-PDC, which is caused or affected both by genetic and environmental component. PDC in Guam is characterized by a marked loss of neurons and deposition of numerous neurofibrillary tangles (NFTs) in the neocortex, hippocampus, amygdale, and brainstem (10). Significant α-synuclein deposition is observed in the amygdalae. These direct observations of the intracerebral pathology of ALS-PDC led us to examine whether ASG may induce α-synuclein aggregation. Protein misfolding of α-synuclein causes α-synuclein fibril formation in the cytoplasm of neuronal cells, subsequently leading to neurotoxicity of progressing severity (11). To demonstrate the potential relationship between ASG and α-synuclein fibril formation in the cytoplasm, we first tested for ASG's effect on aggregation of normal human α-synuclein by the use of an assay system replicating the in vitro conditions of a human subject. We then examined the growth of transformed yeast cells by α-synuclein in the presence or absence of ASG.</p><!><p>The following chemicals were purchased from Sigma Chemical Co. (St. Louis, MO): 1, 2-dipalmitoyl-sn-glycro-3-phosphocholine (DPPC); normal human α-synuclein (recombinant, expressed in Escherichia coli, N-terminal histidine-tagged); and Thioflavin-T (Th T). Acylated steryl-β-glucoside (derived from soybean) was purchased from Matreya. GM1 ganglioside was purified from bovine brain according to a procedure we previously established (12).</p><!><p>SUVs (GM1-SUV and SUV) including GM1/DPPC and DPPC alone were prepared by sonication following previously published methods (13). Briefly, GM1/DPPC (molar ratio 1:1, unless otherwise noted) and DPPC alone were prepared at 10-20 mg/mL lipid per mL solvent (chloroform/methanol (2:1, v/v) by sonication. The solvent was evaporated using a dry nitrogen or argon stream in a fume hood. The lipid film was thoroughly dried to remove residual organic solvent by subjecting the vial or flask to a vacuum pump overnight. The thin film was hydrated in 100 mM NaCl and 20 mM Tris buffer (pH 7.4) with gentle mixing. Hydrated samples were then sonicated at 4 °C with a probe sonicator for 4 min of 10 sec pauses following 10 sec sonication periods. Prior to assay, SUVs were mixed with monomeric α-synuclein. The SUV/α-synuclein mixture was adjusted by a 10:1 mass ratio of lipid to protein.</p><!><p>To induce fibrillization (14), α-synuclein, (80 μM), protein was incubated in either the presence (20 μg/mL) or absence of ASG in KHM buffer (40 mM HEPES-KOH, pH 7.4; 150 mM KCl; 20 mM MgCl2; 1 mM dithiothreitol) plus ATP (10 mM) and an ATP regeneration system (20 mM creatine phosphate and 0.001 mg/mL creatine kinase) (15). The sampling took place for time periods varying from 0–48 hours at 37°C with 80 rotations per minute on a Mini-rotator (Glas-Col, LLC, Terre Haute, IN). Every 8 hours, reactions were supplemented with a fresh ATP regeneration system to maintain ATP levels.</p><p>Fibril amount was determined by ThT fluorescence. ThT in 50 mM glycine (pH 8.5) was added to reach final concentrations of 0.25 μM α-synuclein and 10 μM ThT. Fluorescence at 485 nm was measured after excitation at 450 nm (16).</p><!><p>Saccharomyces cerevisiae W303 (MATa ade2-1 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 can1-100) was transformed using one of two plasmids. The first, plasmid p2HGPD, is an empty vector (control) while the second, p423GPD α-synuclein-YFP, allows for consistent expression of α-synuclein-YFP under the GPD promoter (17). The plasmid transformed cells were streaked onto histidine (SC-H)-free synthetic medium and incubated for 3 days at 30°C. ASG was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/mL, and 100 μL of the solution was streaked onto SC-H plates to achieve a final concentration of 20 μg/mL. To address the toxicity of α-synuclein with ASG, cells were grown on SC-H for 1 day at 30°C then resuspended in water at 107 cells/mL by adjusting to 1.0 value at OD600. The cell suspensions were serially diluted (1:10), and 4 μl of each dilution was spotted onto 2 plates of different media (SC-H and SC-H including 20 μg/mL ASG) and incubated for 2 days at 30°C.</p><!><p>Unless otherwise stated, results are presented as mean ±standard error. Assays were performed in triplicate. The experimental data were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test and Dunnet's test. Statistical analyses were performed using the GraphPad Prism 5.0 software package (GraphPad, San Diego, CA, USA).</p><!><p>ATP is needed for α-synuclein aggregation in the cytoplasm. This necessity is illustrated by our finding that the presence of ATP remarkably enhanced α-synuclein aggregation (Fig. 2A), while in the absence of ATP, α-synuclein aggregation showed only beginning levels of kinetics, even after 48 hours incubation. ASG showed no effect on α-synuclein aggregation. On the other hand, the presence of ATP increased α-synuclein aggregation up to 140 % as compared with aggregation in the absence of ATP. This finding implicates that ASG-induced aggregation in the cytoplasm causes enough fibril formation to lead to cytotoxicity. In contrast, SUV affected α-synuclein aggregation, but ASG did not influence α-synuclein aggregation in the presence of SUV (Fig. 2B). Additionally, GM1-SUV remarkably suppressed α-synuclein aggregation as compared with SUV; however, ASG-induced α-synuclein aggregation did not occur in the presence of GM1-SUV. This finding implies that ASG is not effective in regions or circumstances, such as the plasma membrane, in which GM1 is located abundantly.</p><p>Various factors involved in the plasma membrane and cytoplasm were examined as to their effect on ASG-induced α-synuclein aggregation (Fig. 3A). The presence of ATP or ATP plus SUV affected ASG-induced α-synuclein aggregation with a significant statistical difference (*p<0.01). ASG-induced aggregation was enhanced further by the presence of ATP and SUV as compared with only ATP. Furthermore, to determine whether the interaction between various cytoplasmic factors and ASG-induced α-synuclein aggregation occurs in a time-dependent manner, α-synuclein incubation was extended up to 96 hrs (Fig. 3B). After 48 hrs of incubation, the aggregation plateaued at 72 and 96 hrs. In summarizing these findings, there is no intrinsic difference between α- synuclein aggregation in either the plasma membrane or cytoplasm. Our experiment was able to reconstitute conditions of both compartments. The result was interpreted by varying degrees of aggregation by appearance. ASG-induced α-synuclein aggregation was independent of additive factors after 96 hrs incubation (Fig. 3B). This finding suggests that ASG enhances α-synuclein aggregation but does not change the ultimate endpoint level of α-synuclein aggregates.</p><!><p>The α-synuclein-expressing cells displayed a marked reduction in cell growth compared with vector-expressing cells on the control plate (Fig. 4). On the ASG plate, 20 μg/mL of ASG-treatment caused α-synuclein-expressing cells to exhibit a remarkable decreased growth as compared with non-treated cells on the control plate.</p><!><p>α-Synuclein was originally found as the precursor of the non-Aβ component of amyloid in brains from Alzheimer's disease (AD) patients. Subsequently, α-synuclein was found in fibril inclusions as components of Lewy bodies in Parkinson's disease and multiple system atrophy (18, 19). Under normal conditions, the native structure of α-synuclein is an unfolded monomer reversibly equilibrated to α-helix form. This form of α-synuclein allows the molecule to associate with the surface phospholipids of vesicle membranes. Both this membrane interaction and an increased concentration of α-synuclein can facilitate the formation of α-synuclein dimers in the membrane or in the cytoplasm. Through dimerization, α-synuclein adopts a β-sheet secondary structure, and the membranes associate with α-synuclein monomers or other dimers, leading to oligomer formation. These oligomers in turn lead to fibril formation and are deposited as amyloid in Lewy bodies causing Lewy neuritis (11).</p><p>Consumption of a certain compound in cycad seeds traditionally used for food is assumed to be the risk factor responsible for ALS-PDC being a unique neurological disorder found in natives of Guam (1-5). Steryl glucosides such as ASG have been proposed as the cycad neurotoxin of ALS-PDC(20). Recently, various biological activities of ASG have been reported. These activities, namely anti-diabetic (21, 22), analgesic (23), and anti-hemolytic (24) activities as well as association with macrophage activation (25), implicate ASG as having potential medical use. However, when considering the side effects of ASG, it is important for us to know the relevance of ASG to the ALS-PDC disease state.</p><p>The core of our hypothesis is that ASG-induced α-synuclein aggregation causes cytotoxicity. A new finding in our study was that soybean-derived ASG stimulates aggregation of normal human α-synuclein (Fig. 2) and that subsequent increases in the cytoplasmic concentration of α-synuclein led to cell toxicity. Although the extracellular or plasma membarane functions of α-synuclein remain unknown, the cytoplasmic function of α-synuclein has been clarified. In most of the studies, increased intracellular α-synuclein led to increased aggregate localization and sympathetic nerve membrane binding. In the cytoplasm, ATP is known to be a factor for triggering α-synuclein aggregation (26). In our present experiment, ASG induced a remarkable enhancement of α-synuclein aggregation in the presence of ATP and SUV, but produced no effect on aggregation in the absence of ATP and SUV (Figs. 2a and 3a). Additionally, ASG-induced α-synuclein aggregation did not occur in the presence of GM1-SUV (Fig. 3A). Recently, GM1 was shown to be a mediator in certain essential signaling pathways of the nervous system and other tissues by interactions between GM1 and proteins. The interaction between GM1 and α-synuclein has been focused on Parkinson's disease and shown in animal studies (27, 28). GM1 is localized mostly to a lipid raft within the plasma membranes. This raft associates with α-synuclein binding in a manner that promotes α-helical conformation and prevents pathological cytoplasmic fibrillation (29). Thus, we suggest ASG works by enhancement of α-synuclein aggregation in the cytoplasm.</p><p>In summary, the results of this study have demonstrated that ASG enhances in vitro α-synuclein aggregation and increased cell toxicity via cytoplasmic α-synuclein.</p>

PubMed Author Manuscript

BIAN\xe2\x80\x93NHC Ligands in Transition-Metal-Catalysis: A Perfect Union of Sterically-Encumbered, Electronically-Tunable N\xe2\x80\x93Heterocyclic Carbenes?

The discovery of NHCs (NHC = N-heterocyclic carbenes) as ancillary ligands in transition-metal-catalysis ranks as one of the most important developments in synthesis and catalysis. It is now well-recognized that the strong \xcf\x83-donating properties of NHCs along with the ease of scaffold modification and a steric shielding of the N-wingtip substituents around the metal center enable dramatic improvements in catalytic processes, including the discovery of reactions that are not possible using other ancillary ligands. In this context, although the classical NHCs based on imidazolylidene and imidazolinylidene ring systems are now well-established, recently tremendous progress has been made in the development and catalytic applications of BIAN\xe2\x80\x93NHC (BIAN = bis(imino)acenaphtene) class of ligands. The enhanced reactivity of BIAN\xe2\x80\x93NHCs is a direct result of the combination of electronic and steric properties that collectively allow for a major expansion of the scope of catalytic processes that can be accomplished using NHCs. BIAN\xe2\x80\x93NHC ligands take advantage of (1) the stronger \xcf\x83-donation, (2) lower lying LUMO orbitals, (3) the presence of an extended \xcf\x80-system, (4) the rigid backbone that pushes the N-wingtip substituents closer to the metal center by buttressing effect, thus resulting in a significantly improved control of the catalytic center and enhanced air-stability of BIAN\xe2\x80\x93NHC\xe2\x80\x93metal complexes at low oxidation state. Acenaphthoquinone as a precursor enables facile scaffold modification, including for the first time the high yielding synthesis of unsymmetrical NHCs with unique catalytic properties. Overall, this results in a highly attractive, easily accessible class of ligands that bring major advances and emerge as a leading practical alternative to classical NHCs in various aspects of catalysis, cross-coupling and C\xe2\x80\x93H activation endeavors.

bian\xe2\x80\x93nhc_ligands_in_transition-metal-catalysis:_a_perfect_union_of_sterically-encumbered,

Introduction<!>Heck Cross-Coupling<!>Suzuki-Miyaura Cross-Coupling<!>Negishi Cross-Coupling<!>Sonogashira Cross-Coupling<!>Murahashi Cross-Coupling<!>C\xe2\x80\x93H Arylation<!>C\xe2\x80\x93H Acylation<!>Miscellaneous<!>Buchwald-Hartwig Amination<!>Aminocarbonylation.<!>Palladium\xe2\x80\x93BIAN\xe2\x80\x93NHC-Catalyzed C\xe2\x80\x93S Cross-Coupling<!>Nickel\xe2\x80\x93BIAN\xe2\x80\x93NHC Complexes<!>C\xe2\x80\x93C Cross-Coupling<!>C\xe2\x80\x93N Cross-Coupling.<!>Rhodium\xe2\x80\x93BIAN\xe2\x80\x93NHC Complexes<!>Iridium\xe2\x80\x93BIAN\xe2\x80\x93NHC Complexes<!>Alkene Metathesis<!>C\xe2\x80\x93H Arylation<!>C\xe2\x80\x93S Bond Formation<!>C\xe2\x80\x93N Bond Formation<!>C\xe2\x80\x93O Bond Formation<!>C\xe2\x80\x93C Bond Formation<!>Copper\xe2\x80\x93BIAN\xe2\x80\x93NHC Complexes<!>Conclusions and Outlook

<p>N-Heterocyclic carbenes (NHCs) have found extraordinary utility in organic synthesis and catalysis.[1] Classical NHCs feature at least one nitrogen atom within the heterocyclic system that is directly connected to the carbene center, which renders them powerful ligands in transition-metal-catalysis.[1] Following early studies by Wanzlick and Öfele in the 1960s, resulting in isolation of the first NHC–transition-metal-complexes,[2,3] it was the seminal work by Arduengo and co-workers in 1991 on the stability of a free NHC, 1,3-di(adamantyl)imidazol-2-ylidene,[4,5] that has sparked interest of the chemical community and resulted in a raise of NHCs from academic curiosities to broadly employed species in various fields of science ranging from catalysis to medicine.[6]</p><p>In the last two decades, the organometallic chemistry of N-heterocyclic carbenes has been rapidly developed and a variety of NHC–metal complexes have been explored.[7–10] From a fundamental perspective, N-heterocyclic carbenes represent the most important class of strong σ-donor ligands superseding phosphines[11–20] that have been widely employed in metal coordination chemistry, catalysis, and polymer science. Major advantages of NHCs as ligands include (1) NHC ligands are easy to synthesize and functionalize, in particular in comparison with phosphines, which allows for rapid tuning and modification of catalytic performance; (2) strong NHC–metal bonds provide high complex stability during catalysis and enable isolation of novel metal complexes, including main metals and f-block elements; (3) large structural and stereo-electronic diversity of NHC ligands is available, wherein the ligand properties can be tuned by the backbone or N-wingtip modification; (4) steric shielding of the N-wingtip substituents around the metal center provides efficient control of the catalytic pocket; (5) high stability to oxidative conditions permits for highly successful catalytic applications with metals at higher oxidation state.[21,22]</p><p>In this context, although the research has predominantly focused on the classical NHCs based on imidazolylidene and imidazolinylidene ring systems (Figure 1), recently tremendous progress has been made in the development and catalytic applications of BIAN–NHC (BIAN = bis(imino)acenaphtene) class of NHC ligands (Figure 2). These carbene ligands were first reported by the Çetinkaya group in 2006[23] and feature a naphthalene ring fused to the imidazolylidene ring system. The unique geometric arrangement results in a number of distinct properties of BIAN–NHC ligand resulting from the combination of electronic and steric properties (Figure 2),[24–30] such as (1) stronger σ-donor and better π-acceptor character than the classical imidazolylidenes, which results in enhanced reactivity in both oxidative addition and electrophilic functionalization; (2) the presence of an extended π-system and the rigid backbone that pushes the N-wingtip substituents closer to the metal center by buttressing effect and affords improved control of the catalytic center; (3) significantly improved air-stability of BIAN–NHC–metal complexes at low oxidation state; (4) potential for scaffold modification by naphthalene functionalization, including a related class of perylene-type NHCs; (5) acenaphthoquinone as an NHC precursor enables for the first time for a high yielding synthesis of unsymmetrical NHCs with unique catalytic properties. Overall, these properties result in a highly attractive, easily accessible class of NHC ligands that bring advances in transition-metal-catalysis using various metals and emerge as a leading practical alternative to the classical imidazolylidene and imidazolinylidene NHC ligands. Structures of the most common BIAN–NHC ligands used in catalysis are summarized in Figure 3.</p><p>In this context, an important difference between saturated and unsaturated BIAN–NHC catalysts (e.g. BIAN–IMes vs. BIAN–SIMes) should be noted (Figure 3). In analogy to imidazolinylidenes, saturated BIAN–NHCs are stronger σ-donors,[23] while geometrically the saturated acenaphthylene framework creates a bend-like geometry placing the steric bulk away from the metal center.[24]</p><p>It also worth noting that BIAN–NHC backbone is unique from other benzofused NHC analogues[1–22] due to (1) vastly accessible methods of synthesis, (2) diversity and ease of scaffold functionalization, (3) structural buttressing effects enabled by the acenaphthoimidazolylidene framework, (4) electronic properties enabled by the fused naphthalene system. The closest structural analogue, pyrene-containing ligands, are also included in the review for comparison purposes; however, it should be noted that these systems are less developed than BIAN–NHCs. Since the catalytic performance is the most important aspect of BIAN–NHCs in chemistry, the review covers a detailed description of catalytic results. Comparative studies vs. the classical imidazolylidene NHC ligands have been included in all examples when available. For clarity, all these examples are presented in italicized text. It should be noted that in all these published studies, BIAN–NHCs show superior reactivity to typical NHCs across various reactions, catalytic cycles and metals as demonstrated in this review, establishing broad generality of this ligand class.</p><p>Intriguingly, BIAN–NHCs represent one of the most striking examples of the "flexible steric bulk" concept owing to the buttressing effect of the ring, wherein the ligand can adjust to the steric substrate requirements during catalysis, while enabling high stabilization of the metal center.[22] It is further important to note that during the last decade, a large number of BIAN–NHC ligands[24] and transition-metal-BIAN–NHC complexes, including Ag,[25] Au,[26] Pd,[27] Ni,[28] Ir,[29] Rh,[29] and Pt[30] complexes have been successfully developed, thus demonstrating the facility of synthesis of a broad range of novel BIAN–NHC complexes.</p><p>Herein, we provide a comprehensive overview of BIAN–NHC ligands in transition-metal-catalysis with a focus on both the catalyst structure and the scaffold's role in catalysis. The review is organized by the type of metal used and further by the type of bond being formed. We hope that this review will stimulate the additional use of BIAN–NHC ligands by a range of chemists. In particular, we believe that a combination of the unique stereo-electronic properties, the facile synthesis of novel NHCs and the high air-stability engendered by the BIAN scaffold provide an attractive entryway to applying well-defined metal-BIAN–NHC complexes in modern catalytic processes that are difficult or impossible to achieve using other NHC ancillary ligands.</p><!><p>In 2006, the Çetinkaya group reported the first example of a BIAN-NHC-Pd complex based on naphthyl-fused imidazolinylidene scaffold (Scheme 1).[23] The BIAN–NHC ligand was readily prepared by the sequential reduction-cyclization sequence of the corresponding IMes-BIAN di-imine. The electronic properties indicated that this BIAN–NHC ligand is a stronger σ-donor (νav = 2034 cm−1; TEP = 2047 cm−1 characterized as [RhCl(CO)2(NHC)] complex) than the corresponding SIMes and IMes ligands (SIMes: νav = 2039 cm−1; TEP = 2051 cm−1; IMes: νav = 2041 cm−1; TEP = 2053 cm−1). The catalytic activity of 1 was evaluated in the Heck cross-coupling of aryl halides with alkenes.</p><p>Two classes of catalysts were tested: (1) in situ formed Pd–BIAN–NHC complex generated from Pd(OAc)2 (3 mol%)/1 (3 mol%) in the presence of Cs2CO3; and (2) bis-carbene precatalyst trans-(BIAN–NHC)2PdCl2 (3 mol%). The in situ formed Pd–BIAN–NHC complex showed high reactivity in the cross-coupling of aryl bromides (Scheme 1). Importantly, this seminal study demonstrated the beneficial effect of the BIAN ligand on the Heck cross-coupling as the use of SIMesHCl under the same reaction conditions resulted in lower yields.</p><!><p>In 2010, Green and co-workers reported the synthesis of allyl-supported [Pd(BIAN–NHC)(allyl)Cl] complexes based on naphthyl-fused saturated imidazolinylidene scaffold (2 and 3) (Scheme 2).[31] Interestingly, the X-ray structure of 3 indicated unsymmetrical σ-π bonding of the allyl throw-away ligand, while temperature dependent NMR studies showed η3–η1–η3 isomerisation of the allyl group. The catalytic activity of Pd–BIAN–NHC complexes 2 and 3 was tested in the Suzuki–Miyaura cross-coupling (Scheme 2). It was found that both 2 and 3 are highly active as Pd(II)–NHC precatalysts in the cross-coupling, including highly deactivated and sterically-hindered aryl halides. Impressively, quantitative conversions were obtained even for the challenging cross-coupling between sterically-hindered boronic acids and deactivated aryl chlorides.</p><p>In 2012, Tu and co-workers reported the Suzuki–Miyaura cross-coupling to afford sterically-hindered biaryls using Pd–BIAN–IPr complex 4 (Scheme 3).[32] The most striking feature of this chemistry is that the electronic-modification of the BIAN ligand permitted for a significantly improved reactivity at low catalyst loadings (0.05–0.5 mol%) as compared to the traditional imidazolylidene-based Pd–PEPPSI–IPr catalysts. The reaction was used to prepare extremely sterically-hindered biaryls, including the challenging tetra-ortho-substituted biaryls. This protocol is performed in the presence of t-BuOK as base and dioxane as solvent at 80 °C. The authors proposed that the high reactivity of the Pd–BIAN–IPr complex results from its stronger σ-donor properties than the related Pd–PEPPSI–IPr. The superb efficiency of the method was showcased in multiple one-pot cross-couplings. Furthermore, the protocol showed excellent functional group tolerance, including free hydroxyl and amino groups.</p><p>In 2013, the Peris group reported pyracene-based [Pd(NHC)(allyl)Cl] and Pd–NHC–PEPPSI complexes 6–7 (Scheme 4).[33] These bi-metallic complexes feature a π-extended pyracene-type bis-imidazolylidene group (pyrabim) and are structurally-related to Pd–BIAN complexes, such as Pd–BIAN–PEPPSI complex 4 (Scheme 3) and [Pd(BIAN–IPr)(allyl)Cl] 5 also prepared by the authors. The catalytic properties of complexes 4–7 were evaluated in the Suzuki–Miyaura cross-coupling of aryl bromides under the same reaction conditions. Intriguingly, it was found that bi-metallic complexes 6–7 showed much higher reactivity than their monometallic congeners 4–5, while allyl-based complex 6 [Pd(NHC)(allyl)Cl] was more reactive than PEPPSI-based complex 7. The reactions were performed in the presence of Cs2CO3 at 80 °C in dioxane at 2 mol% Pd loading. The authors proposed that the superior reactivity of bi-metallic complexes results from higher local concentration of palladium, while the extended π-system could be beneficial for substrate preorganization. This interesting study opened up the vistas for the use of polytopic[34] π-extended BIAN-type complexes in catalysis.</p><p>In 2014, Humphrey and co-workers reported mixed NHC/phosphine [Pd(BIAN–NHC)(PPh3)Cl2] complexes 8–9 and evaluated their reactivity in the Suzuki–Miyaura cross-coupling of aryl halides under aqueous conditions (Scheme 5).[35] The performance of catalysts 8–9 was also evaluated in non-polar solvents. Interestingly, it was found that water and toluene promoted catalyst decomposition to yield a new heterogeneous species, while both catalysts were considerably more stable in dichloromethane. Kinetic studies revealed that water significantly enhanced the catalyst activity. As expected, IPr–BIAN catalyst 9 showed higher reactivity than the corresponding IMes–BIAN complex 8.</p><p>In 2016, one of us (F.S.L.) reported the Suzuki–Miyaura cross-coupling of aryl chlorides catalyzed by the backbone modified Pd–BIAN–IPr complex 10 (Scheme 6).[36] In this study, we found that 1,2-di-tert-butyl-substitution of the acenaphthyl group results in a major catalytic improvement over the unsubstituted Pd–BIAN–IPr complex 4. Structural characterization of complex 10 by X-ray crystallography revealed that the bulky t-Bu groups intersect the coordination plane, which likely slows down rotation of N-wingtip substituents. Moreover, the BIAN scaffold leads to the perpendicular arrangement of the N-wingtip substituents. Altogether, these effects result in an enhanced stabilization of the monoligated NHC–Pd(0) and permit to conduct the cross-coupling under aerobic conditions. The catalytic performance was studied by kinetic studies. Interestingly, kinetic profiles showed comparable catalytic efficiency of complexes 10 and 4 at the initial t = 5 min. However, the backbone-modified complex 10 showed much longer life-time than Pd–BIAN–IPr complex 4 and the classical Pd–PEPPSI–IPr, which further confirmed the positive effect of sterically encumbered substituents on the BIAN-backbone. Impressively, this Suzuki–Miyaura cross-coupling could be performed at very low catalyst loading of 0.01–0.05 mol% in the presence of K3PO4 as a weak base in ethanol at 80 °C, and showed excellent functional group tolerance. It is noteworthy that the challenging cross-coupling of 2,6-dimethyl chlorobenzene with ortho-substituted boronic acids was smoothly accomplished to afford tri-ortho-substituted biaryls in close to quantitative yields. The scope was highlighted by the facile synthesis of Boscalid and sartan intermediates.</p><p>The synthesis of tetra-ortho-substituted biaryls has been recognized as one of the most challenging transformations in the field of Pd-catalyzed cross-coupling reactions.[37] Although elegant examples demonstrated the feasibility of the synthesis of tetra-ortho-substituted biaryls using NHCs as ancillary ligands, these reactions have been limited by the necessity for strict air- and moisture-free conditions.[38] In general, the oxidative addition step of di-ortho-substituted aryl halides requires a combination of (1) an electron-rich ancillary ligand, and (2) steric flexibility around the palladium center. Furthermore, while transmetalation using di-ortho-substituted arylboronic acids requires less hindered ligands, it is well established that these less sterically NHC–Pd(0) active species readily decompose to afford unreactive NHC–Pd(O2) complexes in the presence of atmospheric oxygen.[39] Moreover, aerobic conditions favor the formation of homocoupling and oxidation products. Thus, a delicate balance of the flexible steric environment and the use of strongly σ-donating ligands are implicated in the successful design of NHC ligands for the synthesis of tetra-ortho-substituted biaryls.</p><p>In 2017, we reported (F.S.L.) the Suzuki–Miyaura cross-coupling for the synthesis of sterically-hindered biaryls in air using a sterically-flexible Pd–BIAN–IPent catalyst 11 (Scheme 7).[40] The Ir(I) complex [IrCl(CO)2(NHC)] was used to gauge the electronic properties of this new BIAN–NHC carbene ligand, which indicated that both BIAN–IPent (TEP = 2042 cm−1) and BIAN–IPr (TEP = 2042 cm−1) are stronger σ-donors than the corresponding imidazolylidene-based IPent (TEP = 2049 cm−1) and IPr (TEP = 2052 cm−1) ligands. The structure of the Pd–BIAN–IPent complex 11 was confirmed by the X-ray crystallography (% Vbur = 38.2%), implying flexibility of the ligands around to the metal center. We proposed that the high reactivity of BIAN–IPent complex 11 resulted from the strong σ-donation and N-wingtip flexibility to accommodate different steric environment during oxidative addition and transmetallation steps, thus allowing for the synthesis of challenging tetra-ortho-substituted biaryls. The optimized protocol involved 1.0 mol% Pd loading in the presence of t-BuOK in t-BuOH at 80 °C. Notably, other Pd–NHC catalysts, such as Pd–PEPPSI–IPr*, Pd–PEPPSI–IPent or Pd–PEPPSI–IPr resulted in low reactivity, thus clearly showcasing the benefits of the BIAN framework. Importantly, these reaction conditions are also compatible with a range of medicinally-relevant heterocycles, including pyrazines, quinolines, pyridines, thiophenes and furans.</p><p>In 2018, we developed (F.S.L.) a new class of sterically-flexible Pd–BIAN–NHC catalysts such as 12 and applied them in the Suzuki–Miyaura cross-coupling of aryl chlorides at very low catalyst loading in air (Scheme 8).[41] By systematic investigation of the structure-reactivity relationship, we identified complex 12 bearing unsymmetrical 2,4-(CHPh2)2-6-Me-C6H2 N-wingtip substitution as the excellent precatalyst for the Suzuki–Miyaura cross-coupling. The high reactivity of complex 12 was proposed to arise from high N-wingtip flexibility (% Vbur = 41.1%), while leaving significant open space for oxidative addition and transmetallation steps in combination with the strong σ-donation (TEP = 2047 cm−1, Ir(I) complex [IrCl(CO)2(NHC)]. These cross-couplings were conducted at very low 0.025 mol% Pd loading in the presence of K2CO3 as a mild carbonate base in EtOH at 80 °C. Notably, other Pd–NHC precatalysts, including Pd–PEPPSI–IPr, Pd–PEPPSI–IPr* or Pd–PEPPSI–IPent gave no or low yields under the reaction conditions, clearly demonstrating the advantage of BIAN-type backbone and unsymmetrical N-wingtip substitution on the catalytic reactivity. The facile synthesis of NHC salts such as in 12, excellent catalytic efficiency and broad functional group compatibility, including installation of biologically relevant heterobiaryls are attractive features of this class of catalysts.</p><p>In 2017, the Tu group reported the Suzuki–Miyaura cross-coupling of 9-chloroacridines catalyzed by a BIAN-type Pd–NHC precatalyst 13 supported on magnetic nanoparticles (Scheme 9).[42] This reaction showed excellent functional group tolerance to yield biaryl acridine derivatives with potential applications in polymer science and medicinal chemistry at 0.5 mol% Pd loading. An advantage of this approach is that this method allows for catalyst recycling up to 5 times without any loss of catalytic activity.</p><!><p>The Negishi cross-coupling between aryl halides and organozinc reagents catalyzed by the Pd–BIAN–NHC complex 4 was reported by Tu and co-workers in 2013 (Scheme 10).[43] The reaction was catalyzed by Pd–BIAN–IPr PEPPSI-type catalyst, which showed much higher reactivity than the analogous Pd–BIAN–IMes complex as well as the classical imidazolylidene Pd–PEPPSI–IPr. Both aryl bromides and chlorides could be utilized in this process. Likewise, a variety of alkyl and aryl organozinc reagents were compatible, including sterically-hindered and challenging α-branched substrates prone to isomerization. Mild room temperature reaction conditions, low catalyst loading (0.25 mol%) and excellent functional group tolerance, including aldehydes, amines, carbamates, nitriles, are notable features of this protocol. It is especially noteworthy that this protocol efficiently prevents isomerization of 2° alkyl organozinc reagents to the linear products, which could be attributed to the fast reductive elimination vs. the competing β-hydride elimination in the presence of the sterically-encumbered BIAN scaffold.</p><p>Moreover, Browne demonstrated superior reactivity of Pd–BIAN–IPr complex 4 over the classical imidazolylidene Pd–PEPPSI–IPr in the Negishi cross-coupling between aryl bromides and alkyl organozinc reagents under mechanochemical ball milling conditions (not shown).[44]</p><!><p>In 2018, Tu and co-workers reported the Sonogashiracross-coupling of aryl bromides with phenylacetylenes catalyzed by a combination of Pd–BIAN–NHC PEPPSI-type catalyst 4 and the imidazolylidene IPr–Cu–Cl complex 14 (Scheme 11).[45] The reaction was performed at very low Pd loading (0.01 mol%) in the presence of mild carbonate base and 1.0 mol% of Cu in DMSO at 120 °C. Interestingly, the authors found that the BIAN complex 4 gave much higher yields that the analogous allyl complex, [Pd(BIAN–IPr)(allyl)Cl] 5, suggesting non-innocent role of the pyridine throw-away ligand. Similarly, the IPr–Cu–Cl complex 14 showed higher reactivity than CuI and BIAN–IPr–Cu–Cl. The proposed mechanism involves the following steps: (1) oxidative addition of the aryl halide to NHC–Pd(0) to generate NHC–Pd(II)–Ar; (2) transmetallation with NHC–Cu–acetylide; (3) reductive elimination. This important study introduced the use of cooperative catalysis by two distinct transition-metal-NHC complexes involving BIAN scaffold.</p><!><p>In 2019, Feringa, Organ and co-workers reported Murahashi cross-coupling of aryl bromides with alkyl and aryl organolithium reagents catalyzed by Pd–BIAN–IPent catalyst 11 (Scheme 12).[46] Interestingly, this catalyst showed higher reactivity than the classical imidazolylidene-based Pd–PEPPSI–IPr, Pd–PEPPSI–IPent and Pd–PEPPSI–IPentCl, permitting for the first example of the Murahashi coupling at temperatures below −65 °C. The beneficial effect of the BIAN scaffold was also evident in the efficient cross-coupling at very low catalyst loading (0.1 mol%). This chemistry is compatible with a variety of organolithium nucleophiles, including 1° and 2° alkyl organolithium as well as aryl and heteroaryl organolithium reagents. In terms of the electrophile, the cross-coupling is fully selective for bromides in the presence of chlorides. This exquisite selectivity allowed to achieve highly practical one-pot sequential cross-couplings for the synthesis of polyfunctionalized building blocks containing biaryls, amines or sulfides.</p><!><p>In 2018, one of us (F.S.L.) reported the direct C–H arylation of azoles with aryl bromides catalyzed by Pd–BIAN–NHC complex 15 under aerobic conditions (Scheme 13).[47] Interestingly, we found that remote modification of the N-wingtip group by the bulky CHPh2 substitution at the 4-position led to significant improvement in the catalytic reactivity. It was proposed that the strong σ-donation of BIAN-type catalysts would lead to a facile oxidative addition, while the remote N-wingtip substitution would stabilize the reactive Pd species by increased bulk. These C–H activation reactions are performed in air at very low catalyst loading (0.05–0.5 mol%) in the presence of PivOH in DMAc at 130 °C. Complex 15 showed much higher reactivity than the classical imidazolylidene Pd–PEPPSI–IPr or unfunctionalized BIAN-IPr complex 4. Another noteworthy feature is fully regioselective C–H arylation of several classes of heterocycles and excellent functional group tolerance, including aryl bromides and various medicinally-relevant scaffolds, such as imidazoles, pyrazoles, thiazoles, isoxazoles, triazoles, pyridines, pyrimidines and quinolines.</p><!><p>In 2013, the Peris group reported C–H acylation of hydrocinnamaldehyde with aryl halides catalyzed by their bi-metallic pyracene-type Pd–NHC complexes 6–7 and Pd–BIAN–PEPPSI and [Pd(BIAN–IPr)(allyl)Cl] complexes 4-5 (Scheme 14, cf. Scheme 4).[33] The PEPPSI-based catalyst 4 showed the highest reactivity in this acylation. In contrast to the Suzuki–Miyaura cross-coupling (Scheme 4), the mono-metallic catalysts were more reactive than their bi-metallic counterparts.</p><!><p>In addition to the studies outlined above, it is important to note that chiral BIAN-type complexes (see, Section 3.1. and Section 8) have been tested in the Pd-catalyzed enantioselective Suzuki–Miyaura cross-coupling for the synthesis of atropoisomeric biaryls.[48] These Pd–BIAN-ANIPE catalysts showed excellent reactivity; however, resulted in slightly lower enantioselectivity than the analogous imidazolinylidene complexes (80% ee vs. 96% ee). Considering the lower hydrolytic stability of imidazolinylidenes,[1a–d] the use of more stable BIAN-type NHC ligands could provide an attractive avenue for future developments.</p><!><p>In comparison with the numerous phosphine-based ligands in the Pd-catalyzed Buchwald-Hartwig cross-coupling, the development of Pd–NHCs for amination reactions is still in its infancy.[49] In 2010, Green and co-workers reported Buchwald-Hartwig amination of aryl chlorides and bromides using their [Pd(BIAN–NHC)(allyl)Cl] complexes 2–3 based on BIAN imidazolinylidene scaffold (Scheme 15).[31] These reactions were performed using morpholine and N-methylaniline as nucleophiles at 1.0 mol% palladium loading. The use of t-BuOK and dioxane gave optimal results. Impressively, this catalytic system permitted the cross- coupling of deactivated aryl chlorides in excellent yields at room temperature.</p><p>In 2011, Tu and co-workers reported Buchwald-Hartwig amination of aryl chlorides using their Pd–BIAN–NHC catalyst 4 featuring the 3-Cl-pyridine throw-away ligand (Scheme 16).[50] The structure of 4 was confirmed by the X-ray crystallography for the first time, and indicated a shorter Pd–C bond (1.960 Å) than in the classical imidazolylidene-based Pd–PEPPSI–IPr (1.969 Å), consistent with the stronger σ-donor character of the BIAN scaffold. The cross-coupling was performed at very low catalyst loading (0.075–0.5 mol%) using t-BuOK in dioxane at 80 °C. Catalyst poisoning studies indicated that the reaction follows the homogenous mechanism, as expected from the reaction conditions. Notably, the authors demonstrated excellent scalability of the protocol in that the synthesis of 2-(4-morpholinyl)pyridine was performed on 100 mmol scale (16 gram) at 0.075% catalyst loading.</p><p>In 2017, Tu and co-workers reported Buchwald-Hartwig amination catalyzed by Pd–BIAN–NHC catalysts 16a-c supported by the ortho-metallated N,N-dimethylbenzylamine palladacycle throw-away ligand (Scheme 17).[51] They found that the palladacycle catalysts 16a-c showed much higher reactivity in the amination than the PEPPSI-based Pd–BIAN–IPr catalyst 4 and the analogous imidazolylidene-based catalyst 16d. Unexpectedly, both N-mesityl and N-phenyl catalysts 16b-c afforded similar yields to the more sterically-demanding N-2,6-diisopropylphenyl catalyst 16a. The authors proposed that the high activity of complexes 16 results from a high stability of the palladacycle under the reaction conditions; however, kinetic studies were not performed to support this hypothesis. This protocol is compatible with a variety of 3-chloropyridines and related N-heterocycles to provide the cross-coupling products in high yields at 0.50 mol% catalyst loading. Importantly, the use of 16a is highly selective for mono-N-amination under these conditions.</p><p>In 2017, one of us (F.S.L.) reported Buchwald-Hartwig amination of aryl chlorides under practical aerobic conditions catalyzed by unsymmetrical Pd–BIAN–NHC catalyst 17 featuring sterically-differentiated N-Dipp and N-4-MeO-((2,6-Ph2CH)2-C6H2) wingtips (Scheme 18).[52] In general, the synthesis of unsymmetrical imidazolylidene NHC ligands is notoriously challenging due to selectivity issues, the BIAN scaffold enables high yielding introduction of differentiated N-substituents by step-wise condensation with acenaphthoquinone. Kinetic studies established that catalyst 17 is significantly more effective than the classical imidazolylidene-based Pd–PEPPSI–IPr and the analogous Pd–BIAN–IPr catalyst 4 with symmetrical N-Dipp wingtips. Under the tested conditions, Pd–PEPPSI–IPr* was also an efficient catalyst; however, it resulted in lower conversions than that of 17. The electron-donating 4-MeO group in 17 enhances σ-donation of the NHC ligand, while the unsymmetrical arrangement of the N-substituents enables gradual variation of flexible steric bulk protecting monoligated NHC–Pd(0). This combination permits for the highly efficient cross-coupling under aerobic conditions. The optimum conditions involve 17 at 0.1 mol% catalyst loading in the presence of t-AmOK in dioxane at 100 °C. This protocol was compatible with a wide range of sterically-hindered aryl and heteroaryl chlorides, including with 1° and 2° aliphatic and aromatic amines. Furthermore, DFT studies of the catalytic cycle established that oxidative addition is the rate-determining-step.</p><p>In 2018, we reported (F.S.L.) Buchwald-Hartwig amination of sterically-hindered and electronically-deactivated substrates catalyzed by Pd–BIAN–IPent catalyst 18 (Scheme 19).[53] This highly practical cross-coupling was conducted under aerobic conditions using KOt-Bu as a base in dioxane at 100 °C.</p><p>Extensive screen of various Pd–NHC precatalysts indicated that 18 was significantly more reactive than the classical Pd–PEPPSI–IPr, the imidazolylidene-based, sterically-hindered Pd–PEPPSI–IPr* as well as the symmetrical Pd–BIAN–IPr 4 with N-Dipp wingtips and the unsymmetrical Pd–BIAN–NHC 17. Furthermore, pyridine throw-away ligand was found to be more effective than 3-chloro-pyridine. The structure of the catalyst 18 was confirmed by the X-ray crystallography. This allowed to determine % Vbur of this BIAN–IPent ligand (38.2%) to be larger than that of BIAN–IPr (34.7%) and IPr (34.3%) (determined for Pd–PEPPSI complexes), which suggests a more comprehensive protection of the metal center during the cross-coupling. The significant σ-donation is suggested to facilitate oxidative addition. This reaction was compatible with a remarkably broad scope of sterically-hindered aryl chlorides and electron deficient anilines, providing medicinally-relevant heteroaryl amines in excellent yields, including chelating heterocyclic substrates that typically shut down the catalytic cycle through coordination to palladium. It is noteworthy that the use of Pd–BIAN–NHC 18 fully suppressed the formation of diarylated products. However, it should be noted that a limitation of this class of catalysts is the lengthy synthesis of N-IPent aniline (IPent = 2,6-di-isopentyl), which leaves room for future improvements.</p><p>More recently, we reported another strategy for Buchwald-Hartwig cross-coupling of aryl chlorides catalyzed by the sterically-bulky Pd–BIAN–IPr*OMe complex 19 under aerobic conditions (Scheme 20).[54] In contrast to 'bulky-yet-flexible' catalyst design, the most important variation in this class of catalysts is the combination of the π-extended electron-rich BIAN scaffold with the rigid and electron-rich classical IPr*MeO imidazolylidene catalysts. This merger leads to strong σ-donation (TEP = 2048 cm−1, measured for [IrCl(CO)2(NHC)] and large buried volume (% Vbur = 42.6%, measured for Pd–PEPPSI complex) of the BIAN–IPr*OMe ligand in 19. This catalyst allowed for the efficient cross-coupling of deactivated five-membered and six-membered heterocycles, such as thiazoles, benzothiazoles, benzoxazoles, pyridines, quinolines and diazines with heteroaryl amines. The utility of this protocol was showcased in the synthesis of pharmaceuticals, Brexpiprazole and Piribedil. It is important to point out that this class of BIAN–IPr*OMe catalysts benefits from the facile synthesis of N-wingtip anilines as compared with N-IPent-type precursors, while the introduction of strong σ-donor, rigid ligands has a key role in the success of challenging Buchwald-Hartwig aminations.</p><p>In 2017, Bazzi and co-workers reported another approach to Buchwald-Hartwig amination using polyisobutylene-supported Pd–BIAN–NHC catalyst 20 (Scheme 21).[55] A practical advantage of polyisobutylene-tagging is facile catalyst separation in biphasic heptane/acetonitrile mixture. However, under the tested reaction conditions, Pd–BIAN–NHC catalyst 20 showed slightly lower reactivity than the analogous imidazolylidene-based catalyst.</p><!><p>In 2013, Tu and co-workers reported aminocarbonylation of aryl iodides catalyzed by [Pd(BIAN–IPr)(allyl)Cl] complex 5 (Scheme 22).[56[ The X-ray structure of 5 was determined and indicated Pd– C(carbene) bond of 2.048 Å, which can be compared with the Pd–C(carbene) bond of 2.040 Å for the analogous imidazolylidene complex [Pd(IPr)(allyl)Cl].[7] This catalyst showed higher reactivity than BIAN–IPr–PEPPSI complex 4 and the classical imidazolylidene complex [Pd(IPr)(allyl)Cl]. An attractive feature of this protocol is atmospheric pressure of CO, low Pd loading (0.5–1.0 mol%) and broad substrate scope, including the synthesis of medicinally-relevant amides, such as pharmaceutical Tamibarotene. The authors proposed that the strong σ-donation of the BIAN scaffold facilitates oxidative addition in the presence of a large excess of CO.</p><p>Subsequently, the Tu group developed a mild protocol for the double aminocarbonylation of o-dihaloarenes catalyzed by BIAN–IPr–PEPPSI complex 4 at atmospheric CO pressure (Scheme 23).[57] Interestingly, in this reaction the BIAN-supported catalyst 4 showed much higher reactivity than the classical Pd–PEPPSI–IPr, allowing to achieve a TON of 5,000 at 0.10 mol% catalyst loading; however, a very similar reactivity between PEPPSI and allyl-supported BIAN complexes 4 and 5 was reported. This method represents a highly practical approach to various N-substituted phtalimides, including pharmaceuticals Thalidomide and Alrestatin as well as to fluorescent imides.</p><!><p>In 2017, Tu and co-workers reported the direct alkylsulfonylation of boronic acids with halides and potassium metabisulfite (K2S2O5) catalyzed by [Pd(BIAN–IPr)(allyl)Cl] complex 5 (Scheme 24, see also Scheme 36).[58] This transformation represents a rare example of C–S bond cross-coupling catalyzed by BIAN–NHC-metal complexes. In the optimization study, the authors showed that [Pd(BIAN–IPr)(allyl)Cl] complex 5 showed comparable reactivity to its cinnamyl analogue, [Pd(BIAN–IPr)(cin)Cl], and higher than BIAN–IPr–PEPPSI complex 4 and imidazolylidene complex [Pd(IPr)(allyl)Cl]. The scope of this reaction is broad using a-halo-carbonyl, benzylic and even 1° alkyl electrophiles. In terms of the boronic acid component, the reaction tolerates aromatic, heteroaromatic and alkenyl boronic acids; however, a disadvantage of this protocol is the requirement for 5 mol% catalyst loading. A mechanism involving transmetallation between Pd(II) and boronic acid, SO2 insertion to give a dimeric Pd(II) complex, and alkylation was proposed. The authors suggested that TBAF might facilitate the formation of sulfinate from the dimeric Pd(II) species. This transformation appears to be broader in scope than the analogous Au–BIAN–NHC-catalyzed protocol reported by the same group (see Scheme 36).</p><!><p>Recently, Ni-catalysis has emerged as a powerful reactivity manifold in cross-coupling due to high nucleophilicity of nickel, enabling challenging oxidative additions.[59] In this context, Ni–BIAN–NHC complexes have seen major progress as a tool for asymmetric C–H functionalization.</p><!><p>In 2019, the Shi group reported enantioselective C–H alkylation of fluoroarenes with alkenes catalyzed by Ni(cod)2 in the presence of NaOt-Bu and the chiral BIAN ligand 21 (Scheme 25, see also Scheme 41).[60] These same group proposed the buttressing effect of the acenaphthoimidazolylidene ring in enantioselective ligand design in the context of Cu–NHC-catalyzed hydroboration (Scheme 41), pushing the chiral N-wingtip groups closer to the metal center.[79]</p><p>The authors found that in this case, the BIAN-based ligand was more effective for C–H annulation of aryl-substituted olefins, while the analogous imidazolinylidene-based chiral ligand was slightly preferred for cyclization of alkyl-substituted olefins. The chiral ligand 21 showed similar reactivity to BIAN-based (R,R,R,R)-ANIPE ligand (see Scheme 40) developed for a Cu-catalyzed protoboration by the same group (Ar = Ph instead of Ar = 3,5-xyl). Importantly, the authors found that the use of BIAN-based ligand 21 was critical to prevent olefin isomerization, which was the predominant reaction pathway using the classical imidazolinylidene. In this protocol, C–F activation was not observed even though activated polyfluorinated arenes were used as substrates. The authors proposed that the steric-bulk of the ligand accelerates reductive elimination and promotes endo-selective annulation. The scope of this transformation is impressively broad showing tolerance to various functional groups, including alcohols, amides and ketones. It also worth noting that in 2019, Shi and co-workers reported a closely related Ni–NHC catalyzed C–H alkylation of pyridines (not shown).[60b] In this study, they found that BIAN-based ANIPE-type ligands resulted in a superior enantioselectivity than saturated NHC ligands for select challenging substrates.[60b]</p><p>In 2018, Cramer and co-workers reported highly enantioselective C–H annulation of pyridones with alkenes catalyzed by a combination of Ni(cod)2, MAD as a Lewis acid and a sterically-bulky, chiral BIAN ligand 21 (Scheme 26).[61a] The authors found that replacement of the imidazolylidene scaffold in the Gawley's C2-symmetric NHC carbenes[62] with the BIAN framework resulted in a significant improvement in selectivity. The structures of [Ni(BIAN–NHC)CpCl] and [Au(BIAN–NHC)Cl] complexes with BIAN–NHC ligand related to 21 (Ar = Ph instead of Ar = 3,5-xyl) were determined by the X-ray crystallography. Interestingly, both of these complexes showed lower Vbur than the analogous Ni(II) and Au(I) imidazolylidene complexes (Ni: 35.7% vs. 38.7%; Au: 43.5% vs. 49.4%); this steric differentiation could improve steric access to the metal center in BIAN and further indicates flexibility of the bulky BIAN scaffold in adjusting to steric environment. The scope of this C–H annulation is very broad and encompasses various 2- and 4-pyridones as well as uracils and thiopyridones to afford chiral N-containing heterocycles in an attractive C–H functionalization protocol. A very similar study on the Ni–NHC-catalyzed pyridine C–H alkylation was reported.[61b]</p><p>In 2020, Ho and co-workers reported a highly selective cross-dimerizaton of olefins and methylenecyclopropanes catalyzed by Ni–BIAN–NHC complexes (Scheme 27).[63] The authors found that a combination of [Ni(BIAN–IPr)(allyl)Cl] catalyst 22 and NaBArF could be used to achieve high yields and remarkable selectivity in this [3+2] cross-dimerization. The use of BIAN–IPr ligand provided significantly better results than the classical imidazolylidene-based IPr and imidazolinylidene-based SIPr ligands, while the closest, yet lower, reactivity was observed with the difficult to prepare IPentMe ligand. Furthermore, the more sterically-hindered BIAN–IPent ligand provided inferior results. The use of NaBArF as an additive was found necessary for the reaction to occur. The proposed mechanism involves allyl exchange of [Ni(BIAN–IPr)(allyl)BARF] with allyl zwitterion derived from methylenecyclopropane. The high selectivity for cross-dimerization was proposed to originate from the fast exchange and slow β-hydride elimination under the reaction conditions. This reaction is particularly noteworthy for the excellent functional group tolerance, high chemoselectivity and the use of simple unactivated olefins as substrates.</p><p>In 2019, the Ye group has reported another attractive approach in their reductive coupling of alkynes with imines catalyzed by Ni(cod)2 in the presence of BIAN–IPr ligand 23, KOt-Bu as catalytic base and i-PrOH as proton source (Scheme 28).[64] Direct comparison with a collection of phosphine ligands as well as classical imidazolylidene and imidazolinylidene NHC ligands, such as ICy, IPr, SIPr, IPr* and IPrMe indicated that BIAN–IPr is a much superior ligand in this transformation. The authors proposed that the strong σ-donation and the sterically-bulky BIAN backbone are the key to the success of the BIAN–IPr ligand in promoting this challenging reductive coupling. Interestingly, the reaction is highly sensitive to the steric environment as the less bulky BIAN– IMes gave only low yield of the coupling product. This reaction has a very broad substrate scope and is conducted using sustainable isopropanol as a reductant, thus showing the promise for its future applications. Furthermore, the authors demonstrated the capacity of this coupling in an enantioselective fashion to yield chiral allylic amines using ligand 21 (see Scheme 26) under very similar reaction conditions. Labelling studies suggested that isopropanol acts as the hydride source in this new C–C bond forming method.</p><!><p>In 2014, Tu and co-workers reported Buchwald-Hartwig amination of aryl tosylates catalyzed by Ni–BIAN–NHC at low catalyst loading (Scheme 29).[65] The active catalyst was formed in situ from NiCl2·DME and BIAN–IPr salt 23. The use of PhBin and NaOt-Bu as a strong base are required for this reaction. Importantly, BIAN–IPr 23 showed much higher reactivity than the classical imidazolylidene NHCs, such as IPr and IMes. The scope of this amination is broad with respect to the amine component, including 1° and 2° aliphatic as well as aromatic amines; however, the method predominantly requires the use of naphthyl tosylates, which somewhat limits the applicability of this otherwise very interesting transformation. Preliminary results indicated that benzodioxole and even phenyl tosylates might be compatible substrates, which would lead to a more synthetically useful protocol. This reaction could lead to the development of attractive C–O activation methods catalyzed by Ni–BIAN complexes as a practical alternative to Pd.</p><!><p>In 2009, Green and co-workers reported the synthesis of Rh(I) complexes [Rh(BIAN-SIMes)(cod)Cl] 24 and [Rh(BIAN-SIPr)(cod)Cl] 25 based on the saturated imidazolinylidene BIAN scaffold and their application in the hydroformylation of 1-octene (Scheme 30).[66] Both complexes showed similar reactivity at 0.1 mol% loading, favoring aldehyde as the major product. Isomerization to the branched product was observed as the major pathway (l:b = 0.58–0.75), while reduction to the alcohol was typically <5%. The Rh(I) complex 25 was shown to be reactive at 0.01 mol% loading, thus closely matching the most reactive Rh(I)–NHC systems for hydroformylation reactions.</p><!><p>In 2016, Peris and co-workers reported the synthesis of mono- and bi-metallic Ir(III) complexes 26–28 based on pyrene scaffold (Scheme 31).[67] In these NHCs, the rigid aromatic π-system represents an extended homologue of the BIAN scaffold, while the NHC salt is readily prepared from pyrene-4,5-dione (cf. acenaphthoquinone). These complexes feature chloride [Ir(NHC)Cp*Cl2] (26) or carbonate ligands [Ir(NHC)Cp*CO3] (27), [{IrCp*(CO3)}2(μ-NHC)] (27) coordinated to Ir. The authors demonstrated the catalytic activity of 26–28 in β-alkylation and H/D exchange reactions (Scheme 30–31). The bi-metallic complex 28 was found to be the most reactive in the model β-alkylation of 1-phenylethanol with 1° alcohols (Scheme 31). These β-alkylation reactions were performed at 0.50 mol% Ir loading in toluene at 100 °C. The intriguing high activity of complex 28 was proposed to arise from cooperative interaction between the two metal centers.</p><p>The catalytic activity of Ir(III) complexes 26–28 was also tested in H/D exchange reactions (Scheme 32).[67] Although in H/D exchange of acetophenone and styrene monometallic complexes 26–27 appeared to be more reactive than the bi-metallic complex 28, all three complexes were significantly more reactive than phosphine-based Ir(III) complex [Ir(PMe3)Cp*Cl],[68] thus showing the benefit of using π-extended pyrene-based NHC complexes for this process.</p><!><p>In 2012, Merino and co-workers reported the synthesis of a second generation of Hoveyda-Grubbs catalyst based on BIAN–IMes scaffold 29 (Scheme 33).[69] Impressively, compared with the classical imidazolinylidene SIMes and imidazolylidene IMes H-G2 catalysts, complex 29 showed higher reactivity in the ring-closing-metathesis of diethyl diallylmalonate, while a comparable reactivity was observed using N,N'-diallyl-4-methyl-benzenesulfonamide. Complex 29 was stable at room temperature for over 15 days. The high activity of BIAN-based complex 29, in particular, in comparison with the typically used SIMes-based H-G2 catalysts provides an attractive entry to the development of more active catalysts for olefin metathesis.</p><p>In 2016, Bazzi and co-workers reported the synthesis of polyisobutylene-derived BIAN–IMes-based H-G2 catalyst 30 (Scheme 34).[70] The advantage of polyisobutylene tether is that the catalyst can be recycled by performing the reaction in a non-polar solvent and extraction with acetonitrile. Kinetic studies in the ring-closing-metathesis of 2,2-diallylmalonate demonstrated that BIAN–IMes catalyst 30 is more reactive than the classical imidazolinylidene SIMes H-G2 bearing the same polyisobutylene tether. This finding mirrors the study by Merino and co-workers (cf. Scheme 32) in that the BIAN scaffold has a beneficial effect on alkene metathesis. In addition, complex 30 was applied to ring-opening-polymerization of norbornene derivatives, affording high molecular weight polymers.</p><!><p>In continuation of their studies on di-metallic transition-metal-NHC complexes based on pyrene scaffold, in 2014, Peris and co-workers reported the synthesis of Ru(II) complexes 31–32 (Scheme 35).[71] These mono- and di-metallic Ru(II) catalysts were tested in the direct C(sp2)–H arylation of pyridines with aryl halides (Scheme 35) and hydroarylation of alkenes (Scheme 36). Interestingly, both complexes 31–32 showed comparable reactivity in C(sp2)–H arylation, leading exclusively to the formation of bis-arylated products. Likewise, both 31–32 were highly reactive in the hydroarylation of olefins; however, this reaction resulted in the selective mono-hydroarylation of phenylpyridines. This important study demonstrates high activity of π-extended NHC–Ru complexes in the attractive Ru(II)-catalysis C–H activation manifold.[72,20]</p><!><p>In 2017, Tu and co-workers reported the direct alkylsulfonylation of boronic acids using BIAN–IPr–Au–Cl complex 33 as a catalyst (Scheme 37).[73] The reaction is performed with K2S2O5 as the sulfonating reagent. In most examples, activated halides were used; however, a preliminary result indicated the feasibility of 1° alkyl halides as electrophiles. The authors demonstrated that BIAN-based Au(I) catalysts gave significantly higher yields than the classical imidazolylidene-based IPr and IMes (NHC)Au(Cl) complexes. The sterically-demanding BIAN–IPr ligand gave higher yield than the less bulky BIAN–IMes, while both (NHC)Au(Cl) and (NHC)Au(OH) complexes were similarly efficient in this coupling. The authors proposed that the strong σ-donor character of BIAN-scaffold is the key factor in this challenging transformation. The reaction showed broad functional group tolerance, allowing for the convergent synthesis of sulfones. The proposed mechanism involves transmetallation of NHC–Au(I) species with boronic acid, SO2 insertion to afford NHC–AuSO2–Ar, and alkylation via arylsulfinate intermediate.</p><p>More recently, the Tu group reported the synthesis of diaryl sulfones by the direct arylsulfonylation of boronic acids using the same BIAN–IPr–Au–Cl complex 33 as a catalyst (Scheme 38).[74] The key finding of this protocol is that diaryliodonium salts can be used as electrophiles under very similar reaction conditions to alkylsulfonylation (cf. Scheme 37). The authors found that BIAN–IPr ligand is preferred over the classical imidazolylidene ligands, such as IPr and IMes. This protocol tolerates a broad range of functional groups, including ketal, chloro, TMS, and indazole. The reaction could be performed on a gram scale as demonstrated in the synthesis of a farnesyl-protein transferase inhibitor; however, the limitation of this protocol is high catalyst loading. Mechanistically, the key steps involve transmetallation of NHC–Au(I) with aryl boronic acid, SO2 insertion and arylation of the arylsulfinate intermediate by the aryl cation generated from diaryliodonium salt.</p><!><p>In 2018, Peris and co-workers reported the synthesis of a di-gold(I) complex 34 bearing a π-extended pyrene NHC ligand (Scheme 39).[75] The catalytic activity of 34 was tested in hydroamination of phenylacetylene with anilines at low catalyst loading. Impressively, up to 900 TON was obtained at 0.05 mol% loading. Interestingly, a further increase in the catalytic efficiency was observed upon addition of coronene. This effect was explained by π-stacking interactions, which could prevent the formation of inactive dimers. This finding represents a potential future direction in enhancing the reactivity of π-extended NHC ligands.</p><!><p>In 2018, Zuccaccia showed that BIAN–IPr–Au–X complexes are among the most highly active NHC complexes in hydration of 3-hexyne at 0.10 mol% loading (not shown).[76] In this manifold, the reactivity closely depends on the counterion, and showed the following order of reactivity: OTf > OTs > Cl; however, this process was not further explored. Additional studies on the structure and reactivity of BIAN–Au–X complexes have been published.[77,26a,c]</p><!><p>In 2016, Plenio and co-workers reported the synthesis of a BIAN-based Au(I) complex 35 featuring extremely bulky N-pentiptycene wingtip substituents (Scheme 40).[78] This catalyst was tested in the electrophilic cyclization of diethyl 2-allyl-2-(prop-2-ynyl)malonate, affording the desired product in much higher yield and selectivity than the structurally-related (IMes)AuCl.</p><!><p>In 2018, Shi and co-workers reported an impressive protocol for enantioselective protoboration of terminal olefins catalyzed by a chiral BIAN–NHC–Cu complex 36 (Scheme 41).[79] The authors proposed that a combination of strong σ-donation and steric buttressing effect afforded by the BIAN scaffold together with the C2-symmetric chiral wingtip substitution is the key to afford high regio- and enantioselectivity in this process. In agreement with this design, the classical imidazolylidene-based analogue of 36 furnished the product with much lower selectivity. The optimized conditions involve the use of 2 mol% of BIAN–NHC–Cu complex 36 in the presence of B2dmpd2 (dmpd = dimethylpentanediol), MeOH as proton source and t-BuONa as base in n-hexane. The scope of this transformation is very broad, including a range of functional groups, such as ethers, ketones, amines and halides. The proposed mechanism involves the regio- and enantioselectivity determining olefin borylcupration, protonation of the alkylcopper intermediate, and σ-metathesis of the copper alkoxide with the diboron reagent. This important transformation demonstrates the beneficial effect of BIAN–NHC complexes in the essential area of enantioselective Cu–NHC catalysis.[80]</p><!><p>As demonstrated in this review, the recent years have witnessed tremendous advances using BIAN–NHC ligands as pivotal ancillary ligands to promote catalytic transformations. There are several clear benefits of BIAN–NHCs, including (1) stronger σ-donor character, (2) stronger π-acceptance, (3) steric buttressing effect of the scaffold, (4) ease of synthesis of unsymmetrical N-wingtip precursors, (5) high stability of metal-BIAN–NHC complexes to aerobic conditions, (6) capacity to promote reactions at very low catalyst loading, (7) extended π-aromatic system available for coordination, and (8) scaffold modification that permits for the synthesis of diverse analogues. Importantly, the high activity of BIAN–NHCs as privileged ligands in transition-metal-catalysis has been demonstrated with metals across the periodic table, including Pd, Ni, Ir, Rh, Ru, Au and Cu, which provides an attractive springboard for metal-dependent advantages of each transformation.</p><p>Among several exciting developments in this area have been (1) the emergence of asymmetric catalysis by chiral BIAN–NHC ligands; (2) the establishment of Pd-catalyzed cross-coupling reactions at low catalyst loadings; (3) the development of Au- and Pd-catalyzed sulfonation reactions; (4) the discovery of di-metallic complexes with improved selectivity by π-stacking and cooperative catalysis; (5) the development of Pd-catalyzed cross-couplings of challenging electrophiles under exceedingly mild conditions; (6) the establishment of active Ru–BIAN catalysts for alkene metathesis reactions; (7) electrophilic functionalization at low catalyst loading. In these methods, the high catalytic activity of BIAN-transition-metal complexes is retained even at low catalyst loading under aerobic conditions, features improved kinetics, higher selectivity and broader reaction scope compared to the classical imidazolylidene and imidazolinylidene ligands. The "flexible-steric-bulk" BIAN environment established around the metal center allows for adjustment of the ligand architecture to the catalytic reaction pocket. The greater σ-donation of BIAN has been particularly crucial to significantly increase the catalyst activity.</p><p>Nevertheless, despite significant progress, there are several challenges that should be addressed: (1) commercialization of BIAN–NHC ligands would make this scaffold readily available to all researchers and facilitate screening of reaction conditions in a modular fashion compared to the classical imidazolylidene ligands; (2) A major focus should be placed on the discovery of more versatile catalysts and generalization of the reactivity trends observed. This point has already been well addressed in asymmetric catalysis, where the ANIPE-type BIAN-ligands emerged as the privileged class in this family of reactions; (3) BIAN–NHC ligands are ideally poised for the development of new reactions; however, with few exceptions thus far, the majority of methods have focused on improving the established protocols. To fully address the high activity of BIAN–NHC ligands, it is imperative that this platform be advanced to new synthetic methods; (4) The majority of current developments is still limited to Pd–BIAN–NHC, Ni–BIAN–NHC and Au–BIAN–NHC complexes. It is essential that the promising reactivity that has already been observed with other metals is advanced to general and robust reaction pathways. (5) Comprehensive mechanistic studies focusing on both the structure-activity-relationship and the mechanisms underlying catalytic cycles would greatly facilitate the design of new generations of catalysts and stimulate widespread application in various areas of catalysis.</p><p>Most importantly, the demonstrated reactivity already puts the BIAN–NHC ligand class on the same level as the classical imidazolylidene ligands and it is essential that all researches routinely screen BIAN–NHC ligands when developing new NHC-metal-catalyzed protocols.</p>

PubMed Author Manuscript

Electron Paramagnetic Resonance Spectroscopic Identification of the Fe\xe2\x80\x93S Clusters in the SPASM-Domain Containing Radical SAM Enzyme PqqE

Pyrroloquinoline quinone (PQQ) is an important redox active quinocofactor produced by a wide variety of bacteria. A key step in PQQ biosynthesis is a carbon-carbon crosslink reaction between glutamate and tyrosine side chains within the ribosomally-synthesized peptide substrate PqqA. This reaction is catalyzed by the radical SAM enzyme PqqE. Previous X-ray crystallographic and spectroscopic studies suggested that PqqE, like the other members of the SPASM domain family, contains two auxiliary Fe\xe2\x80\x93S clusters (AuxI and AuxII) in addition to the radical SAM [4Fe\xe2\x80\x934S] cluster. However, a clear assignment of the EPR signal of each Fe\xe2\x80\x93S cluster was hindered by the isolation of a His6-tagged-PqqE variant with an altered AuxI cluster. In this work, we are able to isolate soluble PqqE variants by using a less-disruptive strep-tactin\xc2\xae chromatographic approach. We have unambiguously identified the EPR signatures for four forms of Fe\xe2\x80\x93S clusters present in PqqE through the use of multi-frequency EPR spectroscopy: the RS [4Fe\xe2\x80\x934S] cluster, the AuxII [4Fe\xe2\x80\x934S] cluster, and two different clusters ([4Fe\xe2\x80\x934S] or [2Fe\xe2\x80\x932S]) bound in the AuxI site. The RS [4Fe\xe2\x80\x934S] cluster, the AuxII [4Fe\xe2\x80\x934S] cluster and the [2Fe\xe2\x80\x932S] cluster form in the AuxI site, can all be reduced by sodium dithionite, with g tensors of their reduced form determined as [2.040, 1.927, 1.897], [2.059, 1.940, 1.903] and [2.004, 1.958, 1.904], respectively. The AuxI [4Fe\xe2\x80\x934S] cluster that is determined based on its relaxation profile can only be reduced by using low-potential reductants such as Ti(III) citrate or Eu(II)-DTPA to give rise to a g1 = 2.104 signal. Identification of the EPR signature for each cluster paves the way for further investigations of SPASM domain radical SAM enzymes.

electron_paramagnetic_resonance_spectroscopic_identification_of_the_fe\xe2\x80\x93s_clusters_in_the_

INTRODUCTION<!>Plasmids and nomenclature.<!>PqqE expression and purification.<!>EPR sample preparation.<!>X-band CW EPR spectroscopy.<!>Q-band EPR spectroscopy.<!>X-band and Q-band HYSCORE.<!>RESULTS AND DISCUSSION<!>Characterization of the Radical SAM (RS) [4Fe\xe2\x80\x934S]RS cluster.<!>Characterization of the Auxiliary II (AuxII) [4Fe\xe2\x80\x934S] cluster.<!>Auxiliary I (AuxI) [4Fe\xe2\x80\x934S] cluster.<!>Auxiliary I (AuxI) [2Fe\xe2\x80\x932S] cluster.<!>Possible roles of the Fe\xe2\x80\x93S clusters in PqqE catalysis.<!>CONCLUSIONS

<p>Pyrroloquinoline quinone (PQQ) was initially identified as a redox cofactor for prokaryotic dehydrogenases, such as alcohol and sugar dehydrogenases, and plays an important role in bacterial metabolism of alcohols and sugars.1–4 Recently, PQQ has also been recognized as an important nutrient for higher organisms (plants and animals), providing benefits for their growth and oxidative-stress tolerance.5,6 In particular, PQQ has been shown to be beneficial to human health, especially in anti-diabetic, anti-oxidative and neuroprotective actions.6 Higher organisms are not known to synthesize PQQ and therefore rely fully on bacteria to provide this important nutrient.</p><p>The biosynthesis of PQQ is accomplished by the gene products of pqqABCDEFG.7–9 PqqA is a short peptide and is translated by the ribosome and later modified by a series of enzymes encoded by other pqq genes, placing it into the family of ribosomally-synthesized and post-translationally-modified peptides (known as RiPPs). The biosynthesis of PQQ begins with a carbon-carbon crosslink reaction between glutamate and tyrosine side chains within PqqA. This reaction (Figure 1) is catalyzed by the radical S-adenosyl-L-methionine (SAM) enzyme PqqE and employs a chaperone protein PqqD to deliver the peptide substrate PqqA to PqqE.8</p><p>The radical SAM enzyme PqqE contains a canonical CX3CX2C motif10 which provides three cysteine (Cys) residues to ligate a radical SAM (RS) [4Fe–4S] cluster, leaving a site for SAM binding at the fourth site-differentiated Fe.8,11 This RS cluster is responsible for generating a 5'-deoxyadenosine radical (5'-dA•) via the reductive cleavage of SAM; the 5'-dA• radical then initiates the radical chemistry by abstracting a hydrogen atom from the substrate.10 In addition to the N-terminal RS cluster, PqqE is reported to harbor two auxiliary Fe–S clusters (AuxI and AuxII) located in the C-terminal SPASM domain.11,12 The origin of the acronym SPASM comes from the founding enzyme members,12 i.e., AlbA,13,14 PqqE,11,15 anSMEs,16–19 and MftC,20,21 which are involved in the formation of subtilosin A and PQQ as well as the maturation of anaerobic sulfatase and mycofactocin. More recently, an increasing number of new SPASM-domain containing enzymes have been reported and studied in the biosynthesis of RiPPs natural products, such as CteB,22 Tte1186,23 NxxcB24 and HuaB25; these are responsible for sulfur-carbon cross-linkage to form a thioether bond, as well as StrB,26 SuiB,27 AgaB28,29 and WgkB30 that catalyze carbon-carbon cross-linkage between lysine and tryptophan side chains, along the lines of PqqE.</p><p>In addition to the RS cluster, SPASM-domain containing enzymes bind two auxiliary Fe–S clusters in their C-terminal domains through the presence of extra Fe–S cluster binding motifs.12 X-ray crystal structures of a number of enzymes in this superfamily, i.e., anSME,18 SuiB27 and CteB,22 show three Fe–S clusters that are all [4Fe–4S] clusters. As will be discussed in detail below, PqqE is an exception, showing a [2Fe–2S] cluster in the AuxI site.11 Electron paramagnetic resonance (EPR) spectroscopy, which is widely employed to study Fe–S cluster-containing enzymes and reaction intermediates, is challenged in investigating the three Fe–S clusters in SPASM-domain containing RS enzymes because of overlap of the EPR signals and possible inter-cluster dipolar interactions. Furthermore, instability of prepared mutant samples designed to knock out one or two clusters can hinder the reliability of Fe–S cluster EPR signal analyses.11</p><p>Previous investigations of PqqE from Methylorubrum extorquens (M extorquens) provide a good example.11,31,32 Our earlier X-ray crystallographic report showed that the AuxI cluster is a [2Fe–2S] cluster ligated by four cysteine residues (Cys248, Cys268, Cys323, Cys325) and the AuxII cluster is a [4Fe–4S] cluster that is ligated by three cysteine residues (Cys310, Cys313, Cys341) and one aspartate residue (Asp319).11 However, the RS cluster was missing in the structure, and difficulties in obtaining high quality crystals of this enzyme have limited further understanding of both the number and the type of Fe–S clusters in PqqE. Associated EPR spectroscopic studies using wild-type, an RS cluster knockout variant (AuxI/AuxII, see nomenclature in Table 1) and an AuxII cluster knockout variant (RS/AuxT) suggested a mixture of [2Fe–2S] and [4Fe–4S] clusters for the auxiliary clusters. However, we were previously not able to isolate a PqqE variant lacking the AuxI cluster (RS/AuxII), making it difficult to fully assign observed complicated EPR signals or extract a unique g tensor for each cluster. In this prior work, we used Ni-NTA affinity chromatography to isolate both wild-type and PqqE variants containing a hexahistidine (His6) tag, followed by Fe–S cluster reconstitution. However, several desalting steps may disturb the Fe–S clusters.33,34 To mitigate this issue, we now employ a less-disruptive chromatography, i.e., the strep-tactin® approach,33,34 to purify the full range of PqqE variants. This change is in part inspired by our work on the RS enzyme HydG, in which the unique dangler Fe site of the auxiliary [5Fe–4S] cluster is found to be better retained using the strep-tactin® approach as opposed to metal-affinity chromatography.33,34</p><p>Here we report the use of multi-frequency EPR spectroscopy in identifying and characterizing the EPR signatures for four forms of Fe–S clusters present in PqqE enzyme: the RS [4Fe–4S] cluster, the AuxII [4Fe–4S] cluster, and two different clusters ([4Fe–4S] or [2Fe–2S]) bound in the AuxI site. The RS [4Fe–4S] cluster, the AuxII [4Fe–4S] cluster and the [2Fe–2S] cluster form in the AuxI site, can all be reduced by sodium dithionite, revealing the corresponding EPR signals of their reduced forms. However, the AuxI [4Fe–4S] cluster can only be reduced by lower potential reductants, such as Ti(III) citrate or Eu(II)-DTPA. The identification and analysis of the EPR signature for all of the possible clusters within PqqE is an important advance to the EPR characterization of RS proteins, as well as to ongoing efforts to obtain detailed mechanistic understanding of members of the roles of auxiliary sites in SPASM-domain RS enzymes.</p><!><p>The plasmid used in this wok for expressing wild-type PqqE is an N-terminal strep-tag II containing pET-28a vector with a M. extorquens pqqE insert (UniProt number P71517). The PqqE variants used in this work are listed in Table 1. They were generated using Quick-change method, and the primers are listed in Table SI. In order to clarify the clusters in each variant, we denoted each variant using the cluster that presents in the protein sample rather than using the one being knocked out (see Table 1).</p><!><p>The His6-tagged wild-type PqqE was expressed as described previously.35 The strep-tagged wild-type PqqE and its variants were expressed aerobically in an E. coli BL21(DE3) GOLD strain that harbors both an N-terminal strep-tag II containing pET-28a plasmid with a pqqE insert and the suf operon plasmid pPH151. The cells were grown at 31 °C in Terrific Broth (TB) media containing 50 μg/ml of kanamycin, 50 μg/ml of chloramphenicol, 200 μM ammonium ferric citrate and 500 μM MgSO4 to an O.D.600 ≈ 0.6. Then the temperature was decreased to 18 °C; after 1 hour, protein expression was initiated by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 100 μM and cysteine to a concentration of 100 μM. After expression at 18 °C for around 18 hours, the cells were harvested by centrifugation (3,500 rpm at 4 °C for 30 min), flash frozen in liquid nitrogen, and stored in −80 °C.</p><p>The His6-tagged wild-type PqqE was purified as described previously.35 The strep-tagged PqqE was purified anaerobically using a standard manufactory protocol (Germantown, MD) with modifications. The harvested cells were transferred to an anaerobic chamber and suspended in a deoxygenated lysis buffer with the volume ca. five times the mass of cell pastes. The lysis buffer was a HEPES-buffered solution (buffer W, 50 mM HEPES, 150 mM KCl, pH = 7.9) supplemented with BugBuster (Novagen), Benzonase (Novagen) and lysozyme (Novagen). The lysate was then transferred to O-ring sealed tubes and centrifuged at 20,000 rpm for 30 min. The sealed tubes that contained clarified lysate were transferred to the anaerobic chamber. The supernatant was loaded onto the Strep-tactin Superflow Plus resin (QIAGEN, Germantown, MD) with a column volume of 10 mL. The unbound protein fraction was removed by gravity-flowing through the resin and the resin was washed with 2 column volumes of buffer W. The PqqE protein fraction was then eluted by adding buffer W containing 3 mM d-Desthiobiotin, The eluted PqqE protein fraction was gently concentrated by using 30 kDa cutoff Amicon centrifugal filters at 6,000 rpm for 15 min. We used the fresh prepared protein (without freezing) to prepare all the EPR samples in these studies.</p><p>The Strep-tactin Superflow Plus resin was regenerated by using 5 column volumes of buffer W supplemented with 1 mM 2-(4-hydroxyphenylazo)benzoic acid and washed with 5 column volumes of buffer W. The concentration of the protein and the Fe content was determined using the method published previously.35</p><p>For the sample of globally-15N-labled D319H, the protein was expressed in 4 L M9 minimal medium supplemented with 4 g 15NH4Cl and 4 g ISOGRO®-15N medium powder. Instead of using the overnight cell culture to inoculate the M9 minimal media directly, 40 mL overnight cell culture was centrifuged, and washed using autoclaved deionized water first. Then the washed cell that was re-suspended in autoclaved deionized water was used for inoculating the media for expression. 200 μM ferreic chloride was added as extra iron source to replace the ammonium ferric citrate. The other expression and purification procedures were same as the natural abundance PqqE protein.</p><!><p>We used the fresh prepared protein to prepare all the EPR samples in these studies. For dithionate-reduced samples, ≈20 equivalents of sodium dithionate was added to PqqE protein in an X-band or Q-band EPR tube and allowed to incubate for 10 min. Then the EPR samples were transferred outside the anaerobic chamber, and frozen in liquid nitrogen for EPR spectroscopic analysis.</p><p>For cyanide-treated samples, ≈100 equivalents of isotope-labeled K13C15N (Cambridge Isotope Laboratories, Inc) was added to PqqE variant (RS only or AuxI/AuxII) which has already pre-incubated with ≈20 equivalents of sodium dithionate in an X-band or Q-band EPR tube. The samples were then transferred outside the anaerobic chamber, quickly frozen in liquid nitrogen and analyzed by using EPR spectroscopy.</p><p>For Ti(III) citrate-reduced samples,36 fresh Ti(III) citrate (89.7 mM) was prepared by adding 50 μL of a 15% TiCl3 solution to a 500 μL of 0.2 M sodium citrate solution and neutralized with 100 μL saturated sodium carbonate solution. Then ≈45 equivalents of Ti(III) citrate was added to PqqE protein and allowed to incubate for 1 hour. The EPR samples were then transferred outside the anaerobic chamber, quickly frozen in liquid nitrogen and analyzed by using EPR spectroscopy.</p><p>For Eu(II)-DTPA-reduced samples, fresh Eu(II)-DTPA was prepared by anaerobically adding one equivalent Eu(II)Cb powder to Tris-buffered solution (100 mM Tris, pH = 8.0) of DTPA (diethylenetriaminepentaacetic acid).37,38 Then ≈30 equivalents of Eu(II)-DTPA was added to PqqE protein and allowed to incubate for ~30 min. The EPR samples were then transferred outside the anaerobic chamber, quickly frozen in liquid nitrogen and analyzed by using EPR spectroscopy.</p><!><p>X-band (9.37 GHz) continuous-wave (CW) EPR spectra were recorded on a Bruker (Billerica, MA) EleXsys E500 spectrometer equipped with a super-high Q resonator (ER4122SHQE). Cryogenic temperatures were achieved and controlled using an ESR900 liquid helium cryostat in conjunction with a temperature controller (Oxford Instruments ITC503) and a gas flow controller. CW EPR data were collected under slow-passage conditions. The spectrometer settings were as follows: conversion time = 40 ms, modulation amplitude = 0.5 mT, and modulation frequency = 100 kHz; other settings are given in the corresponding figure captions. Spin quantification was determined by comparison of the double integral intensity of the EPR spectra to that of a standard solution of 100 μM CuSO4 with 200 μM HC1, 200 mM NaClO4 and 20% ethylene glycol. Simulations of the CW spectra and the following pulsed EPR spectra were performed using the EasySpin 5.1.10 toolbox39,40 within the Matlab 2014a software suite (The Mathworks Inc., Natick, MA). Euler angles follow the zyz convention.</p><!><p>Q-band two pulse electron spin-echo (ESE)-detected field swept EPR spectra (π/2-τ-π-τ-echo) were collected using a Bruker (Billerica, MA) EleXsys E-580 spectrometer equipped with a 10 W amplifier and a R.A. Isaacson-built cylindrical TE011 resonator mounted in an Oxford CF935 cryostat. Pulse sequences were programmed with the PulseSPEL programmer via the XEPR interface. Experiment parameters were as follows: π/2 = 12 ns, τ = 300 ns, and other settings are given in the corresponding figure captions. The Q-band spectra presented in this work are pseudo-modulated spectra using a modulation amplitude of 3.0 mT.</p><!><p>Hyperfine sublevel correlation spectroscopy (HYSCORE) is a two-dimensional pulse EPR technique, which correlates nuclear spin-flip transition frequencies in one electron-spin manifold to those in another electron-spin manifold. HYSCORE spectra were recorded at 10 K on the Bruker Biospin EleXsys 580 spectrometer by employing a split-ring (MS5) resonator for X-band and the Isaacson cylindrical TE011 resonator for Q-band. The pulse sequence π/2-τ-π/2-t1-π-t2-π/2-τ-echo was programmed with the PulseSPEL programmer via the XEPR interface. The pulse length for inversion pulse (tπ) and the π/2 pulse (tπ/2) was 24 ns. Eight-step phase cycling was used. Time-domain spectra were baseline-corrected (third-order polynomial), apodized with a hamming window, zero-filled to eight-fold points, and fast Fourier-transformed to yield the frequency-domain spectra. Particular spectrometer settings are given in the corresponding figure captions.</p><!><p>Our previous studies supported the view that PqqE enzyme requires all three Fe–S clusters (RS, AuxII and AuxI clusters) to yield the carbon-carbon crosslink activity. Our goal in this work is to identify the EPR signature for each cluster by characterizing various mutant samples (as listed in Tables 1&2) via multi-frequency EPR spectroscopy, as a critical step towards understanding the role each cluster plays in PqqE catalysis. We employed the non-disruptive strep-tactin® approach33,34 to purify all enzyme samples. Also, in order to minimize any potential degradation at the Fe–S clusters in WT and protein variants, all EPR samples were "as-eluted PqqE" that had avoided purification steps capable of damaging the protein such as harsh centrifugation (> 6,000 rpm) or freeze-thaw cycles (see Materials and Methods for details). Figure 2 shows the CW EPR spectrum of the as-eluted wild-type strep-tagged PqqE reduced by dithionite, indicating in this case an EPR spectrum very similar to previously reported EPR data for His6-tagged PqqE. In what follows, we disentangle the EPR spectrum of dithionite-reduced wild-type PqqE—identifying which signal arises from each Fe–S cluster (section 3.1, 3.2 and 3.4). In addition, in section 3.3, a low-potential [4Fe–4S] cluster is observed by using low-potential reductants (Ti(III) citrate or Eu(II)-DTPA), and we discuss these properties in the context of potential mechanisms for carbon-carbon crosslink catalysis (section 3.5).</p><!><p>We first prepared a double-knockout variant (termed as RS only) that only contained the cysteine motif C28X3C32X2C35 for binding the RS [4Fe–4S] cluster. For the deletion of auxiliary clusters, two cysteine residues from each auxiliary cluster were mutated to alanine residues (see Table 1 for details). The corresponding X-band (9.37 GHz) and Q-band (33.91 GHz) EPR spectra of this mutant reduced by dithionite are shown in Figures 3A and 3B, respectively. As expected, the EPR spectra show a single paramagnetic component which is well simulated by employing the g-values = [2.040, 1.927, 1.897], typical for a reduced RS [4Fe–4S]+ cluster.10 The observation of one paramagnetic species is reasonable, as this double-knockout variant presumably only contains the remaining RS cluster.</p><p>To determine the type of this Fe–S cluster, we probed its spin-relaxation properties by examining the signal intensity as a function of both sample temperature and microwave power. As shown in Figures 4A (blue diamonds) and S1C, when the sample temperature was varied between 10 and 50 K, the EPR signal follows the Curie law—the signal intensity is inversely proportional to the temperature; at high temperatures (> 50 K), the signal relaxes too fast to be observed. This temperature dependence of the EPR signals behaves as a typical reduced [4Fe–4S]+ cluster.41 The power dependence of the EPR signals, as shown in Figures 4B (blue filled diamonds) and S1D, yields a half-saturation power (P1/2) ca. 1.0 mW at 10 K, which is close to the P1/2 value (0.8 mW at 12.5 K) of the reduced [4Fe–4S]+ cluster in B. stearothermophilus ferredoxin.41 Therefore, spin-relaxation properties of this paramagnetic species with g-values = [2.040, 1.927, 1.897] match the properties of a reduced [4Fe–4S]+ cluster.</p><p>In order to further confirm that this paramagnetic species corresponds to the reduced RS [4Fe–4S]+ cluster, we sought to record an EPR spectrum after adding SAM to the dithionite-reduced RS-only variant to see if we can observe the corresponding SAM-bound [4Fe–4S]+RS signal which has a typical g tensor [2.01, 1.88, 1.85],10 However, this double-knockout sample was unstable and started to precipitate upon the addition of SAM, preventing us from further EPR analysis. Our previous studies suggested that this RS-only variant conserves the activity of uncoupled SAM-cleavage reaction,11 which could also be a reason for our inability to observe the EPR signal of the SAM-bound [4Fe–4S]+RS complex. As an alternate approach, we employed isotope-labeled cyanide (i.e., K13C15N) as a small molecule probe: cyanide has been shown to bind to the unique Fe site of the RS [4Fe–4S]+ cluster, generating an S = 1/2 13C15N-bound [4Fe–4S]+RS species.42 The EPR sample was prepared by adding ≈100 equivalents of K13C15N to the sample of dithionite-reduced RS-only variant and then analyzed by using both X-band (9.37 GHz) and Q-band (33.90 GHz) EPR spectroscopy, with the corresponding spectra presented in Figures 3A and Figure 3B, respectively. As expected, the addition of K13C15N resulted in a complete conversion of the [4Fe–4S]+ cluster signal with g-values = [2.040, 1.927, 1.897] to a new paramagnetic species with a rhombic g tensor of [2.063, 1.957, 1.913]. This g tensor is similar to the g tensor [2.06, 1.95, 1.93] of the CN-bound [4Fe–4S]+RS species observed in RS enzyme HydG (see Table 3),42 consistent with this new rhombic signal being indicative of the 13C15N-bound [4Fe–4S]+RS species in PqqE.</p><p>Pulse EPR studies confirm this 13C15N-bound [4Fe–4S]+RS species assignment. X-band HYSCORE spectra acquired at multiple magnetic field positions (corresponding to the g-values of 2.060, 1.959 and 1.927, Figure 5A) show obvious two sets of cross-peaks centered at 13C- and 15N-Larmor frequencies, validating the presence of 13C and 15N in the vicinity of the [4Fe–4S]+ cluster. These two sets of cross-peaks are well simulated by using a pseudo-axial hyperfine coupling interaction tensor A for 13C or 15N, with A(13C) = [−4.00, −4.70, 1.80] MHz and A(15N) = [2.00, 0.45, 2.60] MHz, respectively, as shown in Figure 5B. These hyperfine coupling values are comparable to the reported 13C- and 15N-hyperfine values for 13C15N-bound [4Fe–4S]+ clusters (as listed in Table 3) in HydG (A(13C) = [−5.0, −4.7, 0.9] MHz),34 IspH (A(13C) = [−3.9, −3.8, 0.1] MHz and A(15N) = [1.1, 1.1, 2.3] MHz),43 and Pyrococcus furiosus (P. furiosus) ferredoxin (A(13C) = [−4.5, −4.5, 0.1] MHz).44 In the HydG enzyme, in addition to the reported CN-bound [4Fe–4S]+RS species formed in N-terminus (vide supra), cyanide produced from tyrosine is also able to displace the generated [Fedangler(CO)2CN] synthon during HydG reaction by forming an S = 1/2 CN-bound [4Fe–4S]+AUX species in C-terminus.34 The [4Fe–4S] cluster in IspH is ligated by three cysteine residues and thus, has a unique Fe site for cyanide binding to produce a CN-bound [4Fe–4S]+ species described above.43 The [4Fe–4S] cluster in P. furiosus ferredoxin is ligated by three cysteine residues, with the fourth residue as an aspartate (D14) providing a carboxylate ligand, which is able to displaced by CN− to form a CN-bound [4Fe–4S]+ species (vide supra).44</p><p>The observed 13C- and 15N-hyperfine couplings for K13C15N-treated dithionite-reduced RS-only variant confirm the generation of the 13C15N-bound [4Fc–4S]RS+ species with a rhombic g tensor of [2.063, 1.957, 1.913]. These findings provide strong support that the paramagnetic species with the g tensor [2.040, 1.927, 1.897] prior to cyanide addition corresponds to the reduced RS [4Fe–4S]+ cluster with a unique Fe site. Finally, we note that sodium dithionite is able to reduce this RS [4Fe–4S] cluster in PqqE.</p><!><p>In this section, we prepared a single-knockout PqqE variant (termed AuxI/AuxII), in which the three cysteine residues for the RS cluster were replaced by alanine residues (see Table 1 for details). Therefore, presumably only the clusters in the two auxiliary sites remain for this PqqE variant. The X-band and Q-band EPR spectra of dithionite-reduced AuxI/AuxII are shown in Figure 6A and 6B. The major paramagnetic component (≈ 85% via spin quantification of the total signal is well simulated by using a g tensor of [2.059, 1.940, 1.903]. There is also a minor component (≈ 15% via spin quantification) present in the spectra.</p><p>Spin-relaxation properties of the major component were probed by examining the signal intensity (the peak amplitude at g1 2.059 of the major component) as a function of both sample temperature and microwave power. The temperature dependence of the major-component signals, as shown Figures 4A (magenta squares) and S2C, behaves similarly to that of the [4Fe–4S]+RS cluster (vide supra), i.e., when the sample temperature was varied between 10 and 50 K, the major-component signals follow the Curie law; when the temperature was higher than 50 K, the signal of the major component relaxes too fast to be observed, leaving only the minor-component signal to persist at 60 K (see Figure S2C). The temperature-dependence performance of this minor-component signal is similar as that of the [2Fe–2S]+ cluster with slow-relaxation properties.41 In what follows, we focus on probing the major-component signal and will discuss the [2Fe–2S] cluster-related minor component further in section 3.4. The origin and function of this minor component is of interest, with the caveat that it may have arisen from a degraded Fe–S cluster.45 The power dependence of the major-component signals, presented in Figures 4B (magenta filled squares) and S2D, gives a half-saturation power P1/2 ca. 1.1 mW at 10 K, which is similar to the P1/2 value (1.0 mW at 10 K) of the [4Fe–4S]+RS cluster described above. Therefore, spin-relaxation properties of this major component suggest that it corresponds to a reduced [4Fe–4S]+ cluster. Since both auxiliary clusters are present in the AuxI/AuxII sample, this [4Fe–4S]+ cluster could be either the AuxII cluster, or a possible [4Fe–4S] cluster form in the AuxI site (this is in part inspired by the crystal structures of three other enzymes in the SPASM superfamily, i.e., anSME,18 SuiB27 and CteB,22 which show a [4Fe–4S] cluster in the AuxI site.). Also at this point, we could not rule out the possibility that the major-component signal has contributions from both the reduced [4Fe–4S]+AuxII and the [4Fe–4S]+AuxI cluster, which share the same g tensor and identical spin-relaxation properties.</p><p>To address this further we again employed isotope-labeled cyanide (K13C15N), noting that Hoffman et al.44 showed that for the [4Fe–4S] cluster in P. furiosus ferredoxin that is ligated by three cysteine and one aspartate residue (D14), cyanide can displace the carboxylate ligand of the aspartate residue and bind to the Fe site to form an S = 1/2 CN-bound [4Fe–4S]+ species (vide supra). Our previous X-ray structure of PqqE showed that the AuxII [4Fe–4S] cluster is also ligated by three cysteine and one aspartate residue (D319),11 with the binding motif exactly identical to the P. furiosus ferredoxin [4Fe–4S] cluster. Therefore, cyanide should also be able to displace the carboxylate ligand of D319 to form an S = 1/2 CN-bound [4Fe–4S]+AuxII species. In contrast, the AuxI cluster, displayed in the form of a [2Fe–2S] cluster in X-ray structure, was fully ligated by four cysteine residues.11 If a [4Fe–4S] cluster form in the AuxI site exists, it would presumably be fully ligated by the same four cysteine residues that ligate the [2Fe–2S] cluster in the crystal structure. In this case, cyanide-treatment will not alter the [4Fe–4S]+AuxI EPR signal. As shown in Figure 6, by adding ≈100 equivalents of K13C15N to a dithionate-reduced AuxI/AuxII sample, we observed that the major-component signal is completely converted to a new pseudo-axial S = 1/2 signal well simulated with g-values = [2.087, 1.955, 1.941]. The minor component was unaltered by the cyanide treatment (Figure S3). The obtained new g tensor is notably similar to that of CN-bound [4Fe–4S]+Aux in HydG (g-values = [2.09, 1.94, 1.93]) and CN-bound [4Fe–4S]+ in IspH (g-values = [2.08, 1.94, 1.93]), indicating its correspondence to a CN-bound [4Fe–4S]+ species (Table 3). The complete conversion of the reduced [4Fe–4S]+ cluster signal to the CN-bound [4Fe–4S]+ species indicates that the major EPR component corresponds to the reduced [4Fe–4S]+AuxII cluster rather than the [4Fe–4S]+AuxI cluster. Also, the results rule out the possibility that the major-component signal consists of contributions from both of the two auxiliary [4Fe–4S]+ clusters, because if this were the case, we would only be able to see partial signal conversion upon cyanide-treatment.</p><p>We further pursued X-band 13C- and 15N-HYSCORE studies to confirm the assignment of the new pseudo-axial signal as the 13C15N-bound [4Fe–4S]+AuxII species. X-band HYSCORE spectra acquired at multiple magnetic field positions (corresponding to the g-values of 2.080, 1.956 and 1.945, Figure 7A) clearly show two sets of cross-peaks centered at 13C- and 15N-Larmor frequencies, supporting the above assignment. These two sets of cross-peaks are well simulated by using a pseudo-axial hyperfine coupling interaction tensor A for 13C or 15N, with A(13C) = [−4.40, −4.40, 1.00] MHz and A(15N) = [2.10, 2.10, 0.45] MHz, respectively, as shown in Figure 7B. These hyperfine coupling values for both 13C and 15N are comparable to the values of those CN-bound [4Fe–4S]+ species described in section 3.1 (Table 3). Therefore, the obtained 13C15N-bound [4Fe–4S]+AuxII species supports the conclusion that the major-component signal observed from dithionate-reduced AuxI/AuxII arises solely from the reduced [4Fe–4S]+AuxII cluster.</p><p>In addition, we performed site-directed mutagenesis on the gene construct of AuxI/AuxII by replacing the aspartate residue (D319) with a histidine or cysteine residue to generate two new variants, i.e., AuxI/AuxII/D319H and AuxI/AuxII/D319C. By probing whether the g tensor of the [4Fe–4S]+AuxII cluster will be affected upon altering the ligand environment, we can further prove that it is the reduced [4Fe–4S]+AuxII cluster that gives rises to the major-component EPR signal. The X-band and Q-band EPR spectra of dithionite-reduced AuxI/AuxII/D319H and AuxI/AuxII/D319C are shown in Figure 6. In the spectrum of each sample, although there is a minor component which is the residual [2Fe–2S] cluster signal after subtraction (see Figure S4 and section 3.4 for details), a major component with the expected g tensor changes is observed, as presented in Figure 6 and listed in Table 2. We note that the g1 feature is shifted from 2.059 for AuxI/AuxII to 2.046 and 2.042 for AuxI/AuxII/D319H and AuxI/AuxII/D319C, respectively, presenting increasingly smaller g-anisotropy as the aspartate D319 residue is mutated to histidine and cysteine. The trend in the g-anisotropy with the more symmetric ligand environment resulting in less g-anisotropy is consistent with the reported g-tensor changes in the P. furiosus ferredoxin [4Fe–4S]+ cluster,46 where the g-values changed from [2.10, 1.87, 1.79] to a less anisotropic tensor [2.08, 1.93, 1.89] as the aspartate D14 residue was mutated to cysteine residue. The sensitivity of the observed g-tensor changes to the ligand environment further confirms the AuxII [4Fe–4S]+ cluster assignment.</p><p>Of considerable interest, X-band HYSCORE spectrum of dithionate-reduced AuxI/AuxII/D319H acquired at the magnetic position corresponding to g1 value of 2.046 (Figure 8C) shows the correlation ridges in the (−, +) quadrant that are diagnostic of the presence of hyperfine-coupled 14N nucleus in the vicinity of the reduced [4Fe–4S]+ cluster. This contrasts to the remote 14N couplings from protein backbone nitrogens that usually yield weak 14N-hyperfme coupling HYSCORE signals shown in the (+, +) quadrant. As expected, the 14N-hyperfine coupling signals in the (−, +) quadrant were absent in the HYSCORE spectra of dithionate-reduced AuxI/AuxII or AuxI/AuxII/D319C (Figures 8A and 8B), in which the fourth residue of AuxII [4Fe–4S] cluster is the aspartate (D319) and cysteine, respectively, as depicted in Figure 8. The observation of 14N-hyperfine coupling signals in the (−, +) quadrant suggests that the histidine (H319) coordinates to the [4Fe–4S]AuxII cluster, which also supports our assignment of the reduced [4Fe–4S]+AuxII cluster (vide supra).</p><p>To evaluate the 14N-hyperfme coupling arising from the Fe-histidine interaction in the AuxI/AuxII/D319H sample, a globally-15N-labled sample of D319H was prepared by expressing the protein in M9 medium supplemented with 15NH4Cl and ISOGRO®-15N medium powder. This globally-15N-labled sample allows us to extract the strong 15N-histidine hyperfine-coupling tensor A, as the nuclei spin (I = 1/2) of 15N does not have a nuclear quadrupole interaction (in contrast to 14N with I = 1) and will display a simplified HYSCORE spectrum. To be noted, the reason that 15N-labeled D319H was chosen rather than AuxI/AuxII/D319H is due to the higher yield of the former PqqE variant, which therefore can provide enough protein for pulse EPR analysis, and the target 15N-hyperfine coupling from the Fe-histidine interaction will not be altered. Orientation-selected Q-band HYSCORE spectra of dithionate-reduced 15N-labled D319H acquired at multiple magnetic field positions (corresponding to the g-values of 2.044, 1.957 and 1.922) are shown in Figure 9. One set of cross-peaks centered at 15N-Larmor frequencies is observed and is indicative of a strongly 15N-hyperfine interaction between Fe and histidine H319. The HYSCORE spectra are well simulated by using the 15N-hyperfine tensor of A(15N) = [−1.02, −4.55, −1.42] MHz and Euler angle = [55°, 100°, 25°], which corresponds to aiso = −2.33 MHz and T = −1.11 MHz. The spin density delocalized from the reduced [4Fe–4S+AuxII cluster to the ε-nitrogen in the histidine ligand (H319) is ca. 0.36% by using the equation of aiso = a0ρ[1/(1+n)], where ρ is the spin density residing on 15N, a0 (−2540 MHz for 15N)47 is the isotropic hyperfine-interaction for one electron in the 2s orbital of nucleus, and n is the hybridization constant (2spn) that is equal to 3 for an ε-nitrogen.</p><p>With the hyperfine parameters of 15N from the coordinated histidine ligand in hand, we can obtain the corresponding hyperfine parameters of 14N by scaling A(15N) to A(14N) via A(15N)/A(14N) = gn(15N)/gn(14N) = −1.403, resulting in A(14N) = [0.73, 3.25, 1.01] MHz and the same Euler angle = [55, 100, 25]°, which corresponds to aiso = 1.66 MHz and T = 0.8 MHz. By performing spectral simulations, as shown in Figure S5, the quadrupole parameters e2Qq/h = −2.05 MHz and η = 0.55 and the quadrupolar Euler angle = [50, 27, 20]° relative to the hyperfine tensor are determined. As listed in Table 4, the 14N-hyperfine and quadrupole interaction values fall in the range of the typical Fe-14N interactions. Especially, the 14N-quadrupole values are comparable to those of strongly-coupled 14N-histidine from the reduced [4Fe–4S]+ K1 cluster in MaNifB e2Qq/h = −2.1 MHz and η = 0.4) that is ligated by one putative histidine residue as well as three cysteine residues.48</p><p>In this section, we have, thus, clearly identified the EPR signature for the AuxII [4Fe–4S]+ cluster with aspartate D319 as the fourth ligand, confirming the previous X-ray structure study. This assignment is corroborated by the generation of an S = 1/2 CN-bound [4Fe–4S]+AuxII species as well as the corresponding g-tensor shifts once the aspartate ligand (D319) was mutated to a histidine or cysteine residue.</p><!><p>In section 3.2, we have shown that the reduced [4Fe–4S]+AuxII cluster gives rise to the major-component signal in the EPR spectra of dithionate-reduced AuxI/AuxII, indicating that dithionate is able to reduce the AuxII [4Fe–4S] cluster. However, as only the RS cluster was knocked out and the iron content was assayed as ≈10 Fe per AuxI/AuxII enzyme (see Table 5), there presumably should be some [4Fe–4S] AuxI cluster (besides the observed [2Fe–2S] cluster signal that will be discussed specifically in section 3.4) in the AuxI/AuxII sample.18,22,27 Also, as shown from crystal structures of three other enzymes (anSME,18 SuiB27 and CteB22) in the SPASM superfamily, all of these examples indicate [4Fe–4S] clusters at AuxI. We therefore sought alternate explanations for the absence of an EPR signature corresponding to an inferred [4Fe–4S] cluster within the AuxI site of PqqE. It was possible that our inability to observe its EPR signal was due to an inability of dithionate to reduce AuxI, such that it had remained diamagnetic and EPR silent. This suggested that the reduction potential of [4Fe–4S] within AuxI in PqqE may be lower than that of dithionite (−660 mV vs NHE at pH 7.0).</p><p>To test this hypothesis, we employed Ti(III) citrate (≈45 equivalents), a low-potential reductant that has a reduction potential lower than −800 mV at pH 7.0,36,56 to reduce the AttxI/AttxII sample and recorded the EPR spectrum to see whether an additional paramagnetic species could be detected at AuxI. As presented in Figure 10 (blue trace), an additional signal shows up at the magnetic field of 318.1 mT, corresponding to g1 value of 2.104, outside the region around g ~2.0 where the large Ti(III) (S = 1/2, 3d1) EPR signal dominated (orange trace, and also see Figure S6). This new g1 = 2.104 signal is also observed in the spectra of Ti(III) citrate-reduced samples of wild-type PqqE (black trace) and RS/AuxI (red trace), but is absent in the RS/AuxII variant (green trace). Taken together, these results point to the g1 = 2.104 signal arising from a reduced AuxI cluster in PqqE, which can only be EPR-observable by using Ti(III) citrate as the reductant. This assignment is corroborated via cyanide-treatment experiment in which the g1 = 2.104 signal is not affected, suggesting that the signal is due to a fully ligated AuxI cluster with four cysteine ligands (Figure S7). We rule out the possibility that this g1 = 2.104 signal is due to a splitting arising from the dipolar interaction between two reduced clusters as observed in Complex I,57 because the Q-band spectrum shows a signal at the same g-value (see Figure S8).51 If this signal were due to an inter-cluster dipolar interaction that contributes to the spin Hamiltonian, the EPR spectrum is expected to show a microwave-frequency dependence, which is not the case here.</p><p>Therefore, the above results suggest that, in addition to a [2Fe–2S] cluster, the AuxI site of PqqE is also able to accommodate a low-potential Fe–S cluster, with a reduction potential lower than that of dithionite but higher than that of Ti(III) citrate. To be noted, while this AuxI cluster can only be reduced by Ti(III) citrate, the dithionite-reducible [4Fc–4S]RS and [4Fc–4S]AuxII clusters can also be reduced by Ti(III) citrate. Therefore, their corresponding EPR signals should appear in the spectra of Figure 10; however, the overlap with the hugely dominant Ti(III) signals complicates the interpretation of spectra in the g ~2.0 region in Figure 10. This does not invalidate the well characterized spectra of PqqE variants (cf. Figure S9) that verify, for example, the [4Fe–4S]+RS signal in dithionite-reduced RS/AuxI, as well as an [2Fe–2S] cluster signal and the [4Fe–4S]+RS and [4Fe–4S]+AuxII cluster signal in dithionite-reduced RS/AuxII; these results are consistent with the signal assignments described in section 3.1 and 3.2.</p><p>To determine the nature of this low-potential AuxI cluster, we probed its spin-relaxation properties by examining the g1 = 2.104 signal intensity as a function of both sample temperature and microwave power. Both the temperature dependence and power dependence of the signal behave dramatically different from those of either the [4Fe–4S]+RS cluster or the [4Fe–4S]+AuxII cluster (vide supra). As shown in Figures 4A (red circles) and S10A, the signal follows the Curie law, when the sample temperature is varied between 10 and 22 K, and relaxes too fast to be observed when the temperature is higher than 22 K (see Figure S10A).</p><p>The power dependence of the signal, presented in Figure 4B (red filled circles) and S10B, gives a half-saturation power P1/2 ca. 100 mW at 10 K, which is dramatically higher than the P1/2 values (1.0 mW and 1.1 mW at 10 K) for the [4Fe–4S]+RS and [4Fe–4S]+AuxII cluster, respectively. Therefore, the fast spin-relaxation properties of this g1 = 2.104 signal suggest it arises from a fast-relaxing [4Fe–4S] cluster rather than a [2Fe–2S] cluster, because [2Fe–2S] clusters usually relax much slower as shown in Figure 4 (green triangles), with the signal still persistent at 60 K and a much smaller half-saturation power P1/2 ca. 0.04 mW at 10 K. Even in the case of a [2Fe–2S] cluster that relaxes faster than the typical [2Fe–2S] cluster, e.g., the [2Fe–2S] cluster in xanthine oxidase that relaxes faster due to the dipolar interaction with its nearby paramagnetic Mo(V) center, the [2Fe–2S]+ cluster signal is still observable at 60 K.58 In addition, a second low-potential reductant, Eu(II)-DTPA,37–38 with the reduction potential ca. −1.14 V vs NHE at pH 8.0, was employed to verify the g1 = 2.104 signal, as shown in Figures 11 and S11. The observed g1 = 2.104 signal shows the same spin-relaxation properties as that obtained by using Ti(III) citrate (Figure 11), i.e., it is observed at 10 K but not observable at 25 K, suggesting its independence regarding the specific low-potential reductant and confirming its ascription to the reduced AuxI [4Fe–4S]+ cluster in PqqE. We note that the fast-relaxation properties of the AuxI [4Fe–4S] cluster are not altered by the presence or absence of nearby AuxII or RS clusters, as the spin-relaxation properties are similar for the wild-type, AuxI/AuxII and RS/AuxI samples. This is consistent with no inter-cluster dipolar interactions observed in PqqE (vide supra). We also rule out the possible effect of the presence of paramagnetic species, such as Ti(III) or Eu(II). As shown in Figure 11, the EPR spectrum of the wild-type PqqE reduced by Eu(II)-DTPA recorded at 25 K still shows the signals from reduced [4Fe–4S]+RS (g1 = 2.040) and [4Fe–4S]+AuxII clusters (g1 = 2.059), suggesting that the presence of the paramagnetic specie Eu(II) does not alter the spin-relaxation properties of these two clusters as compared to dithionite-reduced samples (vide supra).</p><p>Therefore, we reasoned that it is the local protein environment of the [4Fe–4S] cluster in the AuxI site that causes its fast relaxation. This is the first spectroscopic evidence of low-potential Fe–S cluster in SPAMS-domain containing radical SAM enzymes, which could be relevant to the oxidative cross-link catalysis (see section 3.5).</p><!><p>We have identified a novel low-potential [4Fe–4S] cluster in the AuxI site as described in section 3.3. However, our previous X-ray structure of PqqE revealed a [2Fe–2S] cluster form in the AuxI site.11 In terms of the EPR spectra, the [2Fe–2S]+ cluster signal was observed in both previous EPR studies and this work. As mentioned in section 3.2, a minor component (15%) that has similar slow-relaxation properties as a [2Fe–2S]+ cluster was shown in the EPR spectrum of dithionite-reduced AuxI/AuxII. However, we noticed that the amount of the [2Fe–2S] cluster present in the AuxI/AuxII sample varied from batch to batch. Figure S12 shows the EPR spectrum of dithionate-reduced AuxI/AuxII sample from another batch (batch B, where batch A refers to the sample described in section 3.2); while the AuxII [4Fe–4S]+ signal with g-values = [2.059, 1.940, 1.903] remains the same as that identified from batch A, the [2Fe–2S]+ cluster has higher signal intensity (30% via spin quantification). The reason for varying amounts of the [2Fe–2S] cluster present in AuxI/AuxII could be due to variable lengths of time for purifying the enzyme in the anaerobic chamber; longer purification times led to higher signal intensity of the [2Fe–2S]+ cluster. Also, varying amounts of the [2Fe–2S] cluster were noted when inoculating the expression media with freshly co-transformed E. coli competent cell vs. freeze-thaw glycerol cell stock. In addition, we have observed obvious [2Fe–2S] cluster signals in the samples of dithionite-reduced AuxI/AuxII/D319H and AuxI/AuxII/D319C as well as cyanide-treated AuxI/AuxII, as shown in Figures S3&S4. The minor component shown in these three EPR spectra in Figure 6 is the residual [2Fe–2S] cluster signal after subtraction.</p><p>Besides these AuxI/AuxII-derived PqqE variants, we also detected [2Fe–2S]+ cluster signals in samples of dithionite-reduced wild-type PqqE and RS/AuxI, as shown in Figures 12&S9. Especially for the RS/AuxI sample with the AuxII cluster knocked out, similar to previous studies,11 the [2Fe–2S] cluster signal intensity is dramatically higher than other PqqE variants; the reason for this observation remains unclear. By employing the high-temperature EPR spectra of dithionite-reduced RS/AuxI, we extracted the g tensor [2.004, 1.958, 1.904] for the [2Fe–2S]+ cluster. We also characterized its slow spin-relaxation properties at 10 K for comparative purposes, as shown in Figures S13&4 (green triangles).</p><p>As summarized in Table 5, the only samples that did not display the [2Fe–2S]+ cluster signal are the variants with the AuxI cluster knocked out, i.e., RS/AuxII and RS-only variant. While no low-potential AuxI [4Fe–4S]+ cluster with g1 = 2.104 signal was detected by using Ti(III) citrate to treat either RS/AuxII or RS-only variant (Figures 10&S6), no [2Fe–2S] cluster signal was observed at high-temperature (60 K) EPR spectra of the corresponding dithionite-reduced samples (Figure S9&S1) either. Correspondingly, the remaining reduced [4Fe–4S]+RS and [4Fe–4S]+AuxII signals are shown in the low-temperature (10 K) spectrum of dithionite-reduced RS/AuxII (Figure S9), and only the [4Fe–4S]+RS is observed for RS-only variant (Figure 3).</p><p>To summarize this section, the results of detailed EPR analyses on a myriad of enzyme samples indicate that the Fe–S cluster in the AuxI site of PqqE can exist in the form of either a [2Fe–2S] or a [4Fe–4S] cluster 11,31 Thus, at this juncture, it is not yet possible to conclude with certainty which form is essential for PqqE catalysis.</p><!><p>With multi-frequency EPR spectroscopy we have unambiguously identified four forms of Fe–S clusters present in PqqE. They can be categorized into two groups according to their reduction potentials. One group, reducible by dithionite, includes the RS [4Fe–4S], the AuxII [4Fe–4S] and the AuxI [2Fe–2S] clusters. Hence these three Fe–S clusters when reduced by dithionite give corresponding EPR signals, as described above (see section 3.1, 3.2 and 3.4). The other group is reducible by Ti(III) citrate or Eu(II)-DTPA but not dithionite and contains only the AuxI [4Fe–4S] cluster, which yields the EPR signal with a characteristic g1 = 2.104 feature (see section 3.3). Based on the present categorization, the EPR spectra of dithionite-reduced wild-type PqqE can be well simulated by summing the contributions of three reduced clusters in the first group, with a ratio of 1:1:1 for [4Fe–4S]+RS, [4Fe–4S]+AuxII and [2Fe–2S]+AuxI cluster, as shown in Figure 12. Combining the X-band and Q-band simulations, we did not observe any major spectral contributions arising from inter-cluster dipolar interactions. While precise redox potentials have yet to be been measured for any of the clusters in PqqE, this EPR study shows this to be an important future research goal.</p><p>In PqqE catalysis, the RS [4Fe–4S] cluster is in charge of initiating the reaction via reductive cleavage of the sulfonium C5′–S bond of SAM to yield a cluster-bound methionine and a 5'-deoxyadenosine radical (5'-dA•). The transient 5'-dA• radical abstracts a hydrogen atom from the β-carbon of glutamate residue to generate a glutamyl radical. The RS cluster is essential and is required for carbon-carbon cross-link activity, as suggested by our previous studies.11 In addition to the RS cluster, both auxiliary clusters (AuxI and AuxII) in the SPASM domain play essential roles in PqqE catalysis, as variants lacking either auxiliary cluster abolish the cross-link activity.</p><p>In the AuxI site of PqqE, with a mixture of Fe–S clusters identified, it is not yet possible to ascertain which form ([4Fe–4S] or [2Fe–2S]) is functionally relevant. We note in this context that a low-potential AuxI [4Fe–4S] cluster supports the radical aromatic substitution mechanism catalyzed by StrB/SuiB/AgaB,26,28,29 in which a carbon-carbon bond is formed between the β-carbon of lysine residue and the aromatic C7 of tryptophan residue in the peptide substrate of StrA/SuiA/AgaA, a reaction highly analogous to that performed by PqqE. One proposed requirement for the radical aromatic substitution mechanism is to maintain the AuxI cluster in an oxidized state, poised for acceptance of one electron from an radical anion intermediate generated via de novo carbon-carbon bond formation. Since the PqqE cross-link activity can only be achieved using a biological reducing system (such as NADPH/Flavodoxin A/Flavodoxin reductase11) with a potential much higher than achieved with either the Ti(III) or Eu(II) salts, it is expected that a functional [4Fe–4S] cluster in the AuxI site would remain oxidized during catalytic turnover and, as such could promote radical anion oxidation at an aromatic ring. However, the redox potential of such a radical anion intermediate is expected to be much lower59 than that anticipated for a [2Fe–2S] site at AuxI as well, indicating that other properties are likely, in the end, to rationalize the participation of [2Fe–2S] vs. [4Fe–4S] in the AuxI site of PqqE. The answer to this intriguing question will need to await future studies.</p><!><p>We have unambiguously identified the EPR spectroscopic signatures for four forms of Fe–S clusters present in PqqE: the RS [4Fe–4S] cluster, the AuxII [4Fe–4S] cluster, and two alternative clusters ([4Fe–4S] or [2Fe–2S]) bound in the AuxI site. They can be categorized into two groups according to their reduction potentials. One group is dithionite-reducible, including the RS [4Fe–4S] cluster, the AuxII [4Fe–4S] cluster and the [2Fe–2S] cluster in the AuxI site, with g tensors of their reduced form determined as [2.040, 1.927, 1.897], [2.059, 1.940, 1.903] and [2.004, 1.958, 1.904], respectively. The other group contains the AuxI [4Fe–4S] cluster giving rise to a g1 = 2.104 signal, which can only be reduced by using low-potential reductants, such as Ti(III) citrate or Eu(II)-DTPA. This low-potential AuxI [4Fe–4S] cluster has faster relaxation properties than that of the RS [4Fe–4S] and the AuxII [4Fe–4S] clusters, indicating a specific protein environment of the AuxI site in the SPASM-domain of PqqE. No inter-cluster dipolar interactions are observed. Identification and analysis of the EPR signature for each cluster pave the way for further investigations of PqqE catalysis via EPR spectroscopy. These results also raise interesting, unsolved questions regarding the mechanism of the oxidative carbon-carbon crosslink catalyzed by PqqE.</p>

PubMed Author Manuscript

Simple and Inexpensive Micromachined Aluminum Microfluidic Devices for Acoustic Focusing of Particles and Cells

In this work, we introduce a new method to construct microfluidic devices especially useful for bulk mode acoustic wave (BAW) based manipulation of cells and microparticles. In order to obtain an efficient acoustic focusing, BAW devices require materials that have high acoustic impedance mismatch relative to the medium that cells/microparticles are suspended within and materials with a high-quality factor. To date, silicon and glass have been the materials of choice for BAW based acoustofluidic channel fabrication. Silicon and glass based fabrication is typically performed in clean room facilities, generate hazardous wastes, and can take several hours to complete the microfabrication. To address some of the drawbacks in fabricating conventional BAW devices, we explored a new approach by micromachining microfluidic channels in aluminum substrates. Additionally, we demonstrate plasma bonding of PDMS onto micromachined aluminum substrates. Our goal was to achieve an approach that is both low-cost and effective in BAW applications. To this end, we have micromachined Aluminum 6061 plates and enclosed the systems with a thin PDMS cover layer. These aluminum/PDMS hybrid microfluidic devices use inexpensive materials and are simply constructed outside of a clean room environment. Moreover, these devices demonstrate effectiveness in BAW applications as demonstrated by efficient acoustic focusing of polystyrene microspheres, bovine red blood cells, and Jurkat cells and generating multiple focused streams in flow through systems.

simple_and_inexpensive_micromachined_aluminum_microfluidic_devices_for_acoustic_focusing_of_particle

Introduction<!>Materials<!>Micromachining of the acoustofluidic device<!>Plasma sealing of PDMS onto Al substrate<!>Device preparation for acoustic focusing<!>Surface roughness and device variation analysis<!>Flow and pressure measurements in Al devices<!>Acoustic focusing of microspheres and cells<!>Fluorescence imaging and flow cytometry analysis<!>Construction of the acoustofluidic device in aluminum<!>Device configuration for optimum acoustic focusing<!>Acoustic focusing of microspheres and cells<!>Conclusions

<p>Acoustic standing waves provide a fast, non-contact, and gentle particle manipulation technique in microfluidic conditions. It has emerged as a promising new microfluidic technology for purification, separation, and concentration of beads and biological cell samples [1–10]. Acoustofluidic devices are routinely fabricated on silicon, glass, or poly(dimethylsiloxane) (PDMS) substrates [1, 8–12]. The device material and the format of focusing are mainly governed by the type of acoustic waves, either bulk or surface, being used. In the bulk acoustic wave (BAW) mode, acoustic waves are generated inside the microchannel resonant chamber and in the surface acoustic wave (SAW) mode, acoustic waves are generated on the surface of a substrate that is adhered to the microchannel [13–18].</p><p>In SAW devices, interdigital transducers (IDT) are fabricated on a piezoelectric surface to generate standing waves and often poly(dimethylsiloxane) (PDMS) microchannels are adhered on to the piezoelectric substrate [9, 13, 19]. Because SAW devices do not rely on resonant chambers, soft polymers like PDMS can be used as microchannel material. PDMS microchannels can easily be adhered onto the piezoelectric surface via plasma bonding. However, fabrication of electrodes on the piezoelectric surface needs to be precise and expensive equipment are needed for mask alignment and electrode metal deposition. Often, this equipment is housed in dedicated clean room facilities.</p><p>On the other hand, BAW devices rely on resonant chambers with parallel rigid walls that are capable of reflecting acoustic waves with minimum loss, therefore; the device substrate should possess high acoustic impedance and high Q-value (quality factor) [18]. Soft polymer materials like PDMS are not suitable for BAW, as the polymer walls of such devices lack the necessary difference in acoustic impedance (as compared to the aqueous solution) and acoustic damping is high [11, 20]. Materials with good acoustic properties (higher Q-value, density, speed of sound, and high impedance) are needed to construct effective BAW devices.</p><p>To date, silicon and glass have been the materials of choice for BAW based acoustofluidic channel fabrication. The process has several complex and costly steps that require dedicated instruments like mask aligner and deep reactive ion etcher and/or hazardous materials if chemical etching is used. Many of these steps are carried out in clean room facilities and it is a time consuming and expensive process. Commercially available rectangular or square glass micro-capillaries can also be used to create acoustofluidic devices [12, 21]. However, this approach is not amenable to design complex devices with multiple inlets and outlets and the dimensional tolerances are not precise (typically +/− ~10% of the dimension in question). Recently, microchannels fabricated on thermoplastic (polystyrene) have been utilized in acoustic separation of particles and cells. The biocompatibility of the thermoplastic substrate is attractive for many biological applications, however, due to high acoustic attenuation of the material, acoustic transducers are driven at a very high voltage (~80 V) and these systems need an efficient cooling mechanism [22, 23].</p><p>To address many of the drawbacks in fabricating BAW devices, we explored a new approach by micromachining microfluidic channels in aluminum substrates. Additionally, we demonstrate plasma bonding of PDMS onto micromachined aluminum substrates, which provides a quick and convenient way of enclosing metallic microchannel structures for fluidic applications. Our method does not require photolithography, clean room facilities, or etching thus eliminating long process times, hazardous reagents, and specialized equipment. Microchannels can easily be machined using widely available commercial machining equipment. There has been only one prior report of fabricating a microfluidic device via micromachined aluminum [24]. However, their approach was not simple and comprised of a glass window sandwiched between an epoxy block and a micromachined aluminum block and these layers were assembled using screws. Further, liquid access ports were machined horizontally through the aluminum substrate and rigid plastic connectors were screwed to each liquid port causing limited flexibility in designing more complex microchannel patterns. The device was bulky and not amenable for customization. The process we have developed is rapid, simple, and inexpensive and it can be done at most micromachining facilities. While we expect these devices will be useful in general for microfluidic applications, our implementation in aluminum based devices has specific relevance for BAW applications. Aluminum exhibits a high acoustic impedance relative to aqueous solutions typically of interest in acoustofluidic applications [18, 25]. The efficacy of this new method is demonstrated via acoustic focusing of microspheres and cells in single and multinode acoustic focusing modes.</p><!><p>Aluminum (6061 alloy) plates were purchased from McMaster-Carr (Los Angeles, CA) and Poly(dimethylsiloxane) (PDMS) was purchased from Ellsworth Adhesives (Germantown, WI). Silicone tubing (0.64 mm ID) were purchased from Cole Parmer (Vernon Hills, IL). PZT ceramic transducers were purchased from APC International Ltd (Mackeyville, PA). Acetone, PBS buffer tablets, and casein were purchased from Sigma Aldrich (St. Louis, MO). Bovine red blood cells (B-RBCs) were purchased from Innovative Research (Novi, MI). Nile red polystyrene particles (NR-ps) were purchased from Spherotech (Lake Forest, IL) and CountBright™ absolute counting beads were purchased from Life Technologies (Carlsbad, CA).</p><!><p>Two, 7 cm long microfluidic flow channels with trifurcated outlets (Fig. 1a), one with 250 μm wide main channel and another with 430 μm wide main channel were designed using AutoCAD software (Autodesk, San Rafael, CA). In order to demonstrate the feasibility of using any machining facility for microchannel micromachining, aluminum substrates were milled at two different local machining facilities. The CAD design was feed to the computer connected to a CNC machine (HAAS VF-2-super speed, Haas Automation Inc., Oxnard, CA or Bed Mills with Centroid Control, Birmingham). An 80 μm deep channel was milled into a thin (5 mm) aluminum substrate using a high-speed end mill fitted into the CNC.</p><!><p>The micromachined channel (Fig. 1a) was enclosed for fluid flow by sealing a thin PDMS film to the top of the channel (Fig. 1b). First, a PDMS film with a thickness of ~10–15 μm was prepared by spin coating an uncured PDMS mixture onto a thin transparency plastic (Mylar™) sheet at 500 rpm for 1 minute. The PDMS film was then allowed to cure in an isothermal oven at 65 °C for 15–30 min. Next, the micromachined aluminum substrate (Al device) was thoroughly cleaned with acetone and iso-propanol followed by rinsing with de-ionized water and drying under a stream of compressed nitrogen gas. Next, the device was heated to 140 °C on a hot plate for 2–3 minutes to ensure the complete dryness and the cleaned Al device and the cured PDMS film were exposed to air plasma (Harrick Plasma, Ithaca, NY) for 30 seconds and bonded together. The bonding was allowed to set for about 2–3 hours at ~ 25 °C. Next, the plastic transparency sheet was carefully peeled off from the bonded PDMS film and the film was punched at the two ends of the channel for fluid inlets and outlets using Harris Uni-Core punches (Ted Pella, Redding, CA).</p><!><p>Two-centimeter long silicone tubing were attached to each inlet and outlet of the enclosed microchannel for liquid connection by applying non-cured PDMS as a glue and letting it cure at 65 °C for 2–3 minutes (Fig. 1c). Finally, a lead zirconium titanate (PZT) acoustic transducer (30 mm long, 5 mm wide) with an optimum frequency of 2.91 MHz was super-glued to the top or to the bottom surface of the final device (Fig. 2a–d) to implement acoustic wave fields. The frequency of the PZT was chosen based on microchannel's half width.</p><!><p>The surface roughness and the batch-to-batch variations of the device width were investigated via SEM imaging (Nova NanoSEM™450) while width and depth variation was investigated via optical profilometer scanning (Zygo ZeGage™) (Fig 3a–c). A minimum of three devices was used for the comparison. SEM images were taken at the center of the main channel along the longitudinal direction and the profilometer scanning was taken at three random positions along the main channel.</p><!><p>The maximum flow rate and the pressure that the device can withstand were evaluated by continuous flowing of PBS buffer at an increasing flow rate and pressure. For flow rate measurements, the inlet of the device was connected to a syringe pump (KD Scientific, MA) and fluid was flowed at a set flow rate for about 5 minutes, starting at 25 μL/min. The flow rate was then gradually increased by 25 μL/min increment until the final flow rate of 4 mL/min. We assumed the volumetric flow rate inside the channel was same as the flow rate set at the syringe pump. For pressure measurements, the pressure at the outlet was monitored via a digital pressure gauge (DPG1000B, Omega, Norwalk, CT) connected to the device outlet while the flow rate was gradually increased.</p><!><p>To demonstrate the performance of the aluminum device, a series of typical acoustic focusing experiments were carried out. First, we show the single node focusing of particles by focusing polystyrene particles and biological cells and second, we demonstrate the multinode focusing of particles by focusing particles into two parallel streams. Devices were first rinsed with 1% casein solution followed by 10 mM PBS buffer (pH 7.5) to minimize the non-specific adhesion of particles and cells to the channel walls. A sample of polystyrene particles (105 particles/mL) was prepared by diluting a stock solution of fluorescent NR-ps particles in 20 mL of PBS buffer. For cell samples, Nile red stained Jurkat cells and B-RBCs were used. To stain, 1 mL of Jurkat cells (~106/mL) was mixed with 5 μL of Nile red solution (in DMSO). For B-RBCs, the original sample obtained from the vendor was diluted 200 folds and 1 mL of the diluted sample was stained with 5 μL Nile red without further quantification. Each sample was then diluted to 5 mL with PBS buffer and excess Nile red in the cell suspension was removed by discarding the supernatant after centrifugation. The stained cell sample was then re-suspended in 5 mL of 10 mM PBS buffer. Samples were introduced to the device at a flow rate of 100 μL/min using a syringe pump (KD Scientific, MA). The resonance standing acoustic waves were generated using a waveform generator (RIGOL DG 1022, RIGOL Technologies Inc., OR) and amplified via an RF amplifier (E&I 350L, Electronics & Innovation Ltd., NY). The acoustic performance parameters (frequency, applied voltage, amplitude) were monitored via an oscilloscope (Tektronix TBS 1052B, Tektronix Inc., OR).</p><!><p>Acoustic focusing of particles and cells in the aluminum device was monitored via epi-fluorescence imaging and the extent of the focusing was measured via flow cytometry. The epi-fluorescence images and videos of particle streams were captured near to the end of PZT using an epi-fluorescence microscope equipped with a sCMOS camera (Orca-FLASH 4.0, Hamamatsu, Japan). The obtained images were analyzed by plot profile scanning using ImageJ software (NIH) to compare intensity and resolution profiles of focused and non-focused streams. In addition, 1 mL sample was collected from each outlet and analyzed via a BD FACSCalibur flow cytometer to determine the concentrations of particles in each fluid stream, prior and during the acoustic focusing. In order to determine the particle concentration in each sample, an internal calibration method was employed using flow cytometry standard beads. Briefly, 950 μL of bead sample collected from an outlet was mixed with 50 μL of concentration known CountBright™ absolute counting beads. The concentration of particles or cells was calculated via flow cytometry dot plots, using the following equation.</p><p>The flow cytometry counting beads have a distinct signal from particles used, hence, two regions of particles were appeared in the dot plots (see Electronic Supplementary Material (ESM) Fig. S1). Pure samples of NR-ps particles were first measured to define the regions of interest for test particles in each dot plot and defined the gates accordingly. All particle populations were gated based on their fluorescence intensities (via FL2 fluorescence channel of the flow cytometer that detects the fluorescence at 585 ± 21 nm) and side scattering and the data were presented in the form of dot plots of side scatter (SSC) vs. fluorescence intensity (FL2). All data collection and analysis were performed using BD CellQuest™Pro (BD Bioscience, San Jose, CA) and FCS Express 6 (De Novo Software, Glendale, CA) software respectively. Total of 10,000 events was measured in each measurement and the instrument threshold was set at FL2 to remove unwanted events resulting from non-analytes.</p><!><p>While metal micromachining has not been a common method for microfluidic device construction, the routine availability of mills with very high RPM heads and the development of micro end mills as small as 1 μm cutting diameter is making this approach a compelling method to create micro channels even smaller than 25 μm in width. Among the different grades of aluminum, the alloy 6061 (AL6061) is inexpensive, readily available, corrosion resistant, and highly machinable. The speed of sound in AL6061 is 6320 m/s (vs. 1482 m/s in water), which assures a high acoustic impedance mismatch between microchannel walls (17.06 × 106 kg m−2 s−1) and the aqueous solutions (water ~ 1.48 × 106 kg m−2 s−1) in the channel [18, 25]. This is particularly useful in bulk acoustofluidic devices that require a rigid material to serve as an efficient carrier of acoustic energy that is launched into the channel to create a resonance acoustic field within the fluid. In comparison, silicon substrates commonly used for fabrication of acoustofluidic devices, have an acoustic impedance of 19.79×106 kg m−2 s−1 [18], which is slightly better than Al substrates, however the easy access of micromachining facilities, non-clean room requirement, and the low cost of fabrication will compel the use of aluminum as an alternate for silicon. Further, Al is a better thermal conductor (~167 W/m K) in comparison to semiconductor glass (~12 W/m K) and silicon (~148 W/m K). The thermal stability is important to maintain stable resonance frequency in acoustofluidic systems. Further, micro-machining of aluminum devices is much faster than microfabrication of silicon devices. For example, we could micro-machine the device reported in the current study, in less than an hour, whereas silicon microfabrication of similar device can take up to three hours or more. For comparison, we used two micromachining facilities; a commercial source and the on-campus facility and we did not observe significant differences in quality of the final product or the efficiency of acoustic focusing. The fabricated aluminum device was operated in the transverse resonator mode, where the standing acoustic waves were obtained perpendicular to the incident direction [18].</p><p>To provide a straightforward method to introduce samples and reagents, the liquid ports were integrated perpendicular to the microfluidic channel. This approach is simple and convenient in comparison to the method reported by Lin et al. where liquid access ports were micromachined horizontally through a thick aluminum substrate, which limits the number of liquid ports that can accommodate thus limiting the microfluidic design to simple geometries [24]. Further, a thick substrate is necessary to accommodate horizontal through ports which is not favorable for acoustic applications. Our design is not limited by the substrate thickness and can be further improved by utilizing aluminum sheets as thin as 300–500 μm which is comparable to the thickness of most common silicon wafers. In the current work, the challenge in integrating liquid ports onto metal surfaces is overcome by plasma sealing of PDMS onto Al substrates, a simple process, and it does not deviate from conventional silicon-PDMS plasma sealing procedure. Silicone tubing for liquid access can then be conveniently mounted onto the plasma-bonded PDMS layer. For acoustofluidic applications, it is essential to use a thin film of PDMS in order to minimize attenuation of acoustic forces by the polymer substrates; however, we found out that it is possible to plasma seal thick PDMS slabs as well.</p><p>The presence of smooth microchannel walls is vital to minimize unwanted particle adhesion. The surface smoothness of aluminum devices was compared to that of silicon devices prepared via deep reactive ion etching, simply by comparing the SEM images of channel surfaces of two devices (Fig. 3). Even though visual analysis of SEM images may not be the most perfect way to compare the roughness; the qualitative information we could gather should provide adequate information for the comparison. The two SEM images of the Al (Fig. 3a) and the silicon (Fig. 3b) devices suggest that the surface of the DRIE silicon channel appears smoother than the surface of the aluminum device, however, the aluminum channel surface was smooth enough that we did not observe any impact on the device performance (i.e. no particle sticking, loss of focusing, variation in resonance frequency). The profilometer measurement indicates that more precise micromachining of Al substrate, by having a mean width of three finished channels (433 ±12 μm) near to the target width (430 μm), in comparison to DRIE channel, where, we observed an average of ~12% expansion in the width (+30 μm) of the silicon channel from the target width of 250 μm. We used photomask printed on plastic Mylar™ (20,000 dpi) for photolithography during DRIE. The precision of DRIE channels can be improved if chrome masks are used, however, this can be expensive. Thus, no need of expensive photomask is another attractive feature of aluminum devices. The average depth of aluminum channel was about 80 ± 6 μm (target depth = 80 μm). The microchannels fabricated at the on-campus facility were milled at 3000 rpm. The precision and smoothness of the microchannels can be further improved by optimizing the rpm of the spindle speed and the feed rate. CNC machines that are capable of operating at higher spindle speed can produce high precision and smoother microchannels [26].</p><p>The ability to hold high flow rates and pressures without rupturing the bonded polymer film or delaminating the Al-PDMS composite was evaluated by flowing PBS buffer at increasing flow rates. We tested up to 4 mL/min and we did not observe delamination or rupturing of the PDMS film. The recorded highest hydrostatic pressure at this maximum flow rate was 4.5 atm. The thin film was also robust for repeated usage at high flow rates and the film was not ruptured even after 25-repeated use of the device. Another advantage of having a polymer layer is that it can be taken out by scraping. The device can then be cleaned with organic solvents and rebuild for extended use.</p><!><p>The placement of the acoustic transducer to generate resonance standing waves can be varied [6, 27, 28]. In most silicon based BAW devices, the acoustic transducer is attached to the bottom of the channel. For aluminum devices, we found that this configuration yields very weak focusing of particles. Therefore, we explored several possible configurations of transducer attachment to generate the most effective acoustic focusing. The effectiveness was determined by focusing 10.2 μm NR-ps particles at an applied voltage of 18 VP-P while changing the fluid flow rate. The device was running in single node mode and a frequency scan (2.5–3.5 MHz) was performed to obtain the optimum resonance frequency of the device. Epi-fluorescence images were taken at each tested flow rate at the optimum frequency for the analysis. The focusing efficiency was determined via plot profile scanning of images using ImageJ software. If the full width at half maximum (FWHM) of the focused stream peak was less than 30 μm, the focusing was deemed efficient (data not presented).</p><p>In the first configuration, in addition to the plasma bonded PDMS film, a glass cover slide was also plasma bonded to the top of the PDMS film and the PZT transducer was adhered on top of that using a thin layer of epoxy glue (Fig. 2a). The purpose of having the glass cover slide was that we anticipated the soft PDMS thin film would attenuate acoustic field strength and by having a glass substrate would provide a rigid acoustic reflector that can minimize the acoustic attenuation. However, we did not observe acoustic focusing of particles in this configuration.</p><p>In the second configuration, there was no glass cover slide on the PDMS film and the PZT was attached to the bottom surface of the aluminum substrate, positioned parallel and directly underneath to the channel (Fig. 2b). We observed weak acoustic focusing of particles when the flow rate was low (<15 μL/min). The focusing was not reproducible thus we considered the acoustic focusing as inefficient. The 5 mm thickness and/or mass of the aluminum substrate, which is large compared to silicon wafers that are typically 500 μm thick, could have attenuated the strength of acoustic vibrations reaching the microchannel and resulted inefficient acoustic focusing of particles in this configuration. Additionally, the design of acoustic horns, which the aluminum layer is analogous to here, requires precise calculation to match thickness of the material. This was not done in this configuration and matching the thickness of the aluminum layer to these calculations might improve device performance.</p><p>In the third configuration (Fig. 2c), a similar device as in the second configuration was built and the only difference was the placement of the PZT. The PZT was attached to the top surface of the aluminum layer of the device, positioned adjacent and parallel to the microchannel (Fig. 2c and 2d). Here, we observed stronger, reproducible focusing of NR-ps even at comparatively higher flow rates (~100 μL/min) than in other configurations. We anticipate the improved performance is related to the close proximity of the PZT transducer to the flow channel. The optimum resonance frequency was found to be 3.13 MHz and the driving voltage necessary to acquire efficient focusing was between 18–20 Vp-p. The captured video shows the real time focusing of NR-ps in the device taken at a flow rate of 100 μL/min (ESM Movie S1).</p><!><p>Acoustic focusing performance of the aluminum device was demonstrated via a set of typical acoustic focusing experiments. The data in Figure 4a–c are obtained from the single node acoustic focusing of 10.2 μm diameter NR-ps particles in the 250 μm wide channel, at a flow rate of 100 μL/min. and at an applied voltage of 18.6 Vpp. The Figure 4a (left) shows a stream of particles flowing through the channel in the absence of an applied acoustic field and the plot profile image scan (Fig. 4a, right) shows the appearance of random peaks due to unfocused particles in the fluid stream. In Figure 4b (left), particles are focused into a single streak in the presence of an acoustic field at 3.13 MHz. The plot profile scanning of the epi-fluorescence image shows the appearance of single focused peak with a FWHM of 18 μm (Fig. 4b, right), which is less than a 1/10th of the width of the channel, suggesting a very good focusing of particles into the center of the channel (ESM movie S1). In order to quantify the focusing efficiency, the percentages of particle concentrations in the central stream and the combined lateral streams were compared via flow cytometry analysis. The internal standard beads were utilized to calculate the percentages of particles in each stream as described in the experimental section. Initially, in the absence of acoustic focusing, the two lateral streams contained 54.3% of the total collected particles (Fig. 4c). In the presence of acoustic forces, the central stream is significantly concentrated with particles thus changing the particle composition from 45.7% to 98.2% of total collected. Once we established that our aluminum devices are capable of focusing particles efficiently, we explored the capability of focusing biological samples. For the proof of the concept study we utilized fluorescently labeled B-RBCs (Fig. 5, ESM Movie S2) and Jurkat cells (ESM, Fig. S2 and Movie S3). In the absence of an applied acoustic field, B-RBCs are dispersed across the channel width as shown in the epi-fluorescence micrograph (Fig. 5a) as well as its plot profile scanning (Fig. 5b), once the resonance acoustic field is applied B-RBCs are focused (Fig. 5c and d). The stained B-RBCs have weak fluorescence as indicated by the Figure 5a, however, when focused, the overall fluorescence intensity is greatly enhanced (Fig. 5c). The FWHM of the focused stream is about 34 μm, which is slightly higher than the threshold FWHM (30 μm) of a peak of pre-defined efficient focusing. As the Figure 5c suggests, the B-RBCs are highly concentrated in the tested sample and the focusing could have been improved either by diluting the initial cell sample or by increasing the applied voltage. Nevertheless, data suggest that our aluminum based acoustofluidic devices are capable of focusing biological cells of micrometer size.</p><p>One of the key advantages of acoustic focusing in microfluidic devices is its ability to generate multiple focused streams of particles in a single microfluidic channel without having complex designs, by simply tuning the resonance frequency to generate multiple nodes [12, 21]. To explore the feasibility of generating parallel focused particle streams in our aluminum devices, 10.2 μm NR-ps particles were focused into two streams at a resonance frequency of 3.43 MHz and flow rate of 100 μL/min in a 440 μm wide channel (ESM Movie S4). The multinode focusing was monitored microscopically (Fig. 6a and c) and captured images were analyzed (Fig. 6b and d). The Figure 6d shows that these devices are capable of delivering multi node focusing at commonly used voltages (18–20 V). The two particle streams are focused tightly with FWHM of about 10 μm for both peaks. The data shown in Figure 6c suggests that two nodes are not symmetrically positioned across the width of the microchannel and this non-symmetry can be due to the existence of three dimensional acoustic fields resulting from the vibration of the whole device [29]. It has been reported that the confinement of the resonance wave to a particular channel geometry can be influenced not only by the geometry and acoustic properties of the channel substrate but also by the properties of supporting layers [29, 30]. A thorough study is necessary to characterize acoustic fields in aluminum based devices.</p><!><p>The focus of this work has been to demonstrate the fabrication of aluminum based microfluidic devices and the feasibility of using them in BAW applications. Silicon, glass, and PDMS have been well established as materials of choice for making microfluidic devices. These materials encompass proper properties needed for microfabrication of microfluidic channels. Metals as microfluidic channel substrates are not commonly used. This can be due to the difficulty in the integration of fluidic connection in order to introduce samples and reagents. However, bonding of PDMS substrates to metal surfaces can resolve issues in fluidic integration. We have demonstrated that PDMS can be strongly bonded to micromachined aluminum microchannels. The aluminum alloy 6061 is reactive in strong acidic and basic conditions, however, most microfluidic systems are operated at neutral or near neutral conditions (pH 6–8), where the aluminum 6061 is non-reactive. Aluminum microchannels are especially useful for BAW applications, in addition to good acoustic properties, aluminum micromachining is inexpensive and widely available. The method we have reported here is simple and has fewer steps than silicon microfabrication and it does not require harsh chemicals and does not generate hazardous waste. Complex microchannel structures can be micromachined on AL6061. Further, aluminum devices can be machined at any standard micromachining facility and the overall cost for bulk machining will be less than other microfluidic fabrication methods. Finally, we have shown the usefulness of these devices in acoustofluidic applications, by efficient focusing of polystyrene microspheres and biological cells in single and multinode format. The general availability, the low cost, and the effectiveness for BAW applications will make this approach of value to researchers in a wide variety of fields and for a number of applications that include acoustophoretic separations, acoustic flow cytometry, and acoustic trapping among others. Aluminum based microfluidic devices need to be further explored and improved for a variety of applications and we anticipate this initial work will eventually be expanded.</p>

PubMed Author Manuscript

Structural details of amyloid β oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy

Human PrP (huPrP) is a high-affinity receptor for oligomeric amyloid β (Aβ) protein aggregates. Binding of Aβ oligomers to membrane-anchored huPrP has been suggested to trigger neurotoxic cell signaling in Alzheimer’s disease, while an N-terminal soluble fragment of huPrP can sequester Aβ oligomers and reduce their toxicity. Synthetic oligomeric Aβ species are known to be heterogeneous, dynamic, and transient, rendering their structural investigation particularly challenging. Here, using huPrP to preserve Aβ oligomers by coprecipitating them into large heteroassemblies, we investigated the conformations of Aβ(1–42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy. The disordered N-terminal region of huPrP becomes immobilized in the complex and therefore visible in dipolar spectra without adopting chemical shifts characteristic of a regular secondary structure. Most of the well-defined C-terminal part of huPrP is part of the rigid complex, and solid-state NMR spectra suggest a loss in regular secondary structure in the two C-terminal α-helices. For Aβ(1–42) oligomers in complex with huPrP, secondary chemical shifts reveal substantial β-strand content. Importantly, not all Aβ(1–42) molecules within the complex have identical conformations. Comparison with the chemical shifts of synthetic Aβ fibrils suggests that the Aβ oligomer preparation represents a heterogeneous mixture of β-strand-rich assemblies, of which some have the potential to evolve and elongate into different fibril polymorphs, reflecting a general propensity of Aβ to adopt variable β-strand-rich conformers. Taken together, our results reveal structural changes in huPrP upon binding to Aβ oligomers that suggest a role of the C terminus of huPrP in cell signaling. Trapping Aβ(1–42) oligomers by binding to huPrP has proved to be a useful tool for studying the structure of these highly heterogeneous β-strand-rich assemblies.

structural_details_of_amyloid_β_oligomers_in_complex_with_human_prion_protein_as_revealed_by_solid-s

<!>The N-terminal construct huPrP(23–144) is disordered in solution at mildly acidic and neutral pH<!>The flexible N terminus of huPrP becomes immobilized but remains almost devoid of regular secondary structure upon binding to Aβoligo<!><!>The flexible N terminus of huPrP becomes immobilized but remains almost devoid of regular secondary structure upon binding to Aβoligo<!><!>The flexible N terminus of huPrP becomes immobilized but remains almost devoid of regular secondary structure upon binding to Aβoligo<!><!>The C terminus of huPrP shows changes in α-helices 2 and 3 upon Aβoligo binding<!>High β-strand content of Aβoligo in huPrP(23–144)-Aβ complexes<!><!>High β-strand content of Aβoligo in huPrP(23–144)-Aβ complexes<!>Discussion<!><!>Discussion<!><!>Discussion<!>Aβ<!>huPrP<!>Preparation of Aβ(1–42) stocks<!>Preparation of high-molecular-weight heteroassemblies from amyloid β oligomers and different human prion protein constructs in different molar ratios<!>Characterization by density gradient ultracentrifugation, SDS-PAGE, and RP-HPLC<!>MTT cell viability tests<!>AFM measurements<!>Preparation of solution NMR samples<!>Solid-state NMR experiments<!>Solution NMR experiments<!>Data availability<!>Supporting information<!>Conflict of interest<!>Supporting information

<p>Edited by Wolfgang Peti</p><p>Alzheimer's disease (AD) accounts for an estimated 60 to 80% of all types of dementia (1). One of the hallmarks of AD is the formation of amyloid plaques, which consist mainly of amyloid β (Aβ) peptides comprising 39 to 43 residues (2). Aβ is produced by cleavage of the amyloid precursor protein (APP) by β- and γ-secretases (3). Of the two most abundant species Aβ(1–40) and Aβ(1–42), the latter is more prone to aggregation and its aggregates are more toxic (3). Small to moderately sized Aβ oligomers (Aβoligos) have been identified as the most neurotoxic factor in the pathogenesis of AD, whereas large fibrils are known to be the main component of insoluble plaques (4). Detailed structural information on Aβ(1–42)oligo is thus of paramount interest, and in recent years, structural studies on different oligomer preparations of Aβ(1–42)oligo, Aβ(1–40)oligo (5, 6) (or pyro-Glu-Aβ(3/11–40) oligomers (7)) by solid-state NMR-spectroscopy have been conducted (8, 9, 10, 11, 12, 13, 14, 15). Shape, morphology, and structural details of those oligomers were strongly dependent on preparation conditions, and while all of these oligomers had a high prevalence of β-strand secondary structure, tertiary fold and supramolecular arrangement of β-strands were found to differ strongly between different preparations. While in most mature fibrils β-strands are arranged in parallel in-register β-sheets (16, 17), quaternary structures in oligomers are much more variable, and, depending on the fibrillation pathway, parallel (12), antiparallel (18) β-sheets or even a mixture of both (11) have been found. A major challenge to structural studies of oligomers is their transient nature, and thus, most oligomer preparations exhibit substantial structural heterogeneity. Stabilization of oligomers is essential for long-term structural investigations. In most cases, further aggregation of oligomers was prevented by freeze-trapping with subsequent lyophilization (7, 8, 9, 10, 11, 12, 14). In this study, we used the recombinant human prion protein in its native cellular prion protein (PrPC) conformation to trap Aβ oligomers by coprecipitating them into large heteroassemblies, in which the growth of Aβoligo is prevented, as demonstrated by long-term solid-state NMR measurements over 11 months.</p><p>The PrPC is a high-affinity cell-surface receptor for Aβoligo (19, 20), and PrPC is also able to bind to fibrillar Aβ (21, 22, 23). It has been suggested that binding of Aβoligo to membrane-anchored PrPC mediates Aβ toxicity during AD by mediating synapse damage (24) and the blockade of long-term potentiation by Aβoligo (19, 25) via activation of Fyn-kinase pathways (26, 27) (Fig. S1), but this has also been questioned (28, 29, 30, 31). It has also been described that soluble PrP (32) and its N-terminal fragment PrP(23–111) (33, 34) have a protective role by inhibiting Aβ fibrillation and sequestration of Aβoligo.</p><p>Several in vitro studies on the Aβ-PrP interaction suggest that Aβoligos bind at two Lys-rich parts (residues 23–27 and ≈95–110) on PrP (35, 36, 37, 38, 39, 40), but an additional involvement of the C terminus of PrP has also been suggested (21). Interestingly, the N terminus of human PrP is also able to bind oligomeric α-synuclein with high affinity (41, 42, 43). A structural study of insoluble PrPC-Aβoligo complexes described them as a "hydrogel," in which the Aβ(1−42)oligos were rigid, while PrP still has high molecular mobility (44). Additionally, this study reported a conformational change in the N terminus of PrPC upon complexation with Aβoligo. We recently demonstrated that Aβoligo forms large heteroassemblies with either full-length (huPrP(23–230)) or C-terminally truncated (huPrP(23–144)) membrane-anchorless monomeric PrP (40). These assemblies have a size of a few micrometers as determined by dynamic light scattering and show cloud-like morphologies as seen by atomic force microscopy (40). The Aβ:huPrP stoichiometry of the heteroassemblies depends on the amount of huPrP added to Aβoligo and reaches a value of 4:1 (monomer ratio Aβ:huPrP) if either huPrP(23–144) or huPrP(23–230) is added to the oligomer solution in excess (40). In all these in vitro preparations, Aβ oligomers and early-stage protofibrils are stabilized and prevented from elongation by PrP, which has been shown to preferentially bind to fast-growing fibril and oligomer ends (22).</p><p>Here we exploit this stabilizing effect in an NMR study on different samples of Aβoligo complexed by huPrP. Isotope labeling of either huPrP or Aβ allowed us to characterize both components of the complex separately. While the N-terminal region of huPrP in the complex remains largely devoid of secondary structure and still undergoes fast backbone conformational averaging on the microsecond to millisecond timescale, Aβoligos exhibit a high degree of β-strand conformation. While these Aβoligos are highly heterogeneous, solid-state NMR spectra reveal similarities with the corresponding spectra of all fibril polymorphs published so far (45, 46, 47).</p><!><p>The solution structure of huPrP(23–230) had originally been determined in acetate buffer at an acidic pH of 4.5 and a temperature of 20 °C (48), whereas the huPrP-Aβ(1–42)oligo complex samples for solid-state NMR were prepared at a pH value close to neutral. As a basis for studying the interaction between huPrP and Aβoligo, we therefore first investigated free huPrP(23–144) by NMR spectroscopy in solution at different pH values ranging from 4.5 to 7.0 and at a temperature of 5.0 °C, which is closer to the temperature used for the solid-state NMR experiments. As reported previously, the chemical shifts of the N-terminal amino acid residues 23 to 124 in truncated huPrP(23–144) are almost identical to those of huPrP(23–230), whereas residues 125 to 144, which are part of the well-ordered globular domain of huPrP(23–230), are strongly affected by the truncation at position 144 (40).</p><p>We obtained almost complete sequence-specific 1H, 13C, and 15N backbone resonance assignments for huPrP(23–144) at pH values of 4.5 and 7.0 and a temperature of 5.0 °C using a combination of HNCO, HNCACB, and BEST-TROSY-(H)N(COCA)NH triple-resonance experiments (Fig. S2). The assigned chemical shifts at pH 4.5 and pH 7.0 have been deposited with the Biological Magnetic Resonance Data Bank (BMRB) under accession codes 28115 and 28116, respectively.</p><p>As expected, side-chain titration in this pH range causes significant chemical shift changes for all seven histidine residues and for residues next to histidine. Other than that, the chemical shifts at pH 4.5 and pH 7.0 are very similar to each other and very close to random coil shifts (49). Quantitative analysis reveals that the Random Coil Index (RCI) order parameters (50) SRCI2, which are a measure of how different the backbone chemical shifts are from those of a disordered random coil on a scale of 0 (typical for a random coil) to 1 (typical for a well-ordered backbone conformation), are consistently below ≈0.6 (Fig. S3). This demonstrates conclusively that free huPrP(23–144) in solution at neutral and mildly acidic pH is highly disordered and devoid of any stable secondary structure.</p><!><p>High-molecular-weight heteroassemblies of oligomeric Aβ(1–42) and huPrP(23–144) or of oligomeric Aβ(1–42) and huPrP(23–230) were prepared by adding the respective huPrP construct to a preincubated solution of Aβ(1–42), as described previously (40). Immediately after addition of huPrP to the solution, precipitation of a solid fine white powder was observed.</p><!><p>A, MTT assay of 1 μM Aβoligoand of 1 μM Aβoligoin complex with either 0.5 μM, 0.1 μM, or 0.02 μM of either huPrP(23–230), huPrP(23–144) or huPrP(121–230). Both huPrP(23–230) and huPrP(23–144) reduce Aβoligo toxicity in a concentration-dependent manner. In contrast, the C-terminal fragment huPrP(121–230) does not. None of the huPrP fragments alone reduces cell viability. This reduction of toxicity has been seen for non-membrane-bound huPrP fragments before (32, 36) and is in contrast to toxic effects of membrane-anchored huPrP (19, 24, 25). As our complexes do not exhibit a GPI-anchor, the reduction of toxicity reflects these observations. B, 5 μm × 5 μm AFM image of 440 nM Aβoligo and C, 2 μm × 1 μm AFM image of Aβoligo-huPrP(23–144) coprecipitates generated with 80 μM preincubated Aβ(1–42) and 40 μM huPrP(23–144). The aggregates have sizes up to 1 μm spanning clusters with a smooth surface appearance, whereas Aβoligo are small nm spheres. D, comparison of a PDSD spectrum of huPrP(23–144)∗-Aβ (∗ species is 13C, 15N uniformly labeled) in red with a 13C-13C TOCSY spectrum of monomeric huPrP(23–144) in black. The PDSD spectrum was recorded at a temperature of ≈−6 °C, a spinning frequency of 11 kHz and a mixing time of 30 ms and the TOCSY spectrum at a temperature of 5.0 °C, at pH 6.7. Gray circles indicate some identified amino acid types, dashed lines Pro and Val connections in the PDSD spectrum. Due to broad line widths and a low signal dispersion in the PDSD spectrum several correlations overlap, especially for the residues in the octarepeat region. Nevertheless, spin systems for most of the amino acid types present in the sequence could be identified and an amino-acid-type specific resonance assignment was possible. Differences between the PDSD and TOCSY spectrum are highlighted with blue circles. For an additional PDSD spectrum see Fig. S6, the corresponding double quantum-single quantum correlation spectrum (DQ-SPC5) is shown in Fig. S7.</p><!><p>High-molecular-weight assemblies of Aβ(1–42)oligo and huPrP(23–144) were further analyzed by sucrose density gradient ultracentrifugation (DGC) and subsequent SDS-PAGE and RP-HPLC (40) (Fig. S4). As previously described (40), a molar ratio of Aβ:PrP of 4:1 is obtained in the assemblies if huPrP is added in excess; for higher Aβ:PrP ratios, not all potential PrP-binding sites on Aβoligo are saturated with huPrP(23–144) (as in sample huPrP(23–144)-Aβ∗, ∗ indicates that the Aβ moiety of the complex is 13C, 15N labeled).</p><p>In Figure 1 typical AFM images of Aβoligo alone (Fig. 1B) or in complex with N-terminal huPrP(23–144) (Fig. 1C) are shown. Spherical Aβ oligomers can clearly be identified (Fig. 1B), and no fibrils are observed in the huPrP(23–144)-Aβ condensates (Fig. 1C). Next, we focused on investigating structural features of the complex by NMR spectroscopy.</p><p>To probe the flexibility of the N-terminal construct huPrP(23–144) in the complex, we recorded a 1H-13C insensitive nuclei enhanced by polarization transfer (INEPT)-NMR spectrum as well as dipolar-based 1H-13C and 1H-15N cross polarization (CP)-MAS spectra (51). The INEPT-NMR spectrum of this sample did not show any protein signals at a sample temperature of ≈27 °C (spectrum not shown), whereas in 1H-13C (recorded at a sample temperature of ≈0 °C) and 1H-15N CP spectra (recorded at a sample temperature of ≈−6 °C) strong signals typical for all amino acid types can be seen (Fig. S5). This indicates that huPrP(23–144) in complex with Aβ(1–42)oligo is immobilized and does not undergo rapid isotropic reorientation as in solution.</p><p>In Figure 1D a typical 2D 13C-13C-correlation spectrum obtained with proton-driven spin-diffusion (PDSD) of huPrP(23–144)∗-Aβ (∗ indicates that the huPrP moiety of the complex is 13C, 15N labeled) is overlaid with a 13C-13C total correlation spectrum (TOCSY) of monomeric huPrP(23–144) in solution at pH 6.7. Except for some Val and Ala resonances, most of the peaks align well. This indicates that the natively unfolded N terminus of huPrP does not undergo a major conformational rearrangement upon complex formation with Aβoligo, but conformational averaging of backbone conformations is still possible on the microsecond to millisecond timescale. Due to the lack of secondary structure in the intrinsically unstructured N terminus as well as the repetitiveness of the amino acid sequence in the octarepeats, the signal overlap is so severe that sequence-specific resonance assignment for the solid-state NMR spectra was not possible.</p><!><p>A, amino acid sequence of huPrP(23–230) (48) used in this study. The Aβ-binding regions K23–K27 and T95–K110 (35, 36, 37, 38, 39, 40) and the five octarepeats are indicated above the sequence. B, 3D structure of the natively folded prion domain (residues 125–228) of full-length huPrP(23–230) in solution. β-strands are colored blue, α-helices red. Picture adapted from PDB-File 1QLZ (48). Residues whose entire spin system is missing or shifted in the PDSD spectra (Figs. S10–S12) are highlighted in purple in A and B.</p><!><p>These findings are also supported by analysis of secondary chemical shifts (Fig. S8). Most secondary chemical shifts of huPrP(23–144) in solution are random coil chemical shifts indicative of a lack of regular secondary structure. Likewise, most spin systems of huPrP(23–144) in complex with Aβoligo are typical random coil chemical shifts, with some α-helical shifts found for Ala, Leu, and Val, which are not found for monomeric huPrP(23–144). Notably, almost no β-strand-like secondary chemical shifts were identified for complexed huPrP(23–144). This finding is an indication that huPrP(23–144) did not aggregate into amyloid fibrils. We also compared the chemical shifts of huPrP(23–144) fibrils (52, 53) with our correlation spectrum of huPrP(23–144)∗-Aβ (Fig. S9A). Most of the signals observed for fibrillar huPrP(23–144) do not overlap with the signals in our huPrP(23–144)∗-Aβ spectra. We therefore conclude that the conformations of huPrP(23–144) in huPrP(23–144)∗-Aβ and the huPrP(23–144) fibril are very different, and the interaction with Aβoligo did not induce huPrP(23–144) fibril formation.</p><!><p>Details of the samples used for the solid-state NMR measurements (∗ species is 13C, 15N uniformly labeled)</p><p>13C Direct excitation (DE) and1H-13C CP spectra of huPrP(23–230)∗-Aβ (∗ species is13C,15N uniformly labeled) recorded at temperatures of ≈30, 10, and −10 °C and 11 kHz spinning frequency. Recycle delays of 20 s and 2 s were used for DE and 1H-13C CP experiments, respectively. The signal at 90 ppm is caused by the rotor insert (Delrin) and is cut off for clarity. The signal at around 0 ppm in the 13C DE spectrum belongs to a silicone-based rotor inlet and is likewise cut off for clarity, the broad bump centred at 120 ppm however is the Teflon background of the probe. Both signals are not detected in the CP spectra. Signal intensities were scaled to the number of scans for each spectrum. Even at the lowest temperature the free water in the sample was not completely frozen, as verified by 1H spectra (not shown).</p><p>Comparison of two PDSD spectra of huPrP(23–230)∗-Aβ (∗ species is13C,15N uniformly labeled), shown in black, and huPrP(23–144)∗-Aβ, shown as red contour. Both spectra were recorded at a spinning frequency of 11 kHz and a mixing time of 30 ms, but the black one at a temperature of ≈0 °C and the red one at ≈−6 °C.</p><!><p>We compared our 2D 13C-13C correlation spectrum with the expected correlations between the chemical shifts obtained experimentally for natively folded full-length huPrP in solution at pH 4.5 (48). While the predicted N-terminal cross peaks (residues 23–124) superimpose well with the spectrum of huPrP(23–230)∗-Aβ, some discrepancies between the experimental and the predicted spectrum are observed for the C terminus (residues 125–230) (Fig. S10).</p><p>In particular, correlation signals for Ile, Thr, and Val in α-helical conformation from α-helices 2 and 3 in natively folded huPrP are completely missing in the experimental spectrum (Fig. S11). Instead, correlation signals for Thr and Val with secondary chemical shifts indicative of β-strands that are not observed in natively folded huPrP are clearly visible in the experimental spectrum (Figs. S10–S12). This suggests that at least for a substantial fraction of the huPrP molecules within the complex, some parts of a region between either V121 and I139 and/or V176 and I215 (located in α-helices 2 and 3) have undergone some structural rearrangements including β-strand formation (Fig. 2B). We could not see any fibril formation in huPrP(23–230) within the complexes; nevertheless, we overlaid our spectrum with predicted peaks for two recently published fibrils from huPrP and its fragment huPrP(94–178). The huPrP(94–178) fibril structure exhibits a β-strand in the palindrome region (54), which is likewise not supported by our α-helical-like Ala chemical shifts (Fig. S9B). However, a fibril structure recently published for full-length huPrP (55) (see Fig. S9C) shows a lot of similarities to our spectrum especially for Thr and Val residues, suggesting a rearrangement of the C terminus to more β-sheet-like chemical shifts.</p><!><p>We also investigated the homogeneity and structural characteristics of Aβoligo using two samples containing uniformly 13C, 15N labeled Aβoligo in complex with nonlabeled huPrP(23–144) in two different molar ratios (Table 1). In the first sample (indicated as huPrP(23–144)-Aβ∗), the molar ratio between Aβ monomers and huPrP was roughly 8:1, whereas in the second preparation (indicated as huPrP(23–144)exc-Aβ∗), addition of huPrP(23–144) in excess to the Aβ oligomers resulted in a molar ratio of ≈4:1.</p><!><p>Overlay of a PDSD spectrum of huPrP(23–144)-Aβ∗ (∗ species is13C,15N uniformly labeled), measured at a temperature of ≈0 °C, a spinning frequency of 11 kHz and a mixing time of 50 ms, in red with a13C-13C TOCSY spectrum of uniformly13C,15N isotope labeled Aβ monomers in solution (measured at a temperature of 5.0 °C and pH 7.2 in 30 mM Tris-HCl buffer) in black (the strong resonances at 62.1 ppm and 64.2 ppm with the t1noise are from the Tris buffer). Ala and Ser Cα-Cβ peaks are highlighted with blue circles and six identified Ala spin systems are shown with blue crosses. Chemical shift differences for both residue types between the Aβ monomers (typical random coil chemical shifts) and huPrP(23–144)-Aβ∗ (β-strand-like conformations) can be observed.</p><!><p>A comparison between a solid-state NMR 13C-13C correlation spectrum of Aβoligo in complex with huPrP(23–144) and a 13C-13C TOCSY correlation spectrum of Aβ monomers in solution (Fig. 5) reveals strong chemical shift differences and thus indicates that the Aβ monomer building blocks in oligomers have undergone significant structural changes upon oligomerization. While all signals of the solution spectrum have chemical shifts indicative of a random coil, a strong shift to chemical shifts indicative of β-strand-like secondary structure is observed for almost all spin systems of Aβoligo in the spectrum of the complex. For Cα/Cβ cross peaks of Ala, Ile, Ser, and Val (Fig. 5) in α-helical, unstructured, and β-strand-like conformations, a quantification was possible by integration of the peak regions (see Fig. S19). Hence, these residues are predominantly in a β-strand conformation. For Gly, which is a β-strand breaker, CO/Cα cross peaks are mainly indicative of random coil conformation.</p><p>Due to conformational heterogeneity, inhomogeneous line broadening, and substantial resonance overlap in the 13C-13C and 15N-13C spectra, a full sequential resonance assignment for Aβoligo in complex with huPrP was not possible. However, from a series of PDSD spectra with different mixing times as well as 2D and 3D NCACX and NCOCX spectra, it was possible to identify some interresidual correlations and to obtain site-specific assignments for some parts of Aβ in one predominant conformation (Table S1). However, it is not clear whether all assigned resonances belong to one type of conformer or to different conformers.</p><p>To elucidate whether the stoichiometry of Aβ and huPrP in the heteroassemblies has an influence on the conformations of Aβ molecules, we prepared and investigated a second sample, in which huPrP(23–144) was added in excess to 13C, 15N labeled Aβoligo. In this sample, all potential huPrP-binding sites on Aβoligo should be occupied. Overall there is not much difference between sample huPrP(23–144)-Aβ∗ and huPrP(23–144)exc-Aβ∗ in a PDSD spectrum with a mixing time of 50 ms, except for minor changes (Fig. S20). As there are no major structural changes upon altering the huPrP concentration, we conclude that the conformational heterogeneity is not due to unoccupied huPrP-binding sites in Aβoligo, but rather Aβoligos in complex with huPrP consist of inequivalent conformers and/or Aβoligo assemblies are different from each other.</p><!><p>In this study we investigated the interaction of Aβ(1–42)oligo and huPrP by solid-state NMR spectroscopy. As mentioned above, Aβ(1–42)oligo play a crucial role in AD, as they are neurotoxic (4). Determining structural information of Aβ(1–42)oligo is challenging because of their transient and fast-aggregating nature. Therefore, trapping Aβ(1–42)oligo with huPrP and inhibiting their aggregation is a convenient way to study their structure. The interaction between Aβ(1–42)oligo and huPrP has also a role in AD: Nieznanski et al. and others showed that soluble huPrP is able to inhibit Aβ fibril formation (32, 56), particularly the naturally secreted huPrP fragment N1 (huPrP(23–111)) (33, 34) and sequesters toxic Aβ(1–42)oligo (32). Additionally, soluble huPrP reduces the toxic effects of Aβ(1–42)oligo, as seen by us (see Fig. 1A and (40)) and others (32, 36).</p><p>Aside from the protective role of soluble huPrP in AD, membrane-anchored huPrP is mediating neurotoxicity of Aβ(1–42)oligo (19, 24, 25) via Fyn-kinase (26, 27) or NMDA receptor pathways (57). Although Aβ(1–42)oligo toxicity is not solely dependent on huPrP (28, 29, 30, 31), it has been shown that especially small Aβ(1–42)oligo (58) and high-molecular-weight Aβ(1–42)oligo (59, 60) mediate toxicity by huPrP. To target this interaction efficient inhibitors might prevent Aβ(1–42)oligos' detrimental effects. Indeed, Aβ(1–42)oligo-binding D-enantiomeric peptides (40, 61) and antibodies (19, 62, 63, 64) have been shown to efficiently block the interaction between huPrP and Aβ(1–42)oligo, but more efficient inhibitors are needed. The process of research for efficient inhibitors will be speeded up by detailed knowledge of the binding between Aβ(1–42)oligo and huPrP in terms of structure, because targeted research and rational design of either huPrP- or Aβ(1–42)oligo-binding agents will be possible.</p><p>In this study, high-molecular-weight aggregates were formed by addition of N-terminal or full-length human PrP to preformed Aβ(1–42)oligos. These aggregates formed immediately upon addition of huPrP, visible as the precipitation of a fine white solid powder. The rigidity of this complex was further confirmed by DE and CP NMR spectra recorded at different temperatures (see Fig. 3).</p><p>In a previous study, Kostylev et al. (44) investigated complexes formed between huPrP(23–111) or huPrP(23–230) and oligomeric Met-Aβ(1–42). In that study, the complexes were described as a hydrogel, and PrP molecules exhibited a higher degree of flexibility. The difference between their and our complexes may be explained by differences in the preparation of the complex (different buffer system), and in particular of the Aβ oligomers, which consisted of ≈12 molecules in the study of Kostylev et al. and of on average ≈23 monomers (61) in our study, which most certainly has an effect on their oligomer structure. Also, the Aβ(1–42) species used by Kostylev et al. contained an additional methionine residue at the N terminus, which could lead to different behavior of the Aβ(1–42) oligomers, although Silvers et al. (65) could show that Met-Aβ(1–42) and Aβ(1–42) fibrils exhibit the same aggregation kinetics and, except for a slight change in flexibility of the N terminus, are structurally comparable. Additionally, Kostylev et al. (44) used huPrP(23–111) for the majority of their investigations, whereas we used a slightly longer construct (huPrP(23–144)). This could also account for the different physical behavior in terms of flexibility.</p><p>As just mentioned, we did most of the investigations on an N-terminal construct of huPrP (huPrP(23–144)) for the following reasons: Firstly, the N terminus of huPrP is sufficient for binding Aβ(1–42)oligo, as shown by us (40) and others (35, 36, 37, 38, 39). Further, using huPrP(23–144) instead of huPrP(23–230) drastically reduces signal overlap in the spectra making it more straightforward to draw conclusions for the N terminus. The naturally secreted soluble N1 fragment (although slightly shorter: 23–111) exhibits a protective role in AD by reducing the cytotoxicity of Aβ(1–42)oligo (34). We could show by MTT toxicity tests that also our construct huPrP(23–144) as well as soluble full-length huPrP(23–230) significantly reduced Aβ(1–42)oligo toxicity (Fig. 1A). From a comparison of 2D 13C-13C spectra, we could show that the C terminus of huPrP(23–230) has no impact on the binding of the N terminus (23–144) to Aβ(1–42)oligo, suggesting that the protective effect of soluble huPrP is linked to the N terminus of huPrP. Therefore, the different roles of huPrP in the etiology of AD (i.e., mediation of neurodegeneration versus neuroprotection) might be rather attributed to the place of action (membrane-anchored versus soluble) than to the length of the protein.</p><!><p>Schematic representation showing structural features of the huPrP-Aβoligocomplex for A, low huPrP content as in the sample where huPrP(23–144) is not in excess (huPrP(23–144)-Aβ∗) and B, with high huPrP content as in the sample where huPrP(23–144) was added in excess (huPrP(23–144)exc-Aβ∗). huPrP is shown as orange lines, Aβoligo as blue spheres, α-helices are red, and β-strands blue. Binding regions at huPrP are shown as light green boxes, conformational changes in the C terminus of huPrP as orange dots. Picture adapted from Rösener et al. (40).</p><!><p>For full-length huPrP in complex with Aβoligo we observed some changes for Thr and Val residues from α-helical to random coil or even β-strand-like secondary chemical shifts compared with well-folded monomeric huPrP in solution (48). The residues affected by these chemical shift changes are mainly located in α-helices 2 and 3, thus suggesting that the helical structure of this region is at least partially lost in complex with Aβoligo. For huPrP in a hydrogel with Aβoligo chemical shift changes from α-helical to random coil values were also described for Thr residues, which are mainly located in α-helices 2 and 3 (44). This observation was attributed to a loss of secondary structure during liquid–liquid phase separation of PrP and in the complex with Aβoligo. The loss of secondary structure in the complex with Aβoligo is confirmed by us. This observed change in secondary structure in the C-terminal domain of PrPC upon binding to Aβ oligomers suggests that also the C-terminal domain of PrPC interacts with Aβoligo. On the contrary, the C-terminal domain is not able to bind Aβoligo on its own (40), so chemical shift changes in the C terminus might be some type of steric hindrance, a disfavor of α-helical conformations in close proximity to the β-strand-like Aβoligo or simply a structural change induced by binding of Aβoligo to the N terminus. As we could show that Aβoligo and the C-terminal fragment huPrP(121–230) do not form high-molecular-weight aggregates (40) and that this huPrP fragment does not reduce Aβoligo cytotoxicity (see Fig. 1A), a direct binding of Aβoligo and the C terminus of huPrP is rather unlikely. Consequently the C terminus is free to interact with any secondary (transmembrane)receptors necessary for the signal transduction, because PrPC itself is no transmembrane protein and therefore requires a secondary receptor, such as NMDAR (57) or the metabotropic glutamate receptor 5 (mGluR5) (69) to facilitate Aβoligo-induced neurotoxicity. Indeed the Aβoligo-PrPC-mGluR5 complex has been shown to mediate neurotoxic Fyn-kinase pathways: Um et al. demonstrated that the interaction between membrane-anchored full-length PrPC and mGluR5 is stabilized by Aβoligo. This interaction in turn enables binding to Fyn-kinase and leads to the subsequent Fyn-kinase cascade and independent of that to increased calcium influx into the cell (69). Additionally, the Aβoligo-PrPC-mGluR5 complex enables NMDA and muscarinic-acetylcholine receptor-independent long-term depression (70) and modulates the binding to intracellular proteins (71). It might be attractive to speculate that these interactions are mediated by a structural change in the C terminus of PrPC. This has to be further investigated. In another study (21), PrP constructs encompassing the N-terminal but lacking the C-terminal domain were inactive in inhibiting Aβ polymerization, even though they still bound to fibrils, whereas full-length PrPC completely inhibited fibril elongation. This implied that the C-terminal domain might play some role in inhibiting polymerization. It is thus tempting to speculate that the conformational transition of the C-terminal domain to more β-strand-like structures could also be due to the incorporation into a fibril equivalent surface on Aβ oligomers. This is also supported by the finding that the C-terminal chemical shifts of huPrP overlap well with a recently published full-length huPrP fibril structure (55) (see Fig. S9C). Nevertheless, we should keep in mind that other studies following the aggregation of Aβ in presence of different huPrP constructs suggested the N terminus necessary for inhibiting Aβ aggregation (32, 36, 39) and also our own data argue against a direct binding of Aβoligo to the C terminus of huPrP (40), as stated above.</p><p>Aβoligo in complex with huPrP consists of nonidentical Aβ conformers. This is not surprising given the fact that the complex of huPrP(23–144) and Aβoligo contains four times more Aβ (monomer equivalent) than huPrP(23–144) molecules (40). Not every monomer within the oligomer (containing ≈23 monomer units on average (61)) might be able to bind to huPrP(23–144) in the same way and has therefore the same conformation (40), as described above. These nonidentical conformers can have different origins: (i) different types of monomers within the oligomer, because not every monomer can bind to huPrP (Aβ-huPrP versus Aβ-Aβ interactions); (ii) polymorphism within the oligomer independent of the binding to huPrP (iii) polymorphism between different oligomers; or (iv) a combination thereof.</p><!><p>A PDSD spectrum (measured at a temperature of ≈0 °C, a spinning frequency of 11 kHz and a mixing time of 50 ms, same spectrum as inFig. 5) of huPrP(23–144)-Aβ∗ (∗ species is13C,15N uniformly labeled) in comparison with predicted cross peaks (up to two bonds) for three different fibril types, which are obtained at pH values of 2 (red) (47) or 7.4 (green (46) and blue (45)) and an artificial protofibril (yellow) (13). Separate overlays of this PDSD spectrum with spectra of these fibrils are shown in Figs. S21–S24.</p><!><p>The propensity of huPrP to efficiently bind to Aβoligo and to "freeze" them in a nondynamic and nonelongating state allowed us to investigate the conformers of Aβoligo and the huPrP moiety by NMR over several months without noticeable changes in the sample. It is tempting to speculate whether this property of huPrP is a coincidence, or whether it is part of the long-sought function of PrP. Regardless of whether PrP inhibits elongation of Aβ oligomers and fibrils or whether PrP is a mediator of cytotoxicity of Aβoligos, substances that compete with PrP for Aβoligo binding and which thus can do the same job without the potential of mediating cytotoxicity may be of high therapeutic potential.</p><!><p>For preparation of NMR samples with unlabeled Aβ, synthetic Aβ(1–42) obtained from Bachem AG was used. (For preparation of stocks see below.) Uniformly 13C, 15N labeled Aβ(1–42) was purchased from Isoloid GmbH.</p><!><p>The purification of recombinant full-length huPrP(23–230) and C-terminally truncated huPrP(23–144) either unlabeled or uniformly 13C, 15N labeled, and of recombinant unlabeled huPrP(121–230) was performed as described previously (40).</p><!><p>Synthetic unlabeled Aβ(1–42) (Bachem AG, 1 mg aliquot) was incubated with 700 μl hexafluoro-2-propanol (HFIP) overnight and divided into 108 μg doses in LoBind reaction tubes (Eppendorf AG). Samples were lyophilized in a rotational vacuum concentrator system connected to a cold trap (both Martin Christ Gefriertrocknungsanlagen GmbH). The lyophilizates were stored at room temperature and protected from light.</p><!><p>For sample preparation, Aβ(1–42) lyophilizates (either uniformly 13C, 15N labeled or unlabeled) were dissolved in 30 mM Tris-HCl buffer, pH 7.4, yielding Aβ(1–42) concentrations of 160–300 μM. After 2 h of incubation at 22 °C and 600 rpm shaking to obtain Aβoligo, either huPrP(23–144) or huPrP(23–230) was added to yield concentrations of 40 to 80 μM within the initial mixture leading to the molar ratios mentioned in Table 1. The addition of huPrP resulted in immediate sedimentation of the complex as a powder-like precipitate (40).</p><p>After addition of 0.03% of sodium azide and incubation for 30 min, the samples were centrifuged for 2 to 5 min at 16,100g, and the supernatant was removed. The sediment was washed twice with up to 2 ml of 30 mM Tris-HCl buffer, 0.03% sodium azide, pH 7.4 to remove excess monomeric PrP. After removal of the supernatant, the samples were transferred into 3.2 mm MAS rotors with a Hamilton syringe and centrifuged. In total, four different samples were prepared in which either huPrP or Aβ(1–42) was uniformly 13C, 15N labeled, using different huPrP constructs and molar ratios between huPrP and Aβ(1–42) (Table 1).</p><!><p>For biophysical characterization of e.g., sample huPrP(23–144)-Aβ∗, sucrose density gradient ultracentrifugation (DGC) was performed. To this end, 10 μl of the sedimented but unwashed sample was diluted with 90 μl of 30 mM Tris-HCl buffer, pH 7.4 and applied on a discontinuous sucrose gradient (see (40)) and centrifuged for 3 h at 259,000g and 4 °C. After fractionation, each of the 14 fractions was analyzed by Tris-Tricine SDS-PAGE and RP-HPLC as previously described (40) (Fig. S4).</p><p>RP-HPLC revealed the Aβ:huPrP(23–144) stoichiometry shown in Table 1 as determined in a single measurement. Sample huPrP(23–144)exc-Aβ∗ was not separated by DGC but measured by RP-HPLC and revealed an Aβ:huPrP(23–144) stoichiometry of 3.7 ± 0.12 to 1 after fivefold measurement of the same sample. All stoichiometries represent monomer equivalents.</p><!><p>Potential cell viability rescue of rat pheochromocytoma PC-12 cells (Leibniz Institute DSMZ) from Aβ(1–42)oligo-induced toxicity through addition of soluble huPrP(23–144), huPrP(23–230), or huPrP(121–230) in a concentration-dependent manner was measured in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability tests (40, 61).</p><p>PC-12 cells were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum and 5% horse serum, seeded (10,000 cells in 100 μl per well) on collagen-coated 96-well plates (Gibco, Life Technologies), and incubated in a 95% humidified atmosphere with 5% CO2 at 37 °C for 24 h. Then final concentrations of 1 μM Aβ(1–42)oligo either in the absence or after mixing and further incubation for 30 min at 22 °C with 0.02, 0.1, or 0.5 μM (final concentrations) of the respective huPrP protein were added. In addition, the toxicity of the respective huPrP proteins alone at 0.5 μM final concentrations was also determined.</p><p>After further incubation in a 95% humidified atmosphere with 5% CO2 at 37 °C for 24 h, cell viability was measured using the Cell Proliferation Kit I (MTT) (Roche Applied Science) according to manufacturer's protocol. The MTT formazan product was determined by measuring the absorbance at 570 nm corrected by subtraction of the absorbance at 660 nm in a FluoroStar Optima plate reader (BMG Labtech). The arithmetic mean of five independent measurements per approach ±SD was calculated. All results were normalized to untreated cells grown in medium only.</p><!><p>The samples used for AFM were either Aβ(1–42)oligo or Aβ(1–42)oligo complexed by huPrP(23–144).</p><p>For formation of Aβ(1–42)oligo, monomeric Aβ(1–42) was incubated at a concentration of 80 μM in 30 mM Tris-HCl, pH 7.4 for 2.5 h at 22 °C and 600 rpm shaking. For AFM the sample was then diluted to 0.44 μM with buffer and 50 μl of the solution was transferred to freshly cleaved mica and incubated for 30 min at room temperature for mica adhesion.</p><p>Preparation of the Aβ(1–42)oligo-huPrP(23–144) sample was done as follows: Monomeric Aβ(1–42) at a concentration of 120 μM in 30 mM Tris-HCl, pH 7.4 was incubated for 2 h, 22 °C and 600 rpm shaking. Then huPrP(23–144) was added leading to a final concentration of 80 μM Aβ(1–42) and 40 μM huPrP(23–144) in the sample. The sample was incubated further for 30 min. The generated precipitates are cleared from possibly unbound Aβ(1–42) or huPrP(23–144) by centrifugation at 16,100g and 4 °C for 30 min. The pellet containing the pure Aβ(1–42)oligo-huPrP(23–144) precipitate was washed twice with 100 μl 30 mM Tris-HCl, pH 7.4 with centrifugation steps at 16,100g in between. After resuspension of the condensates in 100 μl of 30 mM Tris-HCl, pH 7.4, then 50 μl of the sample was incubated on freshly cleaved mica for 30 min.</p><p>All samples were washed three times with MilliQ water and dried in a gentle stream of N2. Both samples were measured in a Nanowizard 3 system (JPK Instruments AG) using intermittent contact mode with a resolution of 1024 pixels and line rates of 0.5 to 1 Hz in ambient conditions with a silicon cantilever with nominal spring constant of 26 N/m and average tip radius of 7 nm (Olympus OMCL-AC160TS). Due to the curvature and adhesion of the Aβ(1–42)oligo-huPrP(23–144) condensates, the imaging parameters (amplitude, setpoint, and gain) had to be adapted slightly and the cantilever had to be changed often. The height image of Aβ(1–42)oligo was flattened with the JPK Data Processing software 5.0.69.</p><!><p>For the sequence-specific backbone resonance assignments, samples of 0.36 mM uniformly 13C, 15N labeled huPrP(23–144) with 50 mM sodium acetate buffer in 10% (v/v) D2O (pH 4.5) and 0.30 mM uniformly 13C, 15N labeled huPrP(23–144) with 50 mM HEPES buffer in 10% (v/v) D2O (pH 7.0) were prepared as reported previously (Rösener et al. (40)). 13C-13C "TOtal Correlated SpectroscopY" (TOCSY) NMR measurements in solution were performed on a sample containing 0.33 mM uniformly 13C, 15N labeled huPrP(23–144) monomers (Rösener et al. (40)) with 0.02% (w/v) NaN3 in 30 mM HEPES buffer and 10% (v/v) D2O (pH 6.7) and on a sample of 95 μM uniformly 13C, 15N labeled Aβ(1–42) (Isoloid GmbH) in 30 mM Tris-HCl buffer and 10% (v/v) D2O (pH 7.2) at a temperature of 5.0 °C.</p><!><p>The solid-state NMR measurements were performed either on Varian INOVA NMR spectrometers operating at field strengths of 14.1 T (ω(1H)/(2π) = 600 MHz) for samples huPrP(23–144)∗-Aβ, huPrP(23–230)∗-Aβ and huPrP(23–144)-Aβ∗ or a Bruker AEON 18.8 T (ω(1H)/(2π) = 800 MHz) spectrometer for sample huPrP(23–144)exc-Aβ∗, equipped with 3.2 mm standard (Varian) or wide bore (Bruker) triple-resonance MAS probes. Therefore either 3.2 mm thick wall (25 μl, for samples huPrP(23–144)∗-Aβ and huPrP(23–230)∗-Aβ) or thin wall (36 μl, for sample huPrP(23–144)-Aβ∗) rotors from Varian (Agilent) or 3.2 mm thick wall (46.7 μl, for sample huPrP(23–144)exc-Aβ∗) rotors from Bruker were used. For sample huPrP(23–230)∗-Aβ an insert (signal at ≈90 ppm) was used as a precaution because at the beginning of the study it was not known if PrP in huPrP(23–230)∗-Aβ was present in its pathogenic PrPSc conformation.</p><p>Sample temperatures were indirectly determined with an accuracy of ±5 °C for each spinning speed using nickelocene as an external reference (74). Initial magnetization transfer from protons to 13C or 15N was either achieved by "insensitive nuclei enhanced by polarization transfer" (INEPT) (75) to selectively excite mobile regions via scalar coupling through bond magnetization transfer from 1H to 13C (at ≈20, 27, or 30 °C) or by CP (measured at ≈30, 10, 7, 0, −6, or −10 °C) via dipolar coupling through space transfer for rigid parts. DE experiments for sample huPrP(23–230)∗-Aβ were conducted at ≈30, 10, and −10 °C. In this temperature range, the free water in the samples was not fully frozen, as could be observed from the water signal in 1H spectra (not shown). Additionally, several multidimensional homo- and heteronuclear correlation experiments for the assignment were recorded. Experimental details of all spectra recorded are given in Tables S2–S6. For homonuclear 13C-13C spectra, proton-driven spin diffusion (PDSD) (76) with mixing times between 10 and 300 ms was employed. Homonuclear double quantum correlation spectra were recorded with SPC5 recoupling (77).</p><p>For site-specific assignment 15N-13C correlation spectra were recorded using SPECIFIC-CP (78) for frequency selective polarization transfer from 15N to either 13Cα or 13CO and subsequent DARR-mixing. 2D NCA, NCACX and 3D NCACX and NCOCX spectra were used for the sequential walk through the backbone. During all acquisition and evolution times, high-power broadband proton decoupling with SPINAL phase modulation (79) (radio frequency intensity between 71 and 91 kHz) was used. All spectra were processed with NMRPipe (80) with either squared and shifted sine bell or Gaussian window functions. The line width (FWHM) was estimated in 1D-slices (spectra processed with squared sine bell shifted by 0.35π or 0.40π) of 2D PDSD or NCACX/NCOCX spectra. 13C chemical shifts were externally referenced with adamantane by setting the low-frequency signal of adamantane to 31.4 ppm on the DSS reference scale. 15N chemical shifts were indirectly referenced via the 13C chemical shifts. All resonances were assigned in CCPN (81). Integration of Aβ peaks was done in Topspin via the box sum method in a PDSD spectrum of huPrP(23–144)-Aβ∗, measured at a temperature of ≈0 °C, a spinning frequency of 11 kHz, and a mixing time of 50 ms.</p><!><p>For the sequence-specific backbone resonance assignments of uniformly 13C, 15N labeled huPrP(23–144) in solution at pH 4.5, the following experiments were recorded at 5.0 °C on a Bruker AVANCE III HD 600 MHz NMR spectrometer equipped with an inverse triple-resonance probe: 2D 1H-15N HSQC (82), 3D HNCO (83), and 3D HNCACB (84) (further experimental details are given in Table S7). Sequence-specific backbone resonance assignments at pH 7.0 were obtained from 2D 1H-15N HSQC (82), 3D HNCO (83), 3D HNCACB (84), and 3D BEST-TROSY-(H)N(COCA)NH (85) experiments recorded at 5.0 °C on a Varian VNMRS 800 MHz NMR spectrometer equipped with an inverse triple-resonance probe. Two 2D 13C-13C TOCSY spectra covering either the aliphatic (bandwidth 70 ppm) or full (bandwidth 182 ppm) spectral region with a 13.6 ms 13.9 kHz (aliphatic) or 21.1 ms 15.6 kHz (full) FLOPSY-16 isotropic mixing scheme (86) of 0.33 mM uniformly 13C, 15N labeled huPrP(23–144) at 5.0 °C was recorded on a Bruker AVANCE III HD 700 MHz NMR spectrometer equipped with an inverse triple-resonance probe. Because of the comparatively low protein concentration, a 2D 13C-13C TOCSY spectrum covering the aliphatic region (bandwidth 70 ppm) with a 15.1 ms 15.6 kHz FLOPSY-16 isotropic mixing scheme (86) of 95 μM uniformly 13C, 15N labeled Aβ(1–42) at 5.0 °C was recorded on a Bruker AVANCE III HD 800 MHz NMR spectrometer equipped with a 13C/15N observe triple-resonance probe; a total of 1536 transients was collected over the course of 3 weeks and added up to further improve the signal-to-noise ratio. All triple-resonance probes were cryogenically cooled and equipped with z axis pulsed field gradient capabilities. The sample temperature was calibrated using methanol-d4 (87). The 1H2O resonance was suppressed by gradient coherence selection with water flip-back (88), with quadrature detection in the indirect dimensions achieved by States-TPPI (89) and the echo–antiecho method (90, 91). All solution NMR spectra were processed with NMRPipe (80) software and analyzed with NMRViewJ (92) and CCPN (81). 1H chemical shifts were referenced with respect to external DSS in D2O, 13C and 15N chemical shifts were referenced indirectly (93). RCI (50) backbone order parameters, SRCI2, were calculated from the backbone chemical shifts using TALOS-N (94) with the default parameters.</p><p>To obtain sequence-specific backbone resonance assignments for huPrP(23–144) at different pH values ranging from 4.5 to 7.0 and at a temperature of 5.0 °C, we employed the following strategy: (i) In the first step, as many resonance assignments as possible (see above) were transferred from huPrP(23–230) to the 1H-15N HSQC spectrum of huPrP(23–144) at pH 4.5 and 20.0 °C. (ii) Next, these resonance assignments were propagated along a temperature series of 1H-15N HSQC spectra of huPrP(23–144) at pH 4.5 recorded at temperatures of 15.0 °C, 10.0 °C, and 5.0 °C. (iii) The resulting sequence-specific backbone resonance assignments at pH 4.5 and 5.0 °C were verified and completed using HNCO and HNCACB triple-resonance experiments. (iv) These resonance assignments were then propagated along a pH series of 1H-15N HSQC spectra of huPrP(23–144) recorded at pH values of 5.3, 6.0, and 7.0 at a temperature of 5.0 °C. (v) Finally, the resulting sequence-specific backbone resonance assignments at pH 7.0 and 5.0 °C were verified and completed using HNCO, HNCACB, and BEST-TROSY-(H)N(COCA)NH triple-resonance experiments (Fig. S2).</p><!><p>The assigned chemical shifts of huPrP(23–144) at pH 4.5 and pH 7.0 have been deposited with the Biological Magnetic Resonance Data Bank (BMRB) under accession codes 28115 and 28116, respectively.</p><!><p>This article contains supporting information (13, 19, 24, 25, 26, 27, 32, 33, 34, 40, 41, 47, 48, 53, 54, 55, 75, 76, 77, 78, 95, 96, 97, 98, 99).</p><!><p>The authors declare that they have no conflicts of interest with the contents of this article.</p><!><p>Supplemenatl Figures S1–S24 and Tables S1–S7</p>

PubMed Open Access

Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing

The development of novel affinity probes for cancer biomarkers may enable powerful improvements in analytical methods for detecting and treating cancer. In this report, we describe our use of capillary electrophoresis (CE) as the separation mechanism in the process of selecting DNA aptamers with affinity for the ovarian cancer biomarker HE4. Rather than the conventional use of cloning and sequencing as the last step in the aptamer selection process, we used high-throughput sequencing on an Illumina platform. This data-rich approach, combined with a bioinformatics pipeline based on freely available computational tools, enabled the entirety of the selection process—and not only its endpoint—to be characterized. Affinity probe CE and fluorescence anisotropy assays demonstrate the binding affinity of a set of aptamer candidates identified through this bioinformatics approach.Graphical AbstractA population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.

selection_of_dna_aptamers_for_ovarian_cancer_biomarker_he4_using_ce-selex_and_high-throughput_sequen

<!>Introduction<!>Reagents<!>Capillary electrophoresis and aptamer selection<!><!>PCR amplification<!>Single stranding<!>Sequencing and bioinformatics<!>Affinity probe capillary electrophoresis<!>Fluorescence anisotropy<!>Results and discussion<!><!>Results and discussion

<p>A population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.</p><!><p>The long-term survival of ovarian cancer patients correlates strongly with stage at diagnosis. Local disease, confined to one or both ovaries, responds well to existing treatments, with 5-year survival rates averaging 92 % [1]. By contrast, the 5-year survival rate for patients with metastatic cancer at distant sites is 27 %. These data—and the fact that most ovarian cancers are not diagnosed until metastasis has occurred—provide compelling motivation for the discovery and validation of new ovarian cancer biomarkers that may enable earlier detection.</p><p>Using comparative hybridization assays on an array of 21,500 ovarian cDNAs, Hood and coworkers identified the HE4 (WFDC2) gene as more highly expressed in ovarian cancer tissue than in noncancerous ovarian epithelium [2]. This observation was supported by serial analysis of gene expression, which also found HE4 to be amplified in ovarian cancer [3]. A 2003 study demonstrated that serum HE4 protein is detectable via double-determinant immunoassay and is an ovarian cancer biomarker with sensitivity and specificity comparable to that of CA125, the clinical "gold standard," but with the likely advantage of lower false-positive rates in patients with benign disease [4]. Thorough characterization of protein expression in various tissue types via immunostaining confirmed that normal ovarian epithelium does not express HE4, whereas the protein is strongly expressed on serous and endometrioid tumors, which together constitute the vast majority of ovarian cancer cases [5, 6]. In 2008, the FDA approved the use of a serum HE4 assay for monitoring recurrence in patients with epithelial ovarian cancer. The combination of HE4 and CA125, when used in the Risk of Ovarian Malignancy Algorithm (ROMA) test, is effective at classifying women presenting with a pelvic mass into high- or low-risk categories, which enables the triage of women likely to have ovarian cancer to clinical settings and surgeons with appropriate expertise [7, 8]. The FDA approved this use of ROMA in 2011. Most relevant to the challenge of early detection are the results of ELISA assays performed on banked serum samples collected 1 to 18 years prior to ovarian cancer diagnosis [9]. That study showed the mean concentrations of HE4 (along with serum markers CA125 and mesothelin) in serum samples from cancer patients began to visually increase 3 years before diagnosis, reaching detectable levels 1 year before clinical presentation [9]. Novel analytical approaches to HE4 detection may therefore contribute to early detection of ovarian cancer and associated improvement in patient outcomes.</p><p>To complement existing antibody-based detection strategies, many investigators have explored the use of nucleic acid aptamers [10, 11] as affinity probes. Aptamers share with antibodies the property of high-affinity and high-selectivity binding to a target of interest, while having distinct benefits over antibodies in their greater ease of labeling and facile regeneration of native confirmation upon heat cycling. Along with their entirely in vitro development process—the use of animals or cells is not required—these attributes have made aptamers an attractive recognition element employed in a variety of analytical applications [12–14]. Various modes of aptamer selection, referred to as Systematic Evolution of Ligands by EXponential enrichment (SELEX), have been developed [15, 16]. Briefly, a randomized "library" of oligonucleotides is subject to iterative cycles of (1) incubation with the target, (2) separation to resolve bound oligos from unbound, and (3) amplification to reproduce those oligos possessing desired binding attributes. In our development of DNA aptamers with affinity for HE4, we used a capillary electrophoresis (CE)-based separation mechanism [17, 18]. Owing to the high applied field strength used in CE separations, CE-SELEX enables efficient separation of bound and unbound oligos. This selection method has been shown to converge the unselected library onto functional aptamers in fewer rounds than selection methods based on column chromatography or nitrocellulose filtration [19].</p><p>Traditionally, the final stage of the aptamer selection process has been to clone the selected oligonucleotide pool into a bacterial expression system, sequence these oligos by Sanger methods, and characterize the affinity of the identified sequences for the target. It has been shown, however, that such approaches can fail to identify high-affinity aptamers [20]. The recent proliferation of high-throughput sequencing (HTS) techniques, also known as next-generation sequencing, deep sequencing, or massively parallel sequencing [21, 22], has made it possible to sequence pools of aptamer candidate oligos with significantly greater coverage than a clone-and-sequence approach. HTS enables the full evolutionary path of the SELEX process to be characterized, not only its endpoint [23]. As a result, better aptamers can be selected with fewer rounds of selection [24], even in a single round [25], by removing the need for the pool to fully converge on a consensus sequence or sequences. Advantages of this approach include reducing time and materials required and minimizing the opportunities for the introduction of polymerase chain reaction artifacts [26] that can bias selection, ultimately causing the loss of high-affinity binders [27]. Similarly, sequences identified by their fold enrichment, rather than raw read counts, can locate high-affinity binders that traditional Sanger sequencing can miss [20]. HTS can also reduce the number of sequences needing to be tested in vitro, when coupled with bioinformatics, by identifying clusters of oligos with a common sequence or structure [28]. A novel method of aptamer affinity determination, MPBind [29], uses a statistical analysis of HTS data to determine aptamer affinity and may prove valuable to users in this field.</p><p>Here, we report on our use of CE-SELEX to identify DNA aptamers with affinity for the ovarian cancer marker HE4. Selected DNA was subject to HTS on the Illumina platform. Enrichment and clustering analysis were performed in-house to identify the most promising candidate aptamers for in vitro affinity characterization.</p><!><p>Oligonucleotides—including unselected DNA library, polymerase chain reaction (PCR) and sequencing primers, and labeled aptamers for in vitro testing—were purchased from Integrated DNA Technologies (Coralville, IA). The sequences, previously reported by Bowser and coworkers [23] were forward primer 5′-FAM-AGC AGC ACA GAG GTC AGA TG-3′, reverse primer 5′-biotin-TTC ACG GTA GCA CGC ATA GG-3′, and single-stranded DNA (ssDNA) library 5′-FAM-AGC AGC ACA GAG GTC AGA TG (N)25 CCT ATG CGT GCT ACC GTG AA-3′. Nuclease-free water, 25 mM MgCl2, 5.0 U/μL Taq polymerase (for PCR), and Blue/Orange 6× loading dye (for gel loading) were purchased from Promega (Madison, WI). Deoxyribonucleotide triphosphates (dNTPs, 10 mM stock) were obtained from QIAGEN, Inc. (Valencia, CA). NuSieve GTG agarose was purchased from Cambrex BioScience (Rockland, ME). 5× Tris Borate EDTA (TBE) was made from TRIZMA Base and boric acid purchased from Sigma (St. Louis, MO), and 0.5 M EDTA (OmniPur; Gibbstown, NJ). The buffer used for aptamer selection and CE separation was 25 mM Tris, 192 mM glycine, 5 mM KH2PO4, pH 8.3 (TGK) prepared from Thermo Scientific Tris-Glycine powder (Asheville, NC), and KH2PO4 from Mallinckrodt Chemical Works (St. Louis, MO) using 18.2 MΩ cm water as a diluent. Streptavidin-agarose was purchased from Thermo Scientific Pierce Biotechnology Inc. (Rockford, IL). Bio-Rad columns were purchased from Bio-Rad Technologies (Hercules, CA). Binding and washing (B&W) buffer (10 mM Tris, 2 mM NaCl, 1 mM EDTA at pH 7.6, 2× concentration) was made from NaCl purchased from VWR (Bridgeport, NJ). Absolute ethanol was sourced from AAPER Alcohol and Chemical Co. (Shelbyville, KY). Human recombinant HE4 protein with a glutathione-S-transferase purification tag (HE4-GST), GST protein, and the storage buffer for both proteins (50 mM Tris-HCl containing 10 mM reduced glutathione, pH 8.0) were purchased from Abnova (Taipei, Taiwan).</p><!><p>Capillary electrophoresis aptamer selection was performed on a Beckman Coulter P/ACE MDQ system (Fullerton, CA) with exchangeable UV absorbance and laser-induced fluorescence (LIF) detectors (488 nm excitation, 520 emission). The capillary was 51.3 cm in length and 42.5 cm from inlet to window, with an inner diameter of 50 μm and an outer diameter of 360 μm (Polymicro Technologies Inc., Phoenix, AZ).</p><!><p>Incubation conditions used during rounds of aptamer selection</p><!><p>All PCRs were done using a Mastercycler Personal from Eppendorf AG (Hamburg, Germany). Amplification of selected DNA involved two steps: determination of optimal cycle number and preparative PCR. Master mix was made by combining 484 μL nuclease-free water, 16 μL dNTPs, 20 μL each of forward and reverse primers, 96 μL MgCl2, and 160 μL colorless 6× buffer. After mixing, 149.25 μL of the master mix was removed and combined with 0.75 μL Taq polymerase. To 94.5 μL of this completed master mix we added 5.5 μL of DNA collected during selection. This mixed solution was divided equally over thin-walled tubes that were subject to PCR for different numbers of cycles, where each cycle involved three steps: denaturing (95 °C, 30 s), annealing (53 °C, 15 s), and extension (72 °C, 15 s). The samples, which contained different amounts of amplified product, were resolved on a 4 % agarose gel at an applied voltage of 85 V. Gels were imaged on a Kodak Gel Logic 200 Integrated Illumination Cabinet and Imaging System, and photos were digitally improved using Kodak Molecular Imaging software, version 4.5 (Rochester, NY). The fluorescence of forward primers and amplified product enabled visualization of DNA without ethidium bromide staining. The number of cycles that yielded a visible product band with minimal primer and no by-products was used for preparative PCR. In preparative PCR, 646.75 μL of master mix was combined with 3.25 μL Taq polymerase. Seventy microliters of this completed master mix was combined with 5.5 μL of collected DNA in eight separate vials. Completed master mix without added DNA was run as a negative control. After completing the optimal number of PCR cycles, 10 μL of each sample and the control were visualized on a 4 % agarose gel to confirm yield and purity. Remaining samples were pooled and subject to single stranding.</p><!><p>Double-stranded PCR product was converted to single-stranded DNA using streptavidin columns. For the single-stranding process, 300 μL of streptavidin-agarose slurry was placed in a Bio-Rad chromatography column and washed five times with 500-μL portions of 2× B&W buffer. Pooled PCR product was loaded onto the column with an equal volume of 2× B&W buffer, and the mixture was allowed to incubate at room temperature with gentle vortexing every 5 min for 30 min. The column was then washed ten times, with the following buffers, in order of decreasing ionic strength: four washes with 550 μL 2× B&W buffer; five washes with 550 μL 1× B&W buffer; and one wash with 500 μL ultrapure H2O. Thirty micromoles of NaOH (200 μL of 0.150 M NaOH) was then added to the column, gently vortexed, and incubated at 37 °C for 10 min to denature double-stranded DNA (dsDNA). The column was then gently vortexed and the unretained ssDNA (containing the FAM forward primer) eluted into 30 μmol of acetic acid (200 μL of 0.15 M acetic acid) to neutralize the hydroxide. The solution was buffered by the addition of 40 μL of 3 M sodium acetate, followed by 1000 μL of cold 100 % ethanol to precipitate ssDNA. The NaOH elution process was repeated into a separate collection tube, and the two samples (containing ssDNA in ethanol) were incubated at −20 °C or on wet ice for at least 2 h but not more than 12 h. The two portions of eluted DNA were centrifuged at 13,200 RPM for 45 min at 4 °C. Supernatant was pipetted from each tube, leaving 100 μL DNA-containing solution. One milliliter of cold 70 % ethanol was then added to both tubes. After 20 min of spinning at 4 °C, supernatant was removed, leaving 50 μL. The cold ethanol washing process was repeated; after centrifugation, supernatant was carefully removed, leaving 25 μL. Both portions of eluted DNA were then dried in a Speedvac at medium heat for 10 min, followed by 5 min spinning at room temperature. Each tube was then reconstituted in 15 μL of TGK buffer. The DNA was then combined and divided as follows: 10 μL was archived for sequencing, 10 μL was archived for NanoDrop and bulk affinity measurements, and 10 μL was used for the next round of selection.</p><!><p>After aptamer selection was complete, DNA collected from each round was amplified using Illumina sequencing primers. Archived DNA from each round was diluted to 100 nM using ultrapure water. Each sample was assigned a unique reverse primer containing the index used for barcoding. Master mix containing all PCR reagents except Taq polymerase and the reverse primers was made from 563 μL nuclease-free water, 18 μL dNTPs, 22 μL forward primer, 108 μL MgCl2, and 180 μL colorless buffer. To 97 μL of this mix was added 0.5 μL of Taq polymerase and 2.4 μL of the specific reverse primer. Seventy-four microliters of this mixed solution was added to 1 μL DNA solution; samples and controls were amplified by PCR using an optimized cycle number. PCR products were imaged on a 3 % agarose gel containing 1 μg/mL ethidium bromide to confirm yield and the absence of contaminants or by-products. Samples were sequenced at the University of Wisconsin Biotechnology Center DNA Sequencing Facility.</p><p>Bioinformatic screening of the sequenced DNA used a data pipeline based on freely available software, with the exception of enrichment analysis, which used a Python program written in-house. This program (enrichment.py) has been made available on GitHub at https://github.com/rebeccawhelan/PythonEnrichment. After a preliminary analysis of the FastQC files to ensure the sequencing was successful, data were sent into a Biopieces pipeline. Each round's data was individually read into the pipeline using read_fastqc. Selection primers were removed with remove_primers, using a 5 % mismatch tolerance and a 0 % tolerance for insertions and deletions. All bases beyond the reverse sequence primer (i.e., adaptors and sequencing primers) were removed in this step. These sequences were then filtered using grab to select only sequences with a length of 25 ± 2. These data were processed with uniq_seq, creating one record for each sequence with associated count information. The records were sorted by read count in descending order using sort_records and written to a file as tabular data. From there, the processed data were taken through enrichment analysis, a novel program that determines the fold enrichment for sequences across rounds of selection. Random regions only (with primers excluded) were used in the enrichment analysis to simplify computation; with respect to the enrichment over selection rounds, the primer information is redundant, being identical across all sequences. Fold enrichment has been shown to be a more reliable indicator of binding affinity than read counts [27]. Using CD-HIT-EST [30], the top 1000 most enriched sequences from each round were clustered by sequence homology to determine possible emergent motifs. Sequences were clustered with their primers attached to a sequence identity threshold of 0.8 and assigned to clusters by the highest identity across all clusters.</p><!><p>Affinity probe capillary electrophoresis affinity assays were performed using a Beckman P/ACE MDQ (Beckman Coulter, Fullerton, CA) equipped with an argon-ion laser. An unmodified fused silica capillary (Polymicro Technologies, Phoenix, AZ; ID = 50 μm, OD = 360 μm, total length = 49.5 cm, length from inlet to detector = 39.6 cm) was held at 25 °C. Samples were injected from the outlet end, and negative polarity was applied to minimize the distance from injection to detection (length to detector = 9.9 cm). Each sample contained 10 nM FAM-labeled aptamer (synthesized as a 25mer sequence without primer regions), 20 nM fluorescein (internal standard), and 1 mg/mL BSA. TGK was used both as the diluent in sample preparation and as the electrophoresis buffer. To prepare samples, a bulk solution of aptamer in TGK was heated to 90 °C for 3 min, and then put on ice to cool. Fluorescein and bovine serum albumin (BSA) were then added, and the solution was distributed over an appropriate number of sample tubes. Finally, protein (HE4-GST or GST, in separate experiments) was added to a final concentration ranging from 0 to 240 nM. The volume of protein plus protein buffer was constant in all samples. Pressure injection (0.3 psi, 5 s) was used to introduce the sample onto the capillary; separation was achieved by the application (in negative polarity) of 30 kV. Run time was 3 min. The fluorescence was excited at 488 nm and detected at 520 nm. Peak heights were determined by the instrument control software (32 Karat). The change in the size of the free DNA aptamer peak, relative to the internal standard, was used to indicate the complex formation between aptamer and protein. Data were fit with an isotherm equation:\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ \mathrm{Ratioed}\kern0.3em \mathrm{peak}\kern0.3em \mathrm{height}=rac{\mathrm{constant}}{\left(1+\left({K}_{\mathrm{d}}/\left(T+0.5 imes \left(A+T+{K}_{\mathrm{d}}-{\left({\left(A+T+{K}_{\mathrm{d}} ight)}^2-4 imes A imes T ight)}^{0.5} ight) ight) ight) ight)} $$\end{document}Ratioedpeakheight=constant(1+(Kd/(T+0.5×(A+T+Kd−((A+T+Kd)2−4×A×T)0.5))))where T is protein concentration (varied) and A is aptamer concentration (constant, typically 10 nM), using IgorPro (v. 6.12) graphing software.</p><!><p>Fluorescence anisotropy was measured using a SpectraMax M5 multimode plate reader with polarizing optics (Molecular Devices, Sunnyvale, CA). Tested aptamers were ordered from Integrated DNA Technologies, Inc. (Coralville, IA) with a 5′ TEX615 (Texas Red) fluorophore. One hundred nanomolar DNA aptamer in buffer (TGK) was heated to 95 °C for 3 min, cooled to 4 °C, then allowed to warm to room temperature. Heat-cycled DNA solution was combined with HE4-GST at a range of final concentrations from 0 to 750 nM in the presence of 0.1 mg/mL bovine serum albumin (BSA). The volume of protein plus protein buffer was constant across all samples. After incubating for at least 30 min in the dark at 25 °C, samples were loaded in duplicate (70 μL/well) into a 96-well Fluotrac 200 black immunology plate (USA Scientific, Ocala, FL) and analyzed in the SpectraMax, with temperature held at 25 °C. The λex for fluorescence anisotropy was 585 nm, λem was 635 nm, and the wavelength cut-off was 610 nm. Raw data (fluorescence emission parallel and perpendicular to the exCitation) were blank-corrected before the anisotropy values were calculated. Measurements were run at least in duplicate and fit with an isotherm function:\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$ r-{r}_0=rac{\mathrm{constant}}{\left(1+\left({K}_{\mathrm{d}}/\left(T+0.5 imes \left(A+T+{K}_{\mathrm{d}}-{\left({\left(A+T+{K}_{\mathrm{d}} ight)}^2-4 imes A imes T ight)}^{0.5} ight) ight) ight) ight)} $$\end{document}r−r0=constant1+Kd/T+0.5×A+T+Kd-A+T+Kd2-4×A×T0.5where T is protein concentration (varied), A is aptamer concentration (constant), r is the anisotropy measured in the presence of protein, and r0 is the anisotropy in the absence of protein, using IgorPro (v. 6.12) graphing software.</p><!><p>An unselected DNA library with N = 25 random region was used as the input to the selection process because it provided a good balance of sequence diversity, coverage, and computational tractability. Assuming that each base is equally likely to appear at each position in the random region, there are 425 (~1 × 1015) possible sequences in such a library. In our selection, we used 100 pmol (~6 × 1013 molecules) of DNA as the initial input, giving any individual sequence an expected abundance of 0.05 (a library with N = 23 would give an expected abundance of 1). Using a longer random region would result in lower coverage of sequence space that could result in the loss of useful motifs, whereas a shorter random region might lack the complexity to form relevant secondary and tertiary structures involved in target binding.</p><!><p>a Gel image showing the effect of increasing the number of PCR cycles. b Overlaid capillary electropherograms showing the effect of increasing cycle number on PCR product. The traces have been vertically offset for clarity</p><p>A flow chart showing the steps involved in analyzing high-throughput sequencing data collected after a SELEX experiment</p><p>Characteristics of data resulting from Illumina sequencing</p><p>SELEX round numbers are as described in Table 1. The unselected library is designated "R0"</p><p>HE4 aptamer candidates chosen for in vitro analysis</p><p>n/a not available</p><p>Binding isotherm collected by affinity probe capillary electrophoresis on aptamer A3 and HE4-GST</p><p>Affinity of HE4 aptamer candidates for HE4-GST (K d) determined by fluorescence anisotropy and affinity probe CE</p><p>n/a not available</p><!><p>In conclusion, we have demonstrated the proof of concept for using capillary-based aptamer selection, high-throughput sequencing, and a freely available bioinformatics pipeline to select DNA aptamers with affinity for ovarian cancer biomarker HE4. The validity of this combination has also been demonstrated by another very recent publication in this journal [31]. Our current efforts focus on improving the PCR process to reduce the formation of by-products using emulsion PCR [32] and using a six-histidine-modified HE4 as the target protein in place of the more sterically hindered HE4-GST, with the goal of selecting higher affinity aptamers than those reported here. Aptamers with high binding affinity, reflected by low nanomolar Kd values, are sought for use in bioassays and in eventual clinical application. Inclusion of divalent cations in the selection buffer is also hypothesized to enable greater diversity of secondary structure and therefore greater binding affinity; selection in such a buffer is also in progress.</p>

PubMed Open Access

Reduction in Water Pollution in Yamuna River due to lockdown under COVID-19 Pandemic

The epidemic of Novel COVID-19 was reported in India in January 2020 and increased day by day due to the movement of people from abroad to India and then to the different parts of the country. The COVID-19 has been declared as pandemic because of its high transmission rate and coved more than 2010 countries of the world. Under this scenario when there is no medicine for its treatment, the only solution to this problem is to break the chain of transmission and restrict the count of infected people. To contain a coronavirus (COVID-19) outbreak, the Government of India announced the nationwide lockdown with effect from the midnight of 24 th March 2020 followed by the extension of the lockdown periods and presently it is in its 4 th phase. The various provisions were made under lockdown for closing the industries, transportation, etc. except the essential services. It has been very interesting to note that the behavioural changes in nature are highly positive and atmosphere, hydrosphere, and biosphere are rejuvenating and it gives an appearance that the earth is under lockdown for its repairing work. Under this natural recovery, we tried to look at the improvement in the water quality of the Yamuna River in Delhi, which has been one of the burst polluted rivers. To study this river, the concentrations of pH, EC, DO, BOD, and COD have been measured which showed a reduction by 1-10%, 33-66%, 51%, 45-90%, and 33-82% respectively during the lockdown phase in comparison to the pre-lockdown phase. The Nizamuddin Bridge, Okhla U/s, Najafgarh Drain and Shahdara Drain were the major hotspots responsible for the deterioration of the water quality of Yamuna River while passing by Delhi region. Five major locations of Yamuna River have been analysed in this paper that showed a very impressive recovery of the water quality during the lockdown phase as compared to the pre-lockdown status of water quality.

reduction_in_water_pollution_in_yamuna_river_due_to_lockdown_under_covid-19_pandemic

Introduction<!>Study Area and Methodology<!>Effect of lockdown on Yamuna River water quality<!>pH level in Yamuna River, Delhi<!>Conductivity level in Yamuna River, Delhi<!>Dissolved Oxygen level in Yamuna River, Delhi<!>Biological Oxygen Demand level in Yamuna River, Delhi<!>Chemical Oxygen Demand level in Yamuna River, Delhi<!>Major Pollution hotspots in the Yamuna<!>Conclusion

<p>The origin of the deadly pandemics coronavirus (COVID-19) has been in December 2019 from the City of Wuhan, China (Travaglio et al., 2020;Raibhandari et al., 2020;Chauhan and Singh, 2020) and spread to the almost entire globe. The source of COVID-19 is reported from the novel coronavirus (SARS-CoV-2) and would have been produced from other mammals (Travaglio et al., 2020). The World Health Organisation (WHO) in his report updated on 23 May 2020 at 05:30 GMT said that there are confirmed cases of infection by COVID-19 to 5,206,614 people while 337,736 has lost their life from 216 countries (WHO Report, 2020 dated 24 May 2020).</p><p>Recognizing the rate of spread of this virus with the personal contacts, various countries have imposed complete lockdown in order to maintain forced social distancing and break the chain of the spread of coronavirus. Still if there are some urgent requirements of movement of people, they were asked to under quarantine for 14 days considering the appearance of the symptoms that take about 14 days. The Govt. of India, taking note of the activities adopted by the COVID-19 affected countries, first requested the countrymen to be at home for the entire day and Prime Minister of India gave this a name as Janata (People's) Curfew, which was observed on 22 nd March 2020. On this day of Janta Curfew all flights, trains, bus services, industrial and commercial activities were closed. After its success, an absolute lockdown was imposed on 25 th March 2020 for 21 days to break the chain of COVID-19 (Long, 2020). Further, the lockdown has been extended in phases 2, 3, and 4 till May 31, 2020 to control the spread of infection through a complete halt on the movement. The total lockdown has elevated pandemonium among people but helped in reducing the pace of spreading the virus among society. However, under this lockdown period, Nature started to respond very positively and started giving several signals of improvement to natural parameters of the atmosphere, hydrosphere, and biosphere. It appears that the earth is rejuvenating under the lockdown period and it's a closure for the repairing of earth.</p><p>With the understanding of this natural recovery, we tried to look at the water quality status and improvement, if any, for the Yamuna River in Delhi, which has been famous for its high pollution level in Delhi. The concentrations of pH, EC, DO, BOD and COD have been measured at various hot spots for the pollutions on the bank of river Yamuna. The water quality parameters were compared between the lockdown phase and the pre-lockdown phase. Five major locations of Yamuna River have been analysed in this paper that showed a very impressive recovery of the water quality during the lockdown phase as compared to the pre-lockdown status of water quality. This showed that Nature is flourishing during the coronavirus pandemic followed by the lockdown in the larger part of the world forcing the closure of the sources of anthropogenic pollution. Yamuna river is one of the highly polluted rivers in India, especially in Delhi (Upadhyay et al., 2011) where the recent observations reflected that the water pollution has reduced across the Yamuna River channel during the lockdown phase.</p><p>Hence to quantify the status of water pollution in one of the highly polluted locations of Yamuna river, we have carried out an analysis of pH, Conductivity (EC), DO, BOD and COD at various locations of the Yamuna River, where complete lockdown has been imposed.</p><!><p>The Yamuna River is the second-largest and longest tributary of Ganga which enters Delhi at village Palla. It traverses 22 km to Wazirabad barrage where entire water is impounded to meet the drinking water requirement of Delhi. River Yamuna ceases to exist downstream of Wazirabad Barrage in most of the periods of the year and receives its flow from the Najafgarh drain at Wazirabad downstream. No major fresh water is allowed to flow downstream of Wazirabad barrage except during the monsoon season (Fig. 1). As the river traverses further downstream the flow is blocked by a barrage at Okhla.</p><p>There are a total of 23 drains discharging wastewater in the river Yamuna. Out of 23, a total of 16 drains are discharging wastewater in river Yamuna between Wazirabad downstream to Okhla upstream and 04 drains meet the Yamuna in downstream of Okhla Barrage and 03 remaining drains discharge their wastewater further down at Agra Canal and Gurgaon Canal. There are 05 drains having interception and diversion provision of sewage to the nearby STPs for ensuring further treatment. During the year 2019, the total flow of wastewater was estimated as 3026.24 MLD and BOD load was estimated as 0.10-61.44 TPD (CPCB, 2020).</p><p>The water quality data of various pollutants were collected from the Central Pollution Control Board (CPCB). The data were analyzed for the Yamuna at 5 monitoring stations i.e. Palla, Nizamuddin Bridge, Okhla (U/S), Najafgarh Drain and Shahdara Drain for the period from 1 st March to 7 April 2020 which have been studied in two phases as pre-lockdown phase (11-23 March, 2020) and lockdown phase (24 March -7 April, 2020).</p><!><p>The nationwide lockdown has come in effect since the midnight of March 24 because of the COVID-19 pandemic. Under the lockdown, the major sectors responsible for water pollution like industries, power plants, construction activities, transportation, etc. were put on halt. The academic institutions and hospitality services were also adjourned. Under these circumstances, the improvement in water quality was noticed in the river system of the country. Delhi, which is the hub of air pollution and being counted as number one for most of the time has resulted in a noticeable improvement in the water quality of Yamuna during the lockdown period of the country. Scattered rains in Delhi on 27 th March and during March 28-29, 2020 further helped in improving the water quality of Yamuna River during the lockdown phase. This result has been substantiated while analyzing the data of water pollution and water quality before and after the imposition of lockdown.</p><!><p>The pH of the Yamuna River observed alkaline in nature which varies from 7.1 to 8.7 with a mean value of 7.6 during the pre-lockdown phase (Fig. 2) while it has been observed between 7.1-7.4 in Najafgarh and Shahdara drain during the pre-lockdown phase. However, pH varies from 7.1 to 7.8 with a mean value of 7.3 in the Yamuna during the lockdown phase. The highest pH (8.7) was recorded at village Palla (entry point of Yamuna in Delhi) and lowest (7.1) at Shahdara drain during the pre-lockdown phase. During the lockdown phase, a slight reduction in pH has been observed due to the reduction of industrial activities, the nonfunctioning of essential commercial units, and prevailing weather conditions. The maximum reduction (10%) of pH has been observed at Village Palla during the lockdown phase. The concentration of pH was also correlated with the primary water quality criteria for a bathing water and designated best usable water quality criteria of India (https://cpcb.nic.in/wqm/Primary_Water_Quality_Criteria.pdf).</p><p>These exercises helped in understanding that the concentrations were greater than the threshold limit of pH (6.5-8.5) daily at the village Palla which is vulnerable to the health problem. During the pre-lockdown phase, the pH levels were lower than the threshold limit (6.5-8.5) except at village Palla while it became much lower during the lockdown phase at all locations. The pH drives most of the chemical and biological changes in water. It acts as the driving force in controlling species distributions in aquatic habitats. The varying pH values provides space to different species to flourish within however the optimum pH range is 6.5-8.0 for most of the aquatic organisms. The variability of pH outside this range physiologically put stress on numerous species and may affect decreased reproduction and growth, attack of disease, or even death. Hence beyond the optimum value of pH can adversely affect the biological diversity in water bodies.</p><!><p>In the Yamuna, conductivity varies from 688 to 2485 µS/cm with a mean value of 1526 µS/cm during the pre-lockdown phase (Fig. 3) while it observed between 273-1657 µS/cm during the lockdown phase. The highest conductivity (2485 µS/cm) was recorded at Shadra Drain and lowest (688 µS/cm) at village Palla during the pre-lockdown phase. During the lockdown phase, a slight reduction in conductivity has been observed due to the reduction of industrial activities, the nonfunctioning of essential commercial units, and prevailing weather conditions. The maximum reduction (66%) of conductivity has been observed at the Nizamuddin bridge followed by village Palla (59%), Okhla U/s (43%), and Nazafgarh/Shadra drain (33%) during the lockdown phase (Table 1).</p><p>Discharges into the streams are capable of changing the conductivity depending on their makeup. A failing sewage system raises the conductivity because of the higher presence of chloride, phosphate, and nitrate. It may be noted that 16 drains are discharging wastewater in river Yamuna which are influencing the conductivity of the Yamuna River.</p><!><p>Dissolved oxygen (DO) is one of the most important indicators of water quality on which the survival of aquatic life depends. When DO becomes too low, fish and other aquatic organisms cannot survive. The data for DO was not available at Nizamuddin Bridge and Okhla U/s location during the pre-lockdown phase while it was 17.01 mg/l at village Palla in the same period (Fig. 4). However, DO vary from 1.2 to 8.3 mg/l with a mean value of 3.9 mg/l in the Delhi region of Yamuna during the lockdown phase. During the lockdown phase, improvement in DO has been observed at both the Nizamuddin Bridge and Okhla U/s due to the reduction of industrial activities and rainfall in Delhi. It may be noted that DO was not detected at both Nizamuddin Bridge and Okhla U/s during the pre-lockdown phase due discharge of huge amount of industrial and domestic wastewater. The comparative analysis is given in Table 1. The concentration of DO was also correlated with the Primary Water Quality Criteria for bathing water and designated best use water quality criteria of India. The DO levels were lower than the threshold limit (5 mg/l) except at village Palla during both pre-lockdown and lockdown phase at all locations. Low DO affects most biological processes in water and responsible for lower biological diversity in water bodies.</p><!><p>Biological Oxygen Demand (BOD) is one of the most important indicators of water quality.</p><p>BOD directly affects the amount of dissolved oxygen in water bodies. The greater demand for BOD more rapidly depletes the oxygen in the water bodies making lesser availability of oxygen for higher forms of aquatic life. The consequences of the high BOD are similar to the effect of less oxygen availability putting aquatic life under stress, suffocation and could be lethal. The major sources of increase of BOD in the Yamuna river include dead plants and animals; animal manure; industrial/domestic effluents, wastewater treatment plants, failing septic systems; and urban stormwater runoff.</p><p>BOD vary from 7.9 to 163 mg/l with a mean value of 66.58 mg/l during the pre-lockdown phase (Fig. 5) while it observed between 2-89 mg/l during the lockdown phase. The highest BOD (163 mg/l) was recorded at Shahdara Drain and lowest (7.9 mg/l) at village Palla during the prelockdown phase. However, improvement in BOD (i.e. the reduced demand) has been observed at all locations in the lockdown phase due to the reduction of industrial activities and prevailing weather conditions. The maximum reduction (90%) of the BOD level has been observed at the Nizamuddin Bridge during the lockdown phase followed by Okhla U/s (77%), village Palla (75%), Shahdara drain (45%) and Najafgarh drain (29%). The comparative analysis is given in Table 1. The concentration of BOD was also correlated with the primary water quality criteria for bathing water and designated best use water quality criteria of India. The BOD levels were much higher than the threshold limit (3 mg/l) at all locations during the pre-lockdown phase. A similar trend was also observed during the lockdown phase except for village Palla. Higher BOD affects most biological processes in water and can ultimately lead to reduced biological diversity in streams.</p><!><p>Chemical oxygen demand (COD) is an indicator of contamination that shows the amount of dissolved matter in water susceptible to being oxidized. COD is responsible for the reduction of DO in water bodies. Higher concentration of COD is responsible for quick deterioration of oxygen in water bodies and oxygen availability for higher forms of aquatic life. The major sources that increases the COD in the Yamuna River are industrial/domestic effluents, wastewater treatment plants, failing septic systems; and urban stormwater runoff.</p><p>COD varies from 28 to 574 mg/l with a mean value of 211.6 mg/l during the pre-lockdown phase (Fig. 6) while it observed between 6 to 383 mg/l during the lockdown phase. The highest COD (574 mg/l) was recorded at Shahdara drain and lowest (28 mg/l) at village Palla during the prelockdown phase. However, improvement in COD has been observed at all locations in the lockdown phase due to the reduction of industrial activities, rainfall, and prevailing weather conditions. The maximum reduction (82%) of the COD level has been observed at the Nizamuddin Bridge during the lockdown phase followed by Okhla U/s (81%), village Palla (79%), Najafgarh drain (45%) and Shahdara drain (33%). The comparative analysis is given in Table 1.</p><!><p>During the pre-lockdown period at Nizamuddin Bridge, the results showed pH (7.3), EC (1369 μs/cm), BOD (57 mg/L), DO (not detected), and COD (90 mg/L) whereas in the lockdown period pH (7.2), EC (460 μs/cm), BOD (5.6 mg/L), DO (2.4 mg/L) and COD (16 mg/L) were observed and not complying to the primary water quality criteria for outdoor bathing w.r.t analyzed parameters of DO and BOD which can be attributed to the contribution from mainly 14 drains discharging both treated and untreated sewage, no industrial effluent discharges from the industrial areas or no other human activities such as bathing, throwing of worship materials or solid waste and freshwater discharges from U/s of river Yamuna. A similar trend was also at Okhla U/s where the analysis showed pH (7.2), EC (861 μs/cm), BOD (27 mg/L), DO (not detected), and COD (95 mg/L) during pre-lockdown phase whereas pH (7.1), EC (488 μs/cm), BOD (6.1 mg/L), DO (1.2 mg/L) and COD (18 mg/L) were observed during the lockdown phase and not complying to the primary water quality criteria for outdoor bathing w.r.t analyzed parameters such as DO and BOD which can be attributed to contribution only from two drains carrying both treated or untreated sewage, no industrial effluent discharges and there is a river Yamuna stretch of about 7.5 km (after Nizamuddin Bridge) and might be helping in selfpurification of river Yamuna. The Najafgarh Drain discharges 1938 MLD of wastewater into river Yamuna. During the pre-lockdown period at Najafgarh Drain, the analysis showed pH (7.3), SS (152 mg/L), BOD (78 mg/L), COD (271 mg/L) whereas the analysis showed pH (7.3), EC (1501 μs/cm), BOD (55 mg/L), and COD (150 mg/L) during the lockdown period. While at Shahdara Drain, the results showed pH (7.1), BOD (163 mg/L), COD (574 mg/L) during prelockdown period whereas pH (7.2), EC (1657 μs/cm), BOD (89 mg/L) and COD (303 mg/L) were observed in lockdown phase. The comparative analysis is given in Table 1. Betterment in the water parameters of the Yamuna in Delhi during the lockdown phase is due to no contribution of effluent from all the 23 sources.</p><!><p>During the lockdown period, there has been a general improvement in water quality in the Yamuna River as a result of the restrictions imposed during the lockdown and due to no contribution of effluent from all the 23 sources. The concentrations of pH, EC, DO, BOD and COD showed 1-10%, 33-66%, 51%, 45-90% and 33-82% of reduction in pH, EC, DO, BOD and COD concentrations, respectively during the lockdown phase, as compared to the pre-lockdown phase in Yamuna River. The Nizamuddin Bridge, Okhla U/s, Najafgarh Drain and Shahdara Drain are the major hotspots of effluent in the Yamuna River catchment area in Delhi.</p><p>The Covid-19 lockdown situation in almost the entire world has shown the importance of nature in our day to day life and gave a true picture of the overexploitation of the natural resources and proved that we are responsible for the degradation of nature and putting risk to our wellbeing as well. This lockdown showed that the solution for natures' cleanliness lies in our hands goes through the path of preservation of natural resources and sustainable development. The cleanliness observed in the river Yamuna during the lockdown is much better than the several efforts and actions for Yamuna cleaning where a huge amount of the money was invested but the results were never at the satisfaction and status of the revival of Yamuna at the pre pollution level was not achieved. There are several issues due to the lockdown at the front of social and economic wellbeing which cannot be appreciated at all but some positive lessons related to nature gave us a way forward for restraining from the natural calamities if care for nature is established with honesty.</p>

ChemRxiv

Enhancement of hydrogen evolution reaction kinetics in alkaline media by fast galvanic displacement of nickel with rhodiumfrom smooth surfaces to electrodeposited nickel foams

Energy-efficient hydrogen production is one of the key factors for advancing the hydrogen-based economy. Alkaline water electrolysis is the main route for the production of high-purity hydrogen, but further improvements of hydrogen evolution reaction (HER) catalysts are still needed. Industrial alkaline electrolysis relies on Ni-based catalysts, and here we describe a drastic improvement of HER activity of Ni in alkaline media using several model catalysts for HER obtained upon nickel surface modification in aqueous solution of rhodium salts, when a spontaneous deposition of rhodium takes place based on the chemical displacement reaction 3Ni + 2Rh 3+ = 3Ni 2+ + 2Rh. In the case of smooth Ni-poly electrodes, HER activity surpasses the activity of Pt-poly already after 30 s of exchange with Rh. SEM analysis showed that Rh is uniformly distributed, while surface roughness changes within 10%, agreeing with electrochemical measurements. Furthermore, XPS analysis has shown effective incorporation of Rh in the surface, while DFT calculations suggest that hydrogen binding is significantly weakened on the Rh-modified Ni surfaces. Such tuning of the hydrogen binding energy is seen as the main factor governing HER activity improvements. The same galvanic displacement protocols were employed for nickel foam electrodes and electrodeposited Ni on Ti mesh. In both cases, somewhat longer Rh exchange times are needed to obtain superior activities than for the smooth Ni surface, but up to 10 min. HER overpotential corresponding to −10 mA cm −2 for nickel foam and electrodeposited Ni electrodes, after modification with Rh, amounted to only −0.07 and −0.09 V, respectively. Thus, it is suggested that a fast spontaneous displacement of Ni with Rh could effectively boost HER in alkaline media with minor cost penalties compared to energy saving in the electrolysis process.

enhancement_of_hydrogen_evolution_reaction_kinetics_in_alkaline_media_by_fast_galvanic_displacement_

Introduction<!>Surface Rh exchange and electrochemical measurements<!>Working electrodes preparation<!>Characterization<!>DFT calculations<!>Rh depositionsmooth Ni-poly surface<!>HER mechanism on Rh-modified Ni surfaces<!>Rh exchange on expanded Ni surfaces<!>Conclusions

<p>Faced with the global energy crisis, we strive to reduce fossil fuel consumption and increase the usage of renewable energy sources [1][2][3]. The electrochemical production of hydrogen via hydrogen evolution reaction (HER) is a promising way for obtaining clean and renewable energy [4].</p><p>In alkaline solutions the HER mechanism involves water dissociation and subsequent hydrogen reduction and adsorption (H 2 O + e −  H ads + OH − ), in the Volmer step, which is followed by, either the Heyrovsky step (H ads + H 2 O + e −  H 2 + OH − ) or by the Tafel step (2H ads H 2 ) [5]. Recent improvements in the cost and performance of electrochemical water splitting technologies point towards a more economically viable future for their application at an industrial scale [6]. Currently, the two prevalent methods for obtaining hydrogen are proton-exchange membrane (PEM) electrolysis [7] and alkaline water electrolysis (AWE) [8]. The hydrogen production efficiency of PEM electrolyzers is higher but requires Pt-based catalysts [9]. In the case of AWE, the key goal is high catalytic activity and stability under intermittent polarization in alkaline media [10]. It is well known that the HER rate in alkaline media is inferior to that in acidic ones [11,12] because of the additional water dissociation step [13], precedes the discharge step, but also due to the blockage of active sites by adsorbed hydroxyl ions, but also partly due to hydroxyl ion adsorption resulting in the blockage of active sites [5]. On the other hand, HER in alkaline media leads to less environmental pollution and equipment corrosion [4].</p><p>Nickel and nickel-based electrocatalysts have been thoroughly investigated because of nickel's excellent stability in alkaline media [14]. However, it has been shown that the catalytic activity of Ni largely depends on its morphology and active surface area [15,16]. Therefore, multiple design strategies have been developed to increase the mediocre activity of clean Ni. Alloying Pt with Ni gives rise to two effects that increase the overall activity of the material. The first is the -ligand effect‖, referring to the change in electronic properties of both metals [17]. The other is the -lattice strain effect,‖ which represents the change in Pt-Pt interatomic distance, resulting in a shift of the Pt d-band center [18]. A large number of non-PGM (PGMplatinum group metal) Ni alloys, such as binary and tertiary Ni-Mo alloys [20], NiCu [21], Raney Ni and NiCo [22], exhibit high activity.</p><p>Surface modification of Pt with Ni(OH) 2 is a prime example of a bifunctional electrocatalyst for alkaline HER. As suggested, Ni(OH) 2 promotes water dissociation, and the hydrogen intermediates subsequently adsorb and combine on the Pt surface [19], relying on tailoring catalytically active interfaces. Another approach for improving Ni performance is enhancing interfacial processes related to fast removing adsorbed hydrogen, like interfacing with reduced graphene oxide, which stimulated Hads spillover and improved HER activity [23] while maintaining excellent stability [24]. Despite numerous approaches to boost the HER activity of Ni, which work well in practice, a fundamental understanding of the key interactions in catalytic systems is of utmost importance.</p><p>It is well known that rhodium exhibits high catalytic activity towards both reduction and oxidation reactions [25]. Although several Rh-containing materials have been used in electrocatalysis of HER [26], only a small fraction of these was tested in alkaline media [27]. An example is a Ni 89 Rh 11 alloy which has been experimentally shown to have excellent activity [28], which can largely be attributed to the bifunctionality of the surface. Furthermore, it was discussed that the Ni/Rh phases play the roles of H-adsorption/desorption sites, while the Ni(OH) 2 and Rh 2 O 3 phases catalyze water dissociation, but no direct pieces of evidence were provided for such claims [28]. Moreover, Ni-Rh catalysts were also used for other catalytic reactions [29][30][31][32], showing the versatility of this catalytic system. However, the downside of Rh-containing catalysts is the high price of Rh, caused by its scarcity. With this in mind, it is important to optimize the design of novel Rh-containing electrocatalysts, taking advantage of the high catalytic activity of Rh while maintaining its low concentrations. Finally, it is important to note that the application of Ni/Rh electrocatalysts is potentially highly favorable, since not only does Rh enhance the HER performance [28,33] and the overall water splitting [34], but, most likely, there is an underlying synergistic effect of Ni and Rh which could go beyond HER catalysis.</p><p>This study is aimed to investigate the effects of surface modification of Ni using galvanic displacement with Rh on the HER activity in alkaline media. Using the mentioned approach, only the surface of an electrocatalyst is modified with low amounts of Rh [35], which is highly beneficial for maintaining the low cost of such prepared catalysts while significantly increasing catalytic activity.</p><p>The Rh-exchanged Ni electrodes, starting with smooth Ni surfaces to nickel foam and Ni electrodeposits, were investigated by cyclic voltammetry for different duration of Ni immersion in rhodium salt solutions. In addition, the effects of electrode oxidation pretreatment were investigated to discuss the HER mechanism on modified electrodes. Apart from electrochemical measurements, the effects of the Rh-exchange process on the surface properties of Ni electrodes were also characterized by scanning electron microscopy with energy dispersive X-ray analysis (SEM/EDX) and X-ray photoelectron spectroscopy (XPS). Moreover, Density Functional Theory (DFT) calculations were used to rationalize the enhancement of HER activity upon surface modifications with Rh. We note that this work is focused on the origins of HER activity enhancement on various Ni surfaces upon spontaneous displacement with Rh, while the stability of investigated catalysts is not addressed.</p><!><p>Electrochemical measurements were done using IVIUM Vetex.One or Gamry Interface 1011e potentiostats in one compartment three-electrode electrochemical cell with Saturated Calomel Electrode (SCE) as a reference electrode and a 3×3 cm Ni foam (Goodfellow) as a counter electrode.</p><p>As the electrolytic solution, 1 mol dm −3 KOH solution (Sigma Aldrich) prepared with ultrapure deionized water was used in all experiments. All the measurements were done at room temperature. In this work, potentials are referred to SCE, and to calculate overpotentials for HER, potentials are converted to the Reversible Hydrogen Electrode (RHE) scale as E RHE = E SCE + 0.244 V + 0.059 V × pH (pH = 13.45). Electrolyte resistance was corrected using hardware settings, but only up to 80 % of the resistance value, determined using single-point impedance measurement at −1 V vs. SCE. If a higher percentage of iR drop was corrected, the measurements were unstable. HER measurements were done using cyclic voltammetry at a sweep rate of 10 mV s −1 . Before the potential sweep, the electrode was held −1 V vs. SCE until current dropped below 1 μA cm −2 . No anodic excursions of the working electrode were allowed before the HER measurements unless explicitly stated otherwise.</p><!><p>The first set of measurements relates to smooth Ni-poly rotating disk electrodes (RDE). The Ni disk had a diameter of 3 mm and was inserted in a Teflon cylinder with a 10 mm diameter. The disk was polished to mirror-finish using alumina powder and then sonicated for 1 minute, washed in deionized water, diluted HCl, and again in deionized water. Then it was transferred to the electrolytic cell, and HER measurements were done. Ni/Rh displacement experiments were done by immersing Ni-poly RDE into 0.1 mol dm −3 RhCl 3 solution in 0.1 mol dm −3 HClO 4 for a given amount of time (for the measurements with Ni-poly RDE up to 45 s). After the exchange, the electrode was rinsed in deionized water, 1 mol dm −3 KOH solution, covered with the droplet of the same solution, and quickly transferred into the electrochemical cell. The transfer to the cell typically took under 30 s.</p><p>During the HER measurements, the electrode was rotated at 1800 rpm to remove any bubbles formed on the surface, which could block the electrode surface.</p><p>The second set of the exchange and HER measurements was done using commercial nickel foam (NF) and electrodeposited Ni on Ti mesh (Special Metals, India) (Ni-dep). Before HER and Ni/Rh displacement experiments, NF (5×5 mm was exposed to the electrolyte) was cleaned in HCl, acetone, and deionized water. Exchange with Rh was done in the same way as for the Ni-poly disk.</p><p>Then, HER measurements were done as described above. Ni-dep was produced on Ti mesh, previously cleaned in HCl, acetone, and deionized water. Ni deposition was done on Ti mesh with dimensions of 3×3 mm, from the deposition bath containing 0.2 mol dm −3 H 3 BO 3 , 0.5 mol dm −3 NH 4 Cl, and 0.125 mol dm −3 NiSO 4 . The deposition was done in a 2-electrode electrochemical cell, using Ni foam as a counter electrode, under potentiostatic conditions (2.4 V) for 90 s. Under these conditions, the typical deposition current is around 50 mA. Once the deposition was done, Ni-dep electrodes were thoroughly washed with deionized water and transferred to the electrochemical cell or Rh-exchange solution, the same one as described above.</p><p>We note that here we report currents normalized per geometric surface area. For the measurements with the Ni-poly disk, this is straightforward. For the measurements with NF, we used a geometrical cross-section multiplied by 2. The same is done for Ni-dep electrodes, but we excluded the surface of voids between the Ti wires forming the mesh. It is done because the used mesh is rather sparse, and using a finer mesh would give higher currents within the same geometrical area (3×3 mm).</p><p>As a benchmark, we used a platinized Pt-poly disk (3 mm in diameter), prepared by potentiodynamic cycling of smooth Pt-poly disk in H 2 PtCl 6 solution, as described in ref. [36]. Using H UPD peaks, the roughness factor (RF) of the used platinized Pt-poly disk was evaluated to 7.9. In addition, the activity of the used Pt-disk was also checked in acidic media (0.5 mol dm −3 H 2 SO 4 solution, Figure S1, Supplementary Information) using the same HER measurement protocol as in alkaline solution, confirming that a highly active Pt surface is obtained.</p><!><p>Morphology analysis and chemical composition were probed using SEM-EDX with Phenom ProX Scanning Electron Microscope (Phenom, Netherlands). SEM characterization was done using an acceleration voltage of 10 kV, while the chemical composition was probed with the acceleration voltage of 15 kV. XPS was used to assess the chemical speciation on the samples surfaces after the exchange with Rh. Samples were analyzed using SPECS Systems with XP50M X-ray source for Focus 500 and PHOIBOS 100/150 analyzer. AlKα source (1486.74 eV) at a 12.5 kV and 32 mA was used for this study. Survey spectra (0-1000 eV binding energy) were recorded with a constant pass energy of 40 eV, step size 0.5 eV and dwell time of 0.2s in the FAT mode. High-resolution spectra of Ni 2p and Rh 3d peaks were recorded with a constant pass energy of 20 eV, step size of 0.1 eV and dwell time of 2s in the FAT mode. Spectra were obtained at a pressure of 9×10 −9 mbar. SPECS FG15/40 electron flood gun was used for charge neutralization to minimize the effects of charging at the samples. All the peak positions were referenced to C1s at 284.8 eV. Spectra were collected by SpecsLab data analysis software supplied by the manufacturer and analyzed with a commercial CasaXPS software package.</p><!><p>The first-principle DFT calculations were performed using the Vienna ab initio simulation code (VASP) [37][38][39]. The Generalized Gradient Approximation (GGA) in the parametrization by Perdew, Burk and Ernzerhof [40] combined with the projector augmented wave (PAW) method was used [41]. Cut-off energy of 600 eV and Gaussian smearing with a width of σ = 0.025 eV for the occupation of the electronic levels were used. A Monkhorst-Pack Γ-centered 14×14×1 k-point mesh was used. We modelled Ni(001) and Ni(111) surfaces using the corresponding p(2×2) cells of given surfaces, with 10 and 9 layers slabs, respectively. Rh was inserted in the surface layer (Rh surf ) or the sub-surface layer (Rh sub ), and the top 6 layers of a given surface were allowed to relax fully. Knowing that on Ni HER proceeds under the conditions of high surface coverage [42], the surfaces were completely saturated by H ads (1 monolayer of hydrogen, 4 H ads per cell), and the average hydrogen binding energy (E H,b ) was calculated as:</p><p>where E SURF+H , E SURF , and 4E H stand for the total energy of the surface with a monolayer of hydrogen, the total energy of the clean surface and the total energy of an isolated hydrogen atom. To convert E H,b to the Gibbs free energy for H ads formation (ΔG H,ads ), the procedure of Nørskov et al. [43] was used (ΔG ads (H) = E H,b + 1/2E H2 + 0.24 eV, E H2the total energy of an isolated H 2 molecule).</p><!><p>The clean, polished Rh disk measurements showed rather poor HER activity with η 10 more negative than −0.4 V and the corresponding Tafel slope of −140 mV dec −1 (Figure 1). Even though the Ni-poly disk was polished to a mirror finish, the roughness factor was somewhat higher than 1. By plotting the currents measured at −0.85 vs. SCE as the function of the potential scan rate and using the value of specific capacitance of 20 μF cm −2 (for metallic surfaces [44,45]), the roughness factor was determined to be 2.8. According to the benchmarking by McCrory et al. [46], η 10 for the Ni surface with roughness factor 20±10 was approximately −0.25 V. For the exchange with Rh up to 20 s, we could not see any significant changes in recorded cyclic voltammograms (Figure S2, Supplementary Information), but obtained a significant increase of HER activity. However, for 30 s of exchange with Rh and more, cyclic voltammetry of Rh-exchanged Ni-poly disk shows H UPD regions characteristic for Rh [47] and practically unchanged regions corresponding to Ni 2+ /Ni 3+ oxidation (Figure 1). For 15, 30, and 45 s of exchange with Rh, η 10 amounted to −168, −120, and −110 mV, without significant change of the Tafel slope . These results align with the unchanged surface roughness, evidenced by 3D surface reconstruction using SEM, and a low percentage of Rh on the surface for short Rh-exchange times, with a very uniform distribution. These issues will be discussed later on. We have used platinized Pt-poly as a reference to Rh-modified Ni-poly surfaces. In ref. [46] η 10 for Pt was reported to amount (-100±20) mV for smooth Pt-poly surface (RF 6±2), and ~ −40 mV</p><p>for Pt-poly with RF of 90±20. Our Pt disk has η 10 of -110 mV, which closely agrees with the results of McCrory et al. [46]. Given that the RF values of Ni-poly do not change for more than 10% upon the exchange with Rh (Figures S2 and S3, Supplementary Information), and that the Rh-exchanged electrodes have measured HER current densities (normalized per geometric surface area) close to that of Pt (Figure 1), surface-specific activities of Rh-exchanged Ni-poly actually exceed the activities of Pt (Figure 2). A direct comparison with the results of Nguyen et al. [33], who investigated HER activity of NiRh 3 alloy nanosponges in alkaline media, can be made. With the catalyst loading of 169 μg cm −2 , the authors reported η 10 of −107 mV for NiRh 3 alloy and −119 mV for commercial Pt/C catalyst [33].</p><p>The previous improvements of HER activity were achieved using similar approaches, like spontaneous deposition of Pd and Rh on smooth Pt-poly (RF 2) [48], where Rh-modified Pt-poly showed higher HER activity than Pd-modified Pt-poly, without significant change of the roughness factor (2.1 for Rh-modified surface, and 2.08 for Pd-modified surface). Described catalytic surfaces should also be compared to pure Rh, and recently Rh films on Ni foam and Ti mesh, prepared by aerosol-assisted chemical vapor deposition (AACVD) technique, were investigated as HER catalysts in alkaline media [49]. It was found that Rh film on NF had η 10 of −127 mV, while the one prepared on Ni mesh had the corresponding overvoltage of −67 mV [49]. As there are many studies of HER catalysts in alkaline media, it would be possible to extend the comparison with literature data to a large number of reports. However, here we refer to the review of Bouzek et al. and Rh 3+ and metallic Rh (from Ni 3d high-resolution spectra) [33,50,51]. The results shown are for 10 s exchange with Rh, in which case neither electrochemistry nor SEM shows the formation of welldefined Rh islands, but it is obvious that there are certain parts of the surface containing Rh aggregates. However, after 30 s exchange with Rh, there are clear indications that Rh islands are formed, and the effect is more prominent for higher exchange times. For example, after 45 s of exchange with Rh, the characteristic H UPD region is clearly seen (Figure 1), which is the feature of the Rh phase [47]. This observation unambiguously confirms that longer exchange times lead to the formation of metallic Rh islands on the Ni-poly surface.</p><!><p>In order to rationalize enhanced HER activity of Rh-exchanged Ni surface, we turned to the analysis of H ads energetics on Rh-modified Ni surfaces. We studied Ni(111) and Ni(001) using DFT calculations, and to model modification by Rh, we added Rh in the surface or subsurface layer of these Ni(hkl), effectively modelling the surface of subsurface alloys. We find that surface incorporation of Rh is energetically more favored than subsurface inclusion of Rh on both Ni surfaces, in agreement with previous reports [52]. In the case of Ni(111), surface incorporation of Rh is more favored by −0.20 eV, while this difference for Ni(001) is −0.11 eV. Considering that H ads energetics was shown to give the volcano-type relationship with HER activities in alkaline media [53], just like in acidic media [54], we used the methodology of Nørskov et al. [43] to establish Tafel analysis performed on HER polarization curves of Rh-exchanged smooth Ni-poly</p><p>shows Tafel slopes that are close but usually larger than −120 mV dec −1 (absolute values). Deviation from the theoretical value of −120 mV dec −1 can be ascribed to incomplete correction for the iR drop (see Section 2.1) and the problems with removing H 2 bubbles (despite electrode rotation during the measurements). Thus, although there it is quite inconvincible to determine the mechanism of HER solely based on the Tafel slope, there is enough reliable literature data [42] to conclude that Heyrovski reaction is the rate-determining step (RDS) on Ni-poly surface, at least at higher HER overpotentials where we performed Tafel analysis. The same reference claims that, at lower HER overpotentials, the Tafel reaction is the RDS [42]. However, more recent findings suggest that surface modifications by Ni-oxy-hydroxides enhance HER kinetics through H 2 O dissociation where H ads ends up on the metal surface. At the same time, OH ads is governed by the oxy-hydroxide phase (and ultimately released back to the solution) [19,56]. In the original interpretation, this means that the rate of Volmer reaction is increased, allowing fast recombination of H ads and formation of molecular H 2 via the Tafel step. rate is increased, one can also expect that HER proceeds through with the Heyrovsky reaction as the RDS will be enhanced. Actually, the Ni surface binds H ads strongly, so there is no reason to expect that the Volmer reaction (formation of H ads ) will be RDS, in agreement with ref. [42]. Nevertheless, the HER activity significantly decreases if the Rh-exchanged surface is exposed to the same oxidation protocol (Figure 4). Rh surface binds H ads weaker than Ni surface, but, still, ΔG ads (H) is negative (Figure 4). However, the Rh|Ni interface conversion to Rh|Ni-oxy-hydroxide interface hinders HER.</p><p>As Rh-exchanged Ni surface binds H ads weaker than clean Ni surface, there is no reason to expect that Ni-oxy-hydroxide surface will bind OH ads weaker than on pure Ni surface, Brønsted−Evans−Polanyi relations for water dissociation [57] suggest lower H 2 O dissociation rate. Thus, slower Volmer and Heyrovsky reactions compared to pure Ni surface. However, on pure Ni-poly Tafel reaction is the RDS at low overpotentials due to strong binding of H ads , but this is not the case for Rh-exchanged surface, and the surface is effectively cleaned from H ads by fast recombination/desorption and the formation of molecular H 2 . However, we also observed an additional effect of surface oxidation.</p><!><p>While metallic Rh is extremely expensive [58], significant improvements of HER activity of Rh-exchanged Ni, surpassing the activity of Pt (Figure 3), suggest this approach could be effectively Considering tremendous HER activity improvement, this is a small cost compared to the energy savings achievable using described surface modification of Ni. Thus, we further explore the effects of Rh exchange on the HER activity of extended Ni surface.</p><p>First, we investigate the effect of Rh exchange on commercial Ni foam. The results show that the effects of time of exchange by Rh are not as pronounced as in the case of smooth Ni surface (Figure 6), and the most pronounced effects are seen after several minutes of exchange by Rh. For clean NF η 10 was found to be −0.26 V, and for 2 and 10 min exchange with Rh it reduces to −0.17, and −0.07 V. EDX analysis showed a monotonous increase of Rh surface concentration, without significant alteration of mesh morphology caused by dissolution in acidic Rh-containing solution (Figure 6). Nevertheless, for higher Rh exchange times, we clearly observed the formation of Rh phase on NF surface (Figure S4, Supplementary Information), which we consider mainly responsible for HER activity, as metallic Rh is much more active for HER than Ni. Also, we must note that it was impossible to perform electrode rotation to remove the H 2 bubbles formed on the surface in the case of NF. Thus the measured activity of Rh-exchanged Ni foam is slightly affected by H 2 bubbles blocking the surface. Still, the activities are better than those reported for pure Rh films prepared on NF using AACVD [49], while the modification procedure is greatly simplified. We also note that the improvements of the NF HER activity were previously reported for the case of spontaneous deposition of Ru and Pd [59]. However, the activities reported in that work are much lower than those reported here, not only for the modified NF but also for the clean one. For this reason, we also investigated long Rh exchange time, 5 minutes, and obtained a further improvement of HER activity. In this case, η 10 amounted to −0.09 V, and the Tafel slope was −122±5 mV dec −1 . However, after SEM and EDX analysis, it was observed that the surface is significantly eroded (Figure S7, Supplementary Information), while EDX showed ~20 at.% Rh, in line with clear H UPD peaks after 5 min of exchange with Rh (Figure 7). This finding is in line with recorded cyclic voltammograms of Ni-dep electrodes before and after Rh exchange, showing progressive loss of voltammetric response corresponding to Ni 2+ /Ni 3+ transition with the extension of Rh exchange time (Figure 7). Such erosion of Ni-dep is likely due to fast corrosion of a highly developed Ni surface, and it was not observed for NF or smooth Ni-poly. We ascribe this to the fact that Ni-poly has a low concentration of highly uncoordinated Ni sites, prone to corrosion, unlike Nidep. Moreover, the surface of NF is also rather smooth, much smoother than that of Ni-dep (Figure 6, insets, and Figure 7). We believe this is why no pronounced dissolution and erosion of NF is seen.</p><!><p>Fast galvanic exchange of Ni with Rh, using concentrated acidic Rh solution, leads to tremendous HER activity improvements in alkaline media. In the case of a smooth Ni-poly surface, HER activity surpasses Pt-poly for Rh exchange times of 30 seconds. Our results suggest that there is no significant change in the surface roughness of Rh-exchanged electrodes, and when surface-specific activities of Rh-exchanged smooth Ni-poly are compared to that of Pt-poly, the HER activity of the modified Ni-poly (30 s of exchange) is roughly 2.5 times higher than the activity of Pt-poly. DFT calculations suggest that Rh-modified Ni surface interacts weaker with H ads , making the formation of</p>

ChemRxiv

Peptide late-stage C(sp³)–H arylation by native asparagine assistance without exogenous directing groups

There is a strong demand for novel native peptide motifs for post-synthetic modifications of peptides without pre-installation and subsequent removal of directing groups. Herein, we report an efficient method for peptide late-stage C(sp 3 )-H arylations assisted by the unmodified side chain of asparagine (Asn) without any exogenous directing group. Thereby, site-selective arylations of C(sp 3 )-H bonds at the N-terminus of di-, tri-, and tetrapeptides have been achieved. Likewise, we have constructed a key building block for accessing agouti-related protein (AGRP) active loop analogues in a concise manner.

peptide_late-stage_c(sp³)–h_arylation_by_native_asparagine_assistance_without_exogenous_d

Introduction<!>Optimization of reaction conditions<!>Substrate scope<!>Mechanistic investigation<!>Conclusion<!>Conflicts of interest

<p>Peptides are increasingly important drug candidates, which are largely employed to treat metabolic disorders, cancer, allergy, and immune and cardiovascular diseases. 1 They also represent key tools that modulate biological events mediated by proteinprotein interactions (PPIs). 2 Native peptides usually suffer from poor pharmacological features due to lack of structural diversity or enzymatic degradation, 3 but chemically modied nonnatural peptides could feature higher binding affinities to the target, as well as improved pharmacokinetics, stability, and cell permeability. 4 The late-stage modication represents an effective strategy to obtain structurally diverse peptides and peptidomimetics. Thus, late-stage modication methods of peptides have been achieved in terms of arylations, 5 alkylations, 6 and cycloadditions. 7 Over the past few years, C-H activation has been recognized as an atom-and step-economical pathway towards molecular syntheses, 8 with remarkable applications in materials science, 9 the agrochemical industry, 10 and drug discovery, 11 among others. 12 To the best of our knowledge, studies on latestage functionalizations of peptides via C(sp 3 )-H activation have been scarcely reported. In this context, Yu 13 successfully implemented C(sp 3 )-H activation of peptides using native N,Oor N,N-bidentate coordination without external auxiliary (Fig. 1a). On a different note, Noisier/Albericio 14 reported the synthesis of a novel class of stapled peptides. Likewise, research studies of post-synthetic modication of peptides through C(sp 3 )-H activation by installing exogenous auxiliary assistance have been pursued. In 2017, Ackermann 15 developed a strategy of triazole (Tzl)-assisted C(sp 3 )-H arylations of peptides. In the same year, Chen 16 described 8-aminoquinoline (AQ)-directed C(sp 3 )-H arylation to generate cyclophane-braced peptide macrocycles. Recently, Shi 17 established a palladium-catalyzed site selective g-C(sp 3 )-H silylation and d-C(sp 3 )-H alkylation of amino acids and peptides utilizing picolinamide (PA) auxiliary (Fig. 1b). The installation and subsequent removal of DGs oen implies additional and non-trivial steps. Considering the atom-and step-economy of late-stage modication of peptides, we intended to utilize the natural amino acid embedded in the peptide backbone for chelation assistance. To our knowledge, C(sp 3 )-H functionalizations of peptides assisted by the unmodied side chain of a natural amino acid has not been accomplished thus far.</p><p>Asn is a natural amino acid with a side chain bearing a primary amide and could potentially be exploited as a directing group. This prompts us to survey whether the side chain and backbone of Asn could coordinate with palladium, leading to a bidentate coordinated palladium complex. Therefore, we introduce Asn as an internal bidentate DG to accomplish C(sp 3 )-H activation of peptides. Simultaneously, Asn is a common residue contained in many bioactive peptides, which display a range of biological activities, such as antioxidant activity, 18 blocking the neprilysin activity, 19 and inhibiting ACE activity. 20 Remarkably, Phe-Asn is an essential sequence that exists in some bioactive peptides, for example novel ACE inhibitory peptides, 21 anticancer peptides, 22 and AGRP. 23 Inspired by the signicant work by Ackermann et al., 15,24 we provide a useful strategy employing Asn as an internal directing group for C(sp 3 )-H functionalization of peptides. The unmod-ied side chain of Asn combined with the backbone was utilized as the N,N-bidentate coordination via 5,6-fused bicyclic palladacycles (Fig. 1c) to perform the late-stage peptide C(sp 3 )-H arylation.</p><p>The complex has facilitated the inert C(sp 3 )-H bond arylation in peptides. Thereby, arylated di-, tri-, and tetrapeptides containing Asn have been assembled. The salient features of our approach comprise (a) C(sp 3 )-H activation of peptides assisted by a natural amino acid which circumvent the preinstallation and removal of DGs; (b) the rst unmodied side chain of the natural amino acid as the endogenous auxiliary assistance applied in C(sp 3 )-H activation; and (c) discovery of native bidentate assistance through less-strained 5,6-fused bicyclic palladacycles. 25</p><!><p>To validate our hypothesis, we initiated our studies by exploring reaction conditions for the palladium(II)-catalyzed primary C(sp 3 )-H arylation of N-phthaloyl protected dipeptide 1 with 3-iodotoluene (Tables 1 and S1 in the ESI †). Initial optimization revealed DCE to be the best solvent of choice (Table S1, † entries 1-5), with KF being identied as the optimal additive (entries 1-3). By replacing Pd(OAc) 2 by PdCl 2 as the catalyst the yield of product 2a was excitingly increased to 67% when the amount of AgOAc and KF was increased to 2.5 equivalent (entry 4). Notably, the reaction failed to proceed using AgOTf as the additive (entry 5), while Cu(OAc) 2 gave a dramatically decreased yield (entry 6). Encouraged by the good efficiency of PdCl 2 , other palladium catalysts were further investigated. Gratifyingly, Pd(MeCN) 2 Cl 2 was found to slightly improve the yield of peptide 2a to 72% (entries 7-9). It is noteworthy that other metal catalysts, based on ruthenium, rhodium or cobalt, were ineffective (entries 10-12). The control experiment veried the essential role of the palladium catalyst (Table S1, † entry 20).</p><!><p>With the optimal reaction conditions in hand, the substrate scope of a range of aryl iodides was investigated, and the results are summarized in Scheme 1. Both substrates with electrondonating (Me-, MeO-, and t-Bu-) and electron-withdrawing (F-, Cl-, Br-, CF 3 -, and CO 2 Me-) groups reacted smoothly and afforded the desired products in good yields. Pleasingly, biphenyl and naphthyl moieties were also tolerated, leading to the corresponding products (2m and 2n) in 63% and 64% yields. The reaction performed with good chemo-selectivity.</p><p>Encouraged by the success of the arylation of dipeptides, we next investigated the feasibility of applying this approach to the arylation of tripeptides and tetrapeptides (Scheme 2). Using tripeptide 3a as the substrate, through minor adjustment of the reaction conditions (Table S2 in the ESI †), we were pleased to nd that the arylation of 3a with 1-iodo-4-methoxybenzene 4a could deliver the expected product 5aa in 61% isolated yield.</p><p>Then, the scope of substrates was evaluated under the optimized reaction conditions. Satisfyingly, a wide range of aliphatic amino acids, including Leu, Ala, Val, and Lys, at the Cterminus of the tripeptides are compatible with these conditions. In addition, aryl iodides bearing electron-donating as well as electron-withdrawing substituents were tolerated, affording products 5aa-5ej. Given the feasibility of the tripeptide arylation, we expanded the peptide substrates to structurally complex tetrapeptides. The arylation products of tetrapeptides 6fa-6gh could be obtained in moderate yields (50-58%). Phecontaining tetrapeptide 3h could also be arylated albeit with lower yields (6hg-6hk, 25-36%). While considerable progress has been made in 3 )-H activation, 26 our strategy enabled position-selective arylation of Ala assisted by N,N-bidentate coordination of the Asn in tri-and tetra-peptides.</p><p>To further demonstrate that the reaction coordination site is the primary amide of Asn, the control reaction and competition reaction were investigated under the standard conditions (Scheme 3). First, we removed the Asn side chain of dipeptide 1 and replaced it with a methyl group, while retaining the tertbutyl ester of the dipeptide. Therefore, N-phthaloyl protected dipeptide 7 was independently prepared, and subjected to the optimized reaction conditions. It failed to afford arylated products of arylation of C(sp 3 )-H bonds at the N-terminus (Scheme 3a). Since tripeptides or tetrapeptides both contain Asn bidentate and backbone amide bidentate, it is important to analyze the key role of Asn bidentate in promoting C(sp 3 )-H functionalization. For the competition experiment between tripeptides 3c and 8a, product ratio of approximately (5cg : 9a ¼ 6 : 1) (Scheme 3b) was obtained.</p><!><p>Additionally, we probed the catalyst mode of action by means of computational studies at the PW6B95-D4/def2-TZVP+SMD (DCE)//PBE0-D3BJ/def2-SVP level of theory (Fig. 2). 27 A detailed analysis between the C-H activation and reductive elimination elementary steps provided support for the C-H activation to be the rate-determining step with an activation energy of 19.6 kcal mol À1 , with oxidative addition being energetically more favorable by only 1 kcal mol À1 . An alternative pathway Scheme 1 Scope of ArI C(sp 3 )-H arylation of dipeptides. Reaction conditions: 1 (0.2 mmol), ArI (2.0 equiv.), Pd(MeCN) 2 Cl 2 (10 mol%), AgOAc (2.5 equiv.), KF (2.5 equiv.), DCE (2.0 mL), 130 C in air, 12 h, yields of isolated products.</p><p>Scheme 2 Scope of C(sp 3 )-H arylation of tripeptides and tetrapeptides. a Reaction conditions: 3 (0.2 mmol), ArI (2.5 equiv.), PdCl 2 (15 mol%), AgOAc (3.0 equiv.), KF (2.0 equiv.), DCE (3.0 mL), 110 C in air, 12 h, yields of isolated products. b AgOAc (2.5 equiv.). c KF (1.0 equiv.) where the NH 2 of the terminal amide is deprotonated was also taken into consideration (Fig. S1, see the ESI †). The latter was shown to be overall energetically disfavored, with reductive elimination as the rate-determining step with a high energy barrier of 30.9 kcal mol À1 . These studies provide strong support for the palladium-catalyzed C(sp 3 )-H arylation to occur through a Pd(II/IV) pathway where the NH of the internal, instead of the terminal amide is deprotonated.</p><p>Based on previous reports on palladium-catalyzed amidedirected C-H bond activation and computational studies, we propose a plausible catalytic cycle to be initiated by a facile organometallic C-H activation (Scheme 4). Initially, the palladium catalyst coordinates covalently with the deprotonated NH of the internal amide generating a bidentate coordinated palladium(II) complex A. Subsequently, complex A undergoes slow C(sp 3 )-H bond cleavage to form the 5,6-fused bicyclic palladium complex B. The oxidative addition of the aryl iodide to B affords palladium(IV) intermediate C, which then undergoes reductive elimination followed by protonation leading to the formation of the corresponding arylated product. The silver salt is proposed to accelerate the rate of the oxidative addition or the reductive elimination, while likewise acting as a halide scavenger. 8i,24a,28 Agouti-related protein (AGRP) is a potent orexigenic peptide that antagonizes the melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R). 29 This protein has been physiologically implicated in regulating food uptake, body weight control, and energy homeostasis. 30 In attempts to improve the antagonist activity and selectivity of AGRP active loop, previous studies have applied a substitution strategy to prepare AGRP active loop analogues. 31 The results have indicated that some substitutions of amino acid could increase potency of AGRP. However, the synthesis of AGRP loop analogues requires the introduction of modied unnatural amino acids. Some unnatural amino acids are expensive and difficult to synthesize, such as L-4,4 0 -biphenylalanine (Bip) and 3-(2-naphthyl)-L-alanine (Nal(2 0 )). Through C-H activation, the functional group could be installed directly into native peptides, such an approach is highly efficient, step-and atom-economical. Thus, we attempted to apply our strategy to synthesize new AGRP loop analogues. The arylation products 6 through deprotection of phthaloyl (Phth) gave NH 2 -free tetrapeptides 10 (details see the ESI †). Tetrapeptides 10a and 10b subsequently were coupled with Cbz-DPro-Pro-Arg(Pbf)-Phe-OH to obtain linear octapeptides, which were cyclized to access AGRP loop analogues. AGRP loop analogues 11a and 11b were obtained through this strategy (see the ESI † synthesis of AGRP loop analogues); the introduction of a bromide atom in 11b potentially enables further latestage derivatization of this peptide (Scheme 5).</p><!><p>In conclusion, we have developed an efficient strategy for palladium(II/IV)-catalyzed late-stage C(sp 3 )-H arylations of peptides using unprecedented internal Asn. The protocol avoids the additional requirement for installation and removal of exogenous directing groups. Importantly, our approach has provided a novel synthetic route to access the key building block for the synthesis of AGRP loop analogues.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Highly sensitive interleukin 6 detection by employing commercially ready liposomes in an LFA format

Recent years have confirmed the ubiquitous applicability of lateral flow assays (LFA) in point-of-care testing (POCT). To make this technology available for low abundance analytes, strategies towards lower limits of detections (LOD), while maintaining the LFA’s ease of use, are still being sought. Here, we demonstrate how liposomes can significantly improve the LOD of traditional gold nanoparticle (AuNP)–based assays while fully supporting a ready-to-use system for commercial application. We fine-tuned liposomes towards photometric and fluorescence performance on the synthesis level and applied them in an established interleukin 6 (IL-6) immunoassay normally using commercial AuNP labels. IL-6’s low abundance (< 10 pg mL−1) and increasing relevance as prognostic marker for infections make it an ideal model analyte. It was found that liposomes with a high encapsulant load (150 mmol L−1 sulforhodamine B (SRB)) easily outperform AuNPs in photometric LFAs. Specifically, liposomes with 350 nm in diameter yield a lower LOD even in complex matrices such as human serum below the clinically relevant range (7 pg mL−1) beating AuNP by over an order of magnitude (81 pg mL−1). When dehydrated on the strip, liposomes maintained their signal performance for over a year even when stored at ambient temperature and indicate extraordinary stability of up to 8 years when stored as liquid. Whereas no LOD improvement was obtained by exploiting the liposomes’ fluorescence, an extraordinary gain in signal intensity was achieved upon lysis which is a promising feature for high-resolution and low-cost detection devices. Minimizing the procedural steps by inherently fluorescent liposomes, however, is not feasible. Finally, liposomes are ready for commercial applications as they are easy to mass-produce and can simply be substituted for the ubiquitously used AuNPs in the POCT market.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03750-5.

highly_sensitive_interleukin_6_detection_by_employing_commercially_ready_liposomes_in_an_lfa_format

<!>Introduction<!>Chemicals and consumables<!>Synthesis of sulforhodamine B liposomes<!>Dynamic light scattering (DLS) and ζ-potential measurements<!>Phospholipid concentration<!>Protein coupling to liposomes<!>Lateral flow assay procedure<!>Apparatus<!>Development of photometric and fluorescent liposomes<!><!>Optimization of photometric liposome detection<!>Photometric and fluorescence lateral flow immunoassay<!><!>Stability study and liposome dehydration<!>Conclusion<!>

<p>Open Access funding enabled and organized by Projekt DEAL.</p><!><p>Not only the global pandemic in 2020 emphasized the need and relevance of simple, fast, sensitive, and one-site point-of-care (POC) solution in the medical diagnostic field, but also a market size of USD 29 billion in 2020 (which is estimated to rise to USD 67 billion in 2026, www.reportsanddata.com) [1] depicts its global interest. In this fast-growing market, lateral flow assays (LFA) belong to the major gameplayers, as they are typically very fast and offer low costs and straightforward operation even by non-experts [2]. Aside from the numerous benefits of LFAs, such as amenability to inexpensive mass production, autonomy of additional external equipment, and optical readout, in its simplest form with the human eye, standard LFA platforms still have to face limitations with regard to sensitivity due to their mostly semi-quantitative nature and often provide users only with yes/no answers. A lateral flow assay is typically conducted in either a competitive or sandwich assay format [3]. Here, the test strip consists of a test line typically utilizing a biomolecule directed against the analyte as a capture probe and a control line which is directed against the reporter particle. The sample is added to the sample pad and resolubilizes the reporter particles such as gold nanoparticles (AuNP) or colored latex beads from the subsequent conjugate pad along its capillary force–driven flow throughout the test strip [4]. Depending on the applied format, the analyte-reporter complex binds to the test line yielding an increasing signal with increasing analyte concentration in a sandwich assay format. The test strip consists of various porous materials with each having its unique feature, i.e., assisting in sample transport, containment, and homogenous release of reagents, ensuring homogenous fluid flow, and capturing relevant biomolecules through capture probes via precise test line manufacturing. This general design of the LFA allows vast room for sensitivity enhancement at several stages of the LFA development. Bishop and colleagues [4] critically assessed the adjusting screws which have recently been studied to push the development and potential of lateral flow assays towards sensitivity levels similar to those of laboratory-based test systems. Especially, the applied reagents and envisioned reactions and their realization on an LFA were thoroughly discussed. Key factors for enhancing the sensitivity of an LFA by several orders of magnitude are the development of high-affinity reagents, tweak of transport dynamics for ideal reaction kinetics as well as label and detection optimization of conventional reporter particles, or even integration of signal amplification strategies [4]. Ultimately, investigating a combination of these individual strategies is of special interest. In this study, we focused on the reporter particles and signal amplification strategies by exchanging conventional AuNP with fluorescent dye–loaded liposomes. Liposomes are mainly known as delivery vehicles in medicine and pharmacology but paved their way into analytical and bioanalytical applications as detection particles due to their comparably high surface area, a large internal volume, and flexible surface modification with various biorecognition elements [5, 6]. IL-6 is an important biomarker for immune response and inflammatory processes in the human body. It belongs to the class of pro-inflammatory cytokines currently under evaluation inter alia as a potential biomarker to identify COVID-19 positive patients who are at risk of respiratory failure and death due to severe inflammatory response [7, 8]. Its growing diagnostic relevance as a prognostic marker for infections, especially due to COVID-19, and its presence in low pg mL−1 concentrations in serum [9] (in healthy subjects < 10 pg mL−1) make it an ideal candidate for this study. We herein study sulforhodamine B (SRB)–loaded liposomes with different sizes in a sandwich-based LFA for the detection of interleukin 6 (IL-6) in direct comparison to conventional AuNP. These liposomes were synthesized entrapping SRB, a highly water-soluble fluorescent dye allowing encapsulation of e.g. up to 1.2 million molecules of a 150 mmol L−1 dye solution in a single 300 nm liposome [6] enabling visual and fluorescence readout possibilities when applied to an LFA [10]. The accompanied signal amplification and their double readout feature rise interest in these liposomes for ultrasensitive detection in lateral flow assays. Although these particles have been applied to lateral flow assays previously [10–14], these publications only exploit the colorimetric readout possibility of liposomes focusing on the academic point of view, as the fluorescence of SRB is quenched in intact liposomes. The flexible nature of liposomes with regard to encapsulated dye, size, and surface modification gives additional advantages over standard AuNP such as e.g. multiplexing. We herein demonstrate the evolution of these liposomes to commercially ready detection particles and the gain in sensitivity by applying liposomes with optimized size in direct comparison to a commercial standard AuNP approach and designed for colorimetric and fluorescence readout. Sensitivity improvement by one order of magnitude was already obtained for the colorimetric readout with 350nm-sized liposomes whereas fluorescence measurement can significantly enhance the resolution due to an extraordinary gain in signal intensity. In addition, we show that our protein-modified liposomes remain highly stable for long-term storage in solution and also when dehydrated to the conjugate pad for a ready-to-use LFA. This renders the here demonstrated IL-6 liposome–based LFAs as a model system for any AuNP-based LFA that requires significantly lower LODs to become relevant as POCTs.</p><!><p>All chemicals were commercial analytical reagent grade and were used without purification. Phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (sodium salt) (N-glutaryl-DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt) (biotinyl-DPPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA); 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), purity > 99.5%, was purchased from VWR chemicals (Germany). Sulforhodamine B (SRB) (230162, 75%) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich/Merck (Germany). Fetal bovine serum (FBS) (10270–106) was purchased from ThermoFisher Scientific (Germany), and human serum (HS) was provided by Microcoat Biotechnology GmbH (Bernried, Germany). 2-(N-morpholino)ethanesulfonic acid (MES) and bovine serum albumin (BSA) (T844.2) were obtained from Carl Roth (Karlsruhe, Germany). Custom-made lateral flow test strips as well as anti-digoxigenin and anti-IL-6 conjugates were kindly provided by Microcoat Biotechnology GmbH (Bernried, Germany) as well as recombinant human IL-6 (200–06, Peprotech, Germany), sheep anti-digoxigenin Fab (11214667001 (Roche) Sigma-Aldrich, Germany), LFA running buffer (Art. No. 850003) and LFA 5 X serum buffer (ESS-2913). For all experiments, ultrapure water was used. A more detailed list of standard chemicals and consumable is given in the SI.</p><!><p>Liposomes, containing sulforhodamine B, with 6 mol% carboxy functionalization, were prepared according to an established protocol from Edwards et al. [15] with slight adjustments. Shortly, encapsulant was prepared by dissolving sulforhodamine B (150 mmol L−1) in 4.5 mL 0.02 mmol L−1 HEPES buffer, pH 7.5. DPPC (29.58 mg, 40.3 µmol), DPPG (15.64 mg, 21.0 µmol), cholesterol (19.99 mg, 51.7 µmol) and N-glutaryl-DPPE (6.2 mg, 7.0 µmol) were dissolved in 3 mL chloroform and 0.5 mL methanol and thoroughly sonicated in an ultrasonic bath (VWR ultrasonic cleaner, model USC 300 THD) at 60 °C. Subsequently, 2 mL of preheated (60 °C) encapsulant was added to the lipid solution and emulsified for 4 min at 60 °C, using an ultrasonic bath. After emulsification, the residual solvent was evaporated at 60 °C under reduced pressure. The remaining 2 mL of encapsulant was added after gradual evaporation to 780 mbar and thoroughly vortexed before evaporation was continued to 400 mbar. The remaining solution was extruded at 60 °C successively through 1.0 µm, 0.4 µm, and 0.2 µm membrane using a mini extruder (Avanti Polar Lipids, Inc.) to obtain unilamellar liposomes. Purification was first performed by size-exclusion chromatography with Sephadex® G-50 as stationary phase (column size: 2 cm × 8 cm) and HSS buffer (10 mmol L−1 HEPES, 200 mmol L−1 sodium chloride, 200 mmol L−1 sucrose, 0.01 wt% sodium azide), pH 7.5, osmolality 0.643 osmol kg−1 as mobile phase. Additionally, the liposomes were dialyzed against HSS buffer until the dialysis buffer remains colorless, pH 7.5, osmolality 0.643 osmol kg−1 before the determination of the hydrodynamic diameter via DLS, phospholipid concentration via ICP-OES, and zeta potential was done.</p><!><p>Dynamic light scattering (DLS) and ζ-potential measurements were done on a Malvern Zetasizer Nano-ZS (Malvern Panalytical, Germany). For all measurements, the temperature was set to 25 °C. Size determination was done in semi-micropolymethyl methacrylate (PMMA) cuvettes (Brand, Germany), and ζ-potential was done in disposable folded capillary zeta cells (Malvern Panalytical, Germany). The liposomes were diluted at 1:100 and measured in HSS buffer with the following settings: refractive index (RI) of the material of 1.34, material absorbance of zero, and RI of 1.342 of the dispersant viscosity of 1.1185 mPa s applied for DLS. For ζ-potential, a refractive index of 1.342, a viscosity of 1.1185 mPa s, and a dielectric constant of 78.5 were used. An equilibration time of 60 s was applied before each measurement.</p><!><p>Phospholipid concentration was determined through inductively coupled plasma optical emission spectrometer (ICP-OES) measurements with a SPECTROBLUE TI/EOP (SPECTRO Analytical Instruments GmbH, Kleve, Germany). Phosphorous was detected at 177.495 nm and the device calibrated between 0 and 100 µmol L−1 phosphorous in 0.5 mol L−1 HNO3. Before each measurement, the device was recalibrated with 0.5 mol L−1 HNO3 and 50 µmol L−1 phosphorous. A 1:150 dilution of the liposomes (3 mL) in 0.5 mol L−1 HNO3 was measured. ICP-OES measurements yielded a phospholipid concentration of 6.7 ± 0.04 mmol L−1.</p><!><p>The N-glutaryl modified liposomes were mixed with EDC (10 mg mL−1 in 0.05 M MES buffer, pH 5.5) and NHS (10 mg mL−1 in 0.05 M MES buffer, pH 5.5) and incubated for 1 h at room temperature (RT) while shaking. The respective equivalent of protein (1 mg mL−1 in PBS) was added and incubated for 1.5 h at RT while shaking. A ratio of 1:17:42:0.017 (n(COOH):n(EDC):n(NHS):n(protein)) was applied for antibody coupling. For streptavidin, a ratio of 1:100:180:0.23 was used. Lysine-HCl (1 mol L−1 in ultrapure water) was added to yield a final concentration of 10 mmol L−1 and again incubated for at least 10 min at RT while shaking to quench the reaction. The conjugated liposomes were purified via size exclusion chromatography using Sepharose CL-4B as stationary phase and HSS buffer (10 mmol L−1 HEPES, 200 mmol L−1 sodium chloride, 200 mmol L−1 sucrose, 0.01 wt% sodium azide), pH 7.5 as mobile phase, and a flow rate of approximately 0.5 mL min−1. The conjugates were subsequently characterized by optical density, hydrodynamic diameter, and zeta potential measurements. Optical density was measured at 565 nm of a 1:100 dilution in demineralized water. DLS and zeta potential measurements were done on a Malvern Zetasizer Nano-ZS (Malvern Panalytical, Germany). For all measurements, the temperature was set to 25 °C. The liposomes were diluted 1:500 in demineralized water and measured with the following settings: refractive index (RI) of the material of 1.45, material absorbance of 0.001, and RI of 1.330 of the dispersant viscosity of 0.8872 mPa s applied for DLS. For ζ-potential, a refractive index of 1.330, a viscosity of 0.8872 mPa s, and a dielectric constant of 78.5 were used. An equilibration time of 60 s was applied before each measurement.</p><!><p>The LFA with all reagents in solution was performed according to the following procedure if not stated differently. Thirty-six microliters of the respective dilution of recombinant IL-6 in serum and 9 µL serum buffer were mixed with 5 µL detection solution consisting of detection particles (colloidal gold and 350 nm liposomes (30 mOD per test), 190 nm liposomes (40 mOD per test)) and anti-IL-6 IgG-biotin (50 ng per test) and incubated 5 min at RT in a 2 mL reaction vessel prior to application on the test strip. After 15 min, the test strip was evaluated with a ESEQuant LFR strip reader. The test strip consists of a transparent backed CN150 (colloidal gold and 190 nm liposomes) or CN95 (350 nm liposomes) nitrocellulose membrane (Sartorius, Göttingen, Germany) with a streptavidin (SA) test line (27 mm) and an anti-mouse IgG control line (36 mm). The LFA with the reagent solution applied on the test strip was done by adding the reagent solution (5 µL detection particles (colloidal gold and 350 nm liposomes (30 mOD per test), 190 nm liposomes (40 mOD per test)) and anti-IL-6 IgG-biotin (50 ng per test) to the overlap of sample pad and conjugate pad and placed directly into 45 µL IL-6 dilution including 9 µL serum buffer. The LFA was allowed to run for 15 min and was evaluated photometrically directly after the test run or allowed to dry before lysis with 2 µL absolute ethanol for fluorescence evaluation. Photometric detection was done directly after a test run λmax = 520 nm; fluorescence was measured before and after lysis of the dried strip with 2 µL absolute ethanol with λex = 470 nm, λem = 600 nm if not stated otherwise.</p><!><p>Fluorescence measurements were performed with a Synergy Neo 2 microplate reader from BioTek (Bad Friedrichshall, Germany) for fluorescence measurements of liposomes or with the ESEQuant LFR strip reader from Qiagen (Hilden, Germany) for photometric and fluorescence measurements of the test strips.</p><!><p>In view of a commercial application of liposomes in lateral flow assays, we studied modifications of previously reported SRB liposomes [10] on the synthesis level towards long-term stability in solution and dehydrated on a test strip. We designed them for excellent photometric and fluorescent performance. In theory, the larger the liposome, the more encapsulant is present and hence can contribute to signal recording. Also, the small size distribution of a liposome population enhances their colloidal stability during long-term storage. However, the synthesis of large unilamellar liposomes with small size distribution is difficult [16] and not that applicable to the POCT as it typically involves additional procedural steps and can become quite time-consuming which rapidly increase the cost. We therefore chose the reverse-phase evaporation method that is known for high encapsulation yields followed by size extrusion to quickly generate differently sized liposomes in a very simple, mass-producible manner. Furthermore, by varying the encapsulant concentration, we tailored the liposomes towards photometric (high SRB concentrations) or inherent fluorescent (low SRB concentrations that do not self-quench) detection strategies. A mandatory design feature is the creation of single-step LFA procedures to maintain the dramatic advantage LFAs have over other POCT systems.</p><!><p>Characteristics of small sulforhodamine B liposomes (extruded through 0.2 µm membrane)</p><p>aSize by intensity of a 1:100 dilution; bIintact was obtained by diluting liposomes to 100 µmol L−1 total lipid in HSS buffer (100 µL) and Ilysed by diluting the liposomes in 30 mmol L−1 n-octyl-β-D-glycopyranoside in HSS buffer; cIintact = Iintact/Ilysed × 100, data are presented as mean ± SD with n = 3</p><p>Overview of fluorescence intensities of large and small sulforhodamine B liposomes</p><p>a350 nm liposomes; bIintact was obtained by diluting liposomes to 100 µmol L−1 total lipid in HSS buffer (100 µL) and Ilysed by diluting the liposomes in 30 mmol L−1 n-octyl-β-D-glycopyranoside in HSS buffer; cIintact = Iintact/Ilysed × 100; d190 nm liposomes, data are presented as mean ± SD with n = 3</p><p>Fluorescence performance of large and small liposomes with varying encapsulation concentrations. a Fluorescence performance of intact and lysed large and small liposomes in solution of 100 µL liposome dilution (c (total lipid) = 100 µmol L−1) in HSS buffer (intact) or 30 mmol L−1 n-octyl-β-D-glucopyranoside in HSS buffer (lysed) and b fluorescence performance of intact small 0.1 and 1 mmol L−1 SRB liposomes in an IL-6 LFA, data are presented as mean ± SD (error bar) with n = 3; fluorescence signal was recorded with λex = 550 nm, λem = 600 nm</p><!><p>To fully harness the liposome signaling capability for the photometric approach, they were maximally loaded with SRB by increasing their size and thus the inner volume to increase their overall sensitivity. Furthermore, we were seeking to generate liposomes with small size variation to increase their colloidal stability during long-term storage. Liposomes can be synthesized using the reverse-phase evaporation method and extruded to a desired size range through various sized membranes and extrusion steps (see Table S1 and Fig. S1) which is further supported by Szoka and colleagues [17]. Based on this information, we developed large liposomes in the range of 350 nm (PdI: 0.18) (Table S2) by extrusion through only the 1 µm membrane and small liposomes in the range of 190 nm (PdI: 0.07) (Table 1) by extrusion through 0.4 and 0.2 µm membranes with varying SRB encapsulation concentrations. These liposomes showed in initial characterizations that the larger the liposome and the higher the SRB concentration is, the more SRB is encapsulated within the liposome, as evidenced by the fluorescent measurement of lysed liposomes (Ilysed) (Table 2). As these small and large 150 mmol L−1 SRB liposomes yielded the strongest fluorescence signal and are easy to manufacture, these two types of liposomes were chosen to evaluate their tolerance towards antibody coupling and dehydration and determine their overall performance in a regular LFA. Even larger liposomes could be investigated in the future; however, based on prior experiences (data not shown), it is assumed that more steric hindrance and less colloidal stability could hamper large liposomes.</p><!><p>Previously optimized conditions for coupling streptavidin to liposomes were not directly transferrable to IgG coupling [15]. Thus, we used the relatively inexpensive anti-digoxigenin IgG (< Dig >) to identify the ideal coupling ratio of 1:17:42:0.17 (n(COOH):n(EDC):n(NHS):n(antibody)) and obtained already in this experiment a 4-times steeper slope and an order of magnitude lower detection limit of 1 ng mL−1 vs.10 ng mL−1 with our liposome approach in contrast to commercial AuNPs (Online Resource 1 – ESM 2.3, Fig. S2).</p><p>These coupling conditions were subsequently used for the covalent attachment of anti-interleukin 6 (< IL-6 >) to the liposomes using a coupling ratio of 1:17:42:0.017 (n(COOH):n(EDC):n(NHS): n(antibody)) for anti-interleukin 6 (< IL-6 >). Two different antibodies were tested, where one antibody showed an over 10-times higher sensitivity with over 20-times stronger signals especially for low concentrations (data not shown) which was consequently used in the following experiments.</p><!><p>Illustration of applied analysis principle of developed interleukin 6 lateral flow assay</p><p>Titration of IL-6 with large and small liposome conjugates benchmarked to colloidal gold. a Photometric detection in human serum and b fluorescence detection of liposomes after lysis in human serum benchmarked to colloidal gold (photometric detection). In a, preincubation of liposomes (5 min) with IL-6 and anti-IL-6-biotin IgG in running solution; in b, liposomes on conjugate pad without preincubation. Photometric measurement was done at λmax = 520 nm; fluorescence signal was recorded with λex = 470 nm, λem = 600 nm; data are presented as mean ± SD (error bar) with n = 3; four-parameter logistic fitting with Origin2020 was done within a R2 = 0.9940 (red), R2 = 0.9687 (blue), and R2 = 0.9557 (black) and in b R2 = 0.9493 (red) and R2 = 0.9583 (blue), yLOD = A1 + 3 SDblank</p><p>Recently published techniques for sensitive detection of interleukin 6 with immuno-LFAs</p><p>Fluorescence</p><p>(Eu-NPa)</p><p>70 µL sample</p><p>LFA run 15 min, commercial strip reader</p><p>Fluorescence</p><p>Quantum dots (QD)</p><p>LFA run 20 min</p><p>100 µL sample, multiplex, protype detector with UV-LED</p><p>Photon-upconverion</p><p>(UCP)c</p><p>SERSd</p><p>Au/Au core satellite nanoparticelse</p><p>Fluorescence</p><p>(fluorescent microspheresf)</p><p>7.15 pg mL−1</p><p>48.5 pg mL−1</p><p>Fluorescence</p><p>(Near-infrared dyeg)</p><p>Fluorescence</p><p>Quantum dots (QD)</p><p>Photometry</p><p>(commercial colloidal gold)</p><p>0.025 ng mL−1 (buffer)</p><p>0.081 ng mL−1 (HS)</p><p>36 µL sample</p><p>LFA run 15 min, commercial strip reader</p><p>Photometry</p><p>(dye-loaded liposomes)</p><p>1 pg mL−1 (buffer)</p><p>7 pg mL−1 (HS)</p><p>36 µL sample</p><p>LFA run 15 min, commercial strip reader</p><p>aEuropium(III) chelate–doped nanoparticles; bmolecular weight of 21 kDa for IL-6 was presumed; cup-converting phosphor nanoparticles; dsurface enhanced Raman scattering: ecore functionalized with Raman-active 4-nitrothiophenol for IL-6 or thio-2-naphthol for IL-8; fFluoSpheres®; fluorophore-doped particles (200 nm); gIRDye 800CW (Li-Cor Biosciences)</p><p>Performance test of universal streptavidin-modified liposomes towards direct-coupled small liposomes with anti-interleukin 6 IgG (< IL-6 >), four-parameter logistic fit with R2 = 0.9970 (black) and R2 = 0.9993 (red). Fifty microliters of a mixture of IL-6 and liposomes (40 mOD per test) in running solution were applied to the test strip (< IL-6 > test line), test run for 15 min. Streptavidin-liposomes were mixed with anti-IL-6-biotin (equaling 0.2 µg anti-IL-6-biotin per test) and IL-6; photometric measurement was done at λmax = 520 nm; data are presented as mean ± SD (error bar) with n = 3, yLOD = A1 + 3 × SDblank; slope derived from four-parameter logistic fit function</p><p>Long-term stability of small streptavidin-liposomes in solution a or dehydrated on a test strip b before test run on LFA strips with biotin test line; red line indicates initial response at time point zero. Liposomes were diluted to 25 mOD per test in 90 µL a, test run for 5 min; in b, liposomes with 25 mOD per test were dehydrated on test strip and rehydrated by 50 µL running buffer, test run for 15 min. Photometric measurement was done at λmax = 520 nm; data are presented as mean ± SD (error bar) with n = 5; times marked with an asterisk equals triplicates, reference line indicates initial response</p><p>Evaluation of different conjugate pad materials for small liposome conjugates a obtained signal intensities and b real images. Test strips were prepared with 5 µL liposome dilution in conjugate pad buffer (80 mOD per test); test run for 5 min in 100 µL running buffer; photometric measurement was done at λmax = 520 nm; data are presented as mean ± SD (error bar) with n ≥ 2</p><!><p>Harrigan and colleagues [27] stated that the stability of vesicles critically depends on the vesicle size with the result that smaller systems are most stable. We made similar observations, as currently only the small liposomes straightforwardly tolerate the dehydration process and were hence used for the long-term stability study. Dehydration of the large liposomes is currently under investigation as present data indicates that the liposomes are not fully destroyed (data not shown). Optimization of the drying conditions most likely provides the desired remedy, where we will apply protecting sugars as suggested previously by Martorell and colleagues [28]. They observed decreased recoveries for the larger liposomes as well but to a lesser extent (approx. 60% in contrast to 75% recovery with small liposomes). The results are not directly comparable as support material and size determination vary but it points to prosper when refining the dehydration conditions for our large liposomes.</p><!><p>The established commercial LFA system with colloidal gold for the detection of IL-6 shows already good sensitivity with 0.025 ng mL−1. However, by replacing the gold nanoparticles with refined dye–loaded liposomes, we were able to improve the sensitivity by over one order of magnitude to just 1 pg mL−1 with simple photometric detection. Furthermore, utmost care was taken to ensure that the liposomes are easily mass-producible, could be dehydrated on the LFA membrane itself, and hence could be applied in the same, straightforward, simple, and easy-to-use LFA strategy that is so desirable. Further improvement of the LOD through fluorescent detection approaches, however, is not as easy to accomplish. Inherently, fluorescent liposomes do not provide enough signal intensity and those that require an additional process step, as the dye has to be released from the liposome prior to detection, unfortunately compensate any gained signal intensity at the LOD by higher background signals and less reproducibility due to the additional assay steps. Thus, while in a refined environment such as a microtiter plate, improved LODs can be obtained through fluorescence detection of these liposomes; this is not as easily translated to a robust, commercially ready LFA approach. More experiments are needed to lower background signals, improve analyte-liposome interactions, and hence lower the LOD effectively. However, already now the increased signal intensity afforded by the fluorescent liposomes will assist in the development of less sophisticated detection devices. Expanding on the applicability of these new reporter probes, our universal liposomes, which maintain sensitivity levels of directly conjugated liposomes, show remarkable long-term stability when stored in solution and dehydrated on a test strip of at least 1 year. These adaptable liposomes can easily be transferred to any other analyte of interest manifesting them as a true alternative to standard colloidal gold. With this, a highly flexible and supersensitive toolset is provided for tailored assay development. Furthermore, in light of the importance of IL-6 detection with infectious diseases such as COVID-19, the here presented liposome-based LFA indicates that liposomes will rival the prevalence of colloidal gold as a benchmark in LFA analysis.</p><!><p>Supplementary file1 (DOCX 1162 KB)</p><p>Published in the topical collection Point-of-Care Testing with guest editors Oliver Hayden, Peter B. Luppa, and Junhong Min.</p><p>Publisher's Note</p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p><p>Change history</p><p>3/13/2022</p><p>The original version of this paper was updated to add the missing compact agreement Open Access funding note.</p>

PubMed Open Access

Ions increase strength of hydrogen bond in water

Knowledge of water-water potential is important for an accurate description of water. Potential between two molecules depends upon the distance, relative orientation of each molecule and local environment. In simulation, water-water hydrogen bonds are handled by point-charge water potentials and by polarizable models. These models produce good results for bulk water being parameterized for such environment. Water around surfaces and in channels, however is different from bulk water. Using quantum-mechanical methods, hydrogen bond strength was calculated in the vicinity of different monoions. A simple empirical relationship was discovered between the maximum hydrogen bond and the electric field produced by ion.

ions_increase_strength_of_hydrogen_bond_in_water

I. INTRODUCTION<!>II. METHODS AND DISCUSSION<!>III. CONCLUSION

<p>Water is one of the most important and one of the most abundant compounds on Earth. It played a key role in the development of early life and sustaining all known life forms today. It is also one of the most important compounds in the technological processes and industry. Water has some properties that distinguish it from simpler liquids.[1–5] Some of the most important anomalies of pure water are the following: maximum density at 4 °C in the liquid phase, negative coefficient of thermal expansion below that temperature, high surface tension and viscosity, a minimum in the isothermal compressibility, and a large heat capacity. Used as a solvent, water also has many unusual properties: for nonpolar solutes, a large entropy opposes solvation at room temperature, and a large heat capacity of transfer of apolar solvent into water. Water anomalies are related to the ability of water molecules to form tetrahedrally coordinated hydrogen bonds. To understand the behavior and properties of water and aqueous solutions is therefore crucial to understand hydrogen bonding and its molecular background. A crucial step toward understanding water is therefore a knowledge of water-water potential.</p><p>One can use quantum-mechanical theories to calculate exact properties of water, but the available computers can not handle systems consisting of more than a few molecules. As a consequence, there has been a persistent need to develop various simplified water models that seek a compromise between the accuracy and computational cost. If one wants to run molecular dynamics or Monte Carlo simulations with such models, an intermolecular potential is required as input. Usually simplified point-charge water models like SPC[6], TIP3P[7], TIP4P[8], TIP4P-Ew[9], TIP5P[10] are used. These models have point charges on or near the oxygen and hydrogens, and van der Waals terms. They are parameterized in a such way as to achieve the best agreement of an arbitrarily chosen bulk property with the experimental data. These parameters do not depend on local environment, meaning that they have the same values even if water local environment is far from bulk. There are also some polarizable models like PSPC[11], but are more complex and time-consuming to use in simulations. In recent work by Znamenskiy and Green [18] and our work[19], it was shown that the strength of hydrogen bond between two water molecules depends on the number of water molecules bonded to both water molecules and those on the second coordination shell. The local environment effect on HB strength is not negligible. Similar findings were also reported in other recent theoretical developments, [12–14] which recognize the extended nature of the hydrogen bond and explicitly include three-body interactions. These three-body effects are the same as cooperative effects of hydrogen bonding in water clusters [15]. When two water molecules form a hydrogen bond, the redistribution of charge happens on both of them. The bond formation changes the feasibility of further hydrogen bonding in comparison to an individual water molecule. The water molecule that donates the hydrogen atom in HB has increased electron density on oxygen and this encourages hydrogen bond acceptance. The water molecule that accepts the hydrogen has reduced electron density on hydrogen atoms, which is beneficial for further donating of hydrogen atoms[16]. These electron density changes are the reason for the so-called cooperativity or anticooperativity in hydrogen bond formation in water networks. Further details and references about cooperativity can be found in a review paper by de Olivera[17].</p><p>The goal of this contribution is to show the extensive nature of the hydrogen bond, spreading its influence beyond the hydrogen-bonded pair due to long-range, charge-density shifts accompanying formation of the bond. This was done with calculations of energies of clusters consisting of water molecule pair and different monovalent cations and anions, using post Hartee-Fock calculations.</p><!><p>Calculations were performed using the Gaussian 09[20] program suite, employing MP2(full) method[21] with def2-QZVP basis sets[22, 23]. Full geometry optimization of the water molecule with MP2(full) method for this basis set yielded the OH bond length 0.9562Å and the HOH angle 104.42° which is very close to the experimental data (OH bond length 0.9572 Å, HOH angle 104.52°)[24] for an isolated water molecule in a vacuum. Water-water potential for two molecules in a vacuum was also calculated with this method and basis set. Electronic energies are reported as the difference between the energy of dimer and the energy of two isolated water molecules at the infinite distance. Calculated maximum interaction is -5.22 kcal mol−1 and the oxygen-oxygen distance 2.8935Å when geometry optimization was done for all distances and angles, and -5.21 kcal mol−1 and 2.8957Å when geometry of water molecule was kept fixed. This confirmed our assumption that we can get good results for the strength of hydrogen bond when internal degrees of freedom of water molecules are kept fixed. Both energies compare favorably with the experimentally measured value, giving an estimate of the electronic interaction energy of -5.4 ± 0.7 kcal mol−1[25, 26] and both distances with the experimental oxygen-oxygen distance. The experimental study by Odutola and Dyke[27] yielded an equilibrium oxygen-oxygen distance of 2.946 Å. Various basis sets have been used in initial calculations and def2-QZVP was ultimately chosen as the best compromise between the computational complexity and the accuracy of the results for the set of monoions. All calculations were carried out with and without basis set superposition error (BSSE), but there was only small difference in results (less than 5%). As a first step, we calculated the bonding energies and distances for ion and single water molecule. In Tables 1 and 2, we presented the equilibrium ion oxygen distance and the bonding energy of systems for different cations and anions and one water molecule. In the vicinity of a cation, water dipole is parallel to electric field of the ion, while in case of an anion, one hydrogen atom points towards the ion. Then we attached the ion to one of the water molecules in a water dimer, either on the water donating hydrogen in hydrogen bond or on the water accepting hydrogen, and calculated the energy of the system while varying oxygen-oxygen distance. This system consists of two waters (particle 1 and 2) and ion (particle 3). In Figure 1 minimum energy geometry of the water dimer and ion is shown. The total energy E of the system depends on all three particles. In physics, it is always possible to split potential of many bodies into the sum over interactions that involve not just two, but clusters of three or more nearby atoms. In our case the total energy of three body system can be split into a three-body term, E3(1, 2, 3), three two body terms, E2(i, j), and electronic energy of single particle, E1(i), as (1)E=E3(1,2,3)+E2(1,2)+E2(1,3)+E2(2,3)+E1(1)+E1(2)+E1(3), with 1 and 2 standing for the water molecules and 3 for the ion. We have approximated the strength of the hydrogen bond in the proximity of an ion as sum of three-body energy term and two-body term between the water molecules (2)EHB=E3(1,2,3)+E2(1,2). In Figure 2, we plotted the distance dependence of the hydrogen bond strength between two water molecules in the presence of lithium and fluoride ions attached to donor and acceptor water, respectively and also in case of the absence of ions. When a cation is attached to the water donating hydrogen, this cation attracts electrons from water molecule, making hydrogen atom more positive and leading to increase of the strength of HB. When cation is attached to the water acceptor, this cation attracts the electrons from water, making the oxygen atom less negative and leading to a decrease of the strength of HB. With an increase of the strength, the minimum shifts to smaller distances and vice-versa. For anions we have the opposite effect. When an anion is attached to the water donating hydrogen in dimer, this anion causes an increase of partial charges on the hydrogen, which leads to a weaker hydrogen bond. When it is attached to the water acceptor, the negative charge of oxygen becomes more negative, which leads to a stronger hydrogen bond. Figure 3 shows a distance dependence of HB for geometries where HB increases in comparison with an isolated dimer. Figures are plotted for series of cations and anions. Smaller ions have a bigger impact on the strength of HB because strength of electric field of the ion is bigger on position of water dimer. In the presence of lithium and fluoride ions, the HB is the strongest and shifted to smallest distance for all ions. This effect decreases with an increase of radius of ion. Correlation between the maximum HB strength and distance between waters, when this happens, is shown in Figure 4. This relation is approximately linear and can be described as (3)−EHB=A⋅rOO+B with the parameters A = −24.03 kcal·mol−1Å−1 and B = 74.90 kcal·mol−1, which is in agreement with our previous calculations [19] for clusters of water molecules. This correlation allows us to focus our further research of local environment effects on the HB strength only since by knowing the strength we know also distance. From Figure 3 we saw that the strength of HB depends on type of ion and the type of ion we associated with strength of electric field produced by this ion on position of oxygen atom of water to which ion was attached. Strength of the electric field is proportional to 1/r2Oi where rOi is the distance between ion and oxygen atom of water to which ion is attached. Figures 5 shows this correlation, which can be described with the following relation (4)−EHB=CrOi2+D with the parameters for cations Cc = 10.69 kcal·mol−1Å2, Dc = 6.06 kcal·mol−1 and for anions Ca = 16.70 kcal·mol−1Å2, Da = 5.91 kcal·mol−1. The asymmetry for cations and anions arises because of different bonding of water to single ion, as seen in Figure 1. Furthermore, we also checked the relationship between the transfer of charge from ion to closest water molecule,Δe, and the strength of HB. This relationship is almost linear. We approximated it as (5)−EHB=Ae⋅Δe+Be with the parameters Ae = 12.18 kcal·mol−1 and Be = 6.67 kcal·mol−1. Charges were calculated by Mulliken population analysis. This is plotted on Figure 6a while on figure 6b we plotted the same results, but obtained by Natural Bond Orbital analysis (NBO)[28]. Here we have the same linear relationship, but different function for cations and anions. For anions we have parameters Ae = 34.66 kcal·mol−1 and Be = 6.03 kcal·mol−1 and for cations Ae = 178.7 kcal·mol−1 and Be = 6.64 kcal·mol−1.</p><!><p>We have calculated the dependence of HB strength in water on the presence of different monoions in the local environment by performing quantum chemical calculation. Calculations have shown that the introduction of ions to the local environment increases or decreases the HB strength depending on type of ion and to which water molecule an ion is bonded. In other words, if a water molecule is donor or acceptor of hydrogen in HB, the strength changes. When an increase in the strength of HB is observed, this increase linearly depends on the strength of the electric field produced by an ion and on the transfer of charge. This result provides new insight into understanding the nature of hydrogen bonding. Local environment appears to play far greater role in the HB strength and length than previously thought. Simple water models take into account only pairwise potential interactions between water molecules. Our calculations confirmed that this is not enough. Using only pairwise interactions for water close to surfaces and in channels does not suffice since the presence of charges in additional molecules of surrounding walls considerably changes the potential between two molecules. One way to address this is to use polarisable models of water where interactions are not pairwise. Another option is to construct water models that use pairwise and three-particle interactions [29].</p>

PubMed Author Manuscript

3D printed multifunctional PEEK bone scaffold for multimodal treatment of osteosarcoma and osteomyelitis

In this work, we developed the first 3D PEEK based bone scaffold with multi-functions targeting challenging bone diseases such as osteosarcoma and osteomyelitis. 3D printed PEEK/graphene nanocomposite scaffold was deposited with drug laden (antibiotics and/or anti-cancer drugs) hydroxyapatite coating. The graphene nanosheets within the scaffold served as effective photothermal agents that endowed the scaffold with on-demand photothermal conversion function under NIR laser irradiation. The bioactive hydroxyapatite coating significantly boosted the stem cell proliferation in vitro and promoted the new bone growth in vivo. The presence of antibiotics and anti-cancer drugs enabled eradication of drug resistant bacteria as well as ablation of osteosarcoma cancer cells, the treatment efficacy of which can be further enhanced by the on-demand laser induced heating. The promising results demonstrate the strong potential of our multi-functional scaffold in applications such as bone defect repair as well as multimodal treatment of osteosarcoma and osteomyelitis.

3d_printed_multifunctional_peek_bone_scaffold_for_multimodal_treatment_of_osteosarcoma_and_osteomyel

Introduction<!>Preparation of HA coated PG scaffold<!>Materials characterization<!>Antibacterial testing<!>Tumor ablation experiments<!>Cytocompatibility and Bone Regeneration<!>Bone regeneration in vivo<!>Statistical analysis<!>Results and discussion<!>Cancer ablation in vitro<!>In vitro and in vivo osteogenic differentiation<!>Conclusion

<p>Osteosarcoma (OS) is the most common type of primary bone cancer and it usually occurs in children and adolescents aged 10-19 years [1]. It is mostly found in the long bones of the lower extremity and metastasis is diagnosed in 20% of patients [2]. Osteomyelitis (OM) on the other hand, is a bacterial induced bone infection which also targets a considerable number of patients, particularly children, elderly, and patients with comorbidities (e.g. diabetics). The disease can lead to serious functional impairment, long-lasting disability, permanent handicap and even life threatening conditions. Both bone diseases are challenging clinical conditions, and the treatment protocol usually involves resection of the diseased bone tissue, followed by use of anti-cancer or antibiotic drugs. The treatment would inevitably result in large bone defect beyond the bone's self-healing ability (critical size bone defect), and the patients often suffer from recurrence of the diseases due to the residual cancer cell or bacteria.</p><p>Multimodal therapy, such as chemo-photothermal therapy (chemo-PTT) [3], photothermal/photodynamic therapy (PTT/PDT) [4], PDT/chemotherapy therapy [5] etc, are emerging strategies for the treatment of osteosarcoma and osteomyelitis. For instance, in chemo-PTT of bone cancer, cancer drugs can be delivered to the tumorous tissue in the targeted area, and the near-infrared (NIR) laser induced heating can enhance the sensitivity of tumor cells towards chemotherapy, leading to reduced drug dosage and improved treatment efficacy [6]. Combined use of laser heating and antibiotics is also known to enhance bacterial eradication at the infection site [7], which can be potentially used for osteomyelitis treatment.</p><p>To date, chemo-PTT is mainly achieved through local delivery of nanomaterials such as CuFeSe 2 [8], Fe-CaSiO 3 [9], graphene oxide [10], etc, which act as drug delivery vehicles and photothermal (PT) conversion agents. Some researchers also attempted to introduce PT conversion agents and drugs into hydrogels (such as pNIPAAm-co-pAAm) [11] or 3D porous ceramic (such as β-TCP) [10] or polymer (such as chitosan) scaffolds [12] for bone repair and chemo-PTT therapy. However, the above mentioned materials cannot provide sufficient mechanical properties, which would impede their applications, particularly in load-bearing bone repair [13,14].</p><p>In the past decade, polyetheretherketone (PEEK) has attracted increased attention in the biomedical field. PEEK has outstanding properties such as biocompatibility [15], X-ray/thermal/chemical stability [16], and mechanical properties (elastic modulus) similar to that of the human bone [7]. Being a FDA approved biomaterial, PEEK has been successfully deployed in applications such as artificial knee joints [17], spine fusion [18], skull [19], and orthopedic implants [20], etc. Its thermoplastic nature also allows it to be 3D printed into tissue scaffold with be-spoke geometry [21]. One major limitation of PEEK is its lack of bioactivity, which impedes its application for bone regeneration [22,23]. To address this issue, various strategies (such as surface coating or bulk reinforcement with bioactive agents) have been adopted to enhance the bioactivity of PEEK implants, a detailed review of which can be found in [24].</p><p>In the present study, we created a highly functional 3D printed PEEK/graphene composite scaffold with drug laden bioactive coating. The graphene nanosheets within the scaffold act as strong photothermal conversion agent, while the loaded drugs (such as antibiotics stearyltrimethylammonium chloride (STAC), and/or cancer drug cisplatin (DDP)) enabled effective cancer and/or bacteria eradication with the aid of NIR laser induced heating. The scaffold has been sucessfully demonstrated for bone regeneration, as well as multimodal treatment (chemo-PTT) of cancer ablation and bacterial eradication. We believe our multi-functional bone scaffold can potentially serve as a new platform for the management of challenging bone diseases such as osteosarcoma and osteomyelisis. wt% G were dispersed in ethanol and sonicated at 25 for 30 min. After filtration ℃ and drying, the power mixture was spun into filaments for further 3D printing (Funmat HT, Intamsys, Shanghai, China) of PEEK/G composite (PG) scaffold. 3D printed scaffolds (100 mm × 100 mm × 4 mm) with simple cubic lattice structure and different pore size (200 μm and 500 μm, respectively) were prepared. The PG scaffold were then plasma treated to modify the surface with oxygen rich functional groups, which can facilitate the subsequent electrophoretic deposition process. 3D printed pristine PEEK (P) scaffold was also prepared for comparison.</p><!><p>Hydroxyapatite (HA) was synthesized by hydrothermal method according to established procedures [25,26]. Positively charged stearyltrimethylammonium chloride (STAC) (Aladdin, China) was loaded onto HA particles through physisorption and uniformly dispersed STAC/HA suspension was used for subsequent electrophoretic coating deposition.</p><p>Electrophoretic deposited STAC/HA coating was deposited onto the 3D PEEK scaffold following [7] under a DC voltage of 50 V for 60 min with a carbon rod being the anode and PG scaffold being the cathode. The coated scaffold was named as PGH.</p><p>The coated scaffold was then immersed in sodium chloride solution of cisplatin (DDP, 1 mg/mL) following [27] for anti-cancer drug loading, and the final drug laden scaffold is named as PGHD .</p><!><p>Mechanical testing coupons (10 mm × 10 mm × 4 mm) and electrical conductivity testing coupons (100 mm × 100 mm × 1 mm) were produced from hot pressed P and PG sheet. Compressive tests were performed using universal mechanical testing machine (MTS, model E45, USA) at a speed of 1 mm/min, following GB/T 1041-2008/ISO 604:2002. Four-point probes method (RTS-8, Tianjin Nuleixinda Technology Co., Ltd., China) was used for electrical conductivity measurements. X-ray photoelectron spectroscopy analysis (XPS, XSAM800, Kratos, England) was performed to confirm the elemental state of PG before and after plasma treatment. Zeta potential (Zetasizer Nano ZS, Malvern, England) was used to determine the surface charge of the STAC-HA particles. Scanning electron microscope (SEM, JSM-7500F, JEOL, Japan) and EDS (JSM-7500F, JEOL, Japan) were used to analyze the morphology and elemental information of HA and the coating deposited on PGH.</p><p>The photothermal conversion effect of all samples was analyzed in air and in phosphate buffered solution (PBS), respectively. A 808 nm NIR laser (0-2 W/cm 2 , Richeng Science and Technology Development Co. LTD, Shanxi, China) was used at different laser power densities (0.05 W/cm 2 , 0.15 W/cm 2 , 0.30 W/cm 2 ) and the temperature of the scaffold was monitored in real time using an infrared thermal imaging system (TiS20+, Fluke, USA). The temperature data were analyzed using FLUKE software.</p><p>For drug release analysis, PGHD (10 mm ×10 mm × 4 mm) was immersed in 5 ml deionized water and placed in a shaking incubator under 37 .</p><p>℃ The cumulative release of DDP (Pt element) was detected by Inductively Coupled Plasma Emission Spectroscopy (ICP-OES, AXIS Ultra DLD, Kratos, UK) at different time intervals (1 h, 3 h, 10 h and 24 h). XPS was performed to detect the state of Pt element.</p><!><p>Gram-negative Escherichia coli (E. coli, ATCC25922) and gram-positive</p><p>Methicillin-resistant Staphylococcus aureus (MRSA, ATCC29213) were used to evaluate the antibacterial properties of the P, PG, PGH and PGHD scaffolds. Ten-fold dilution method was used to quantitatively measure the bactericidal rate (BR) defined by Eq.1 [28]. Briefly, bacterial strain was incubated in culture medium for 24 h and the subculture from the second passage was used as the pre-made bacterial fluid. 50 μL pre-made bacterial fluid was drop-casted onto scaffold samples and incubated for 2 h followed by topping of 4 mL physiological saline. The mixture of bacterial fluid and physiological saline extracted from each sample was subsequently diluted 10 4</p><p>times. Finally, 50 μL of each diluted fluid sample was inoculated on nutrient Luria-Bertani agar plate. The number of bacterial colonies up to 30~300 CFU (colonyforming unit) was counted.</p><p>Where n 0 is the number of colonies in the control group, n is the number of colonies in the experimental group.</p><p>To investigate the effect of photothermal conversion on bacteria eradication, a separate set of scaffold samples were irradiated by NIR laser for 10 min before the ten-fold dilution method was applied. The bacterial fluid irradiated by NIR laser was used as the control (named NIR only).</p><!><p>MG-63 cells were seeded in a 48-well plate at a density of 1.0 ×10 were measured every two days using a vernier caliper. The tumor volume (V t ) was calculated following:</p><p>Where L is the tumor length, W is the tumor width, V s is the scaffold volume (5 mm 3 ).</p><p>Relative tumor volume is defined as</p><p>Where V t0 = V t (day 0) -V s .</p><p>The body weight of the mice was also recorded every two days. Whole-body fluorescent imaging was performed on day 0 and day 10, respectively. On day 11, the mice were sacrificed and the tumors were harvested along with the heart, liver, spleen, lungs and kidney to evaluate the potential side effect of the scaffolds and/or the laser treatment. The tissue and organs were infiltrated with 4% paraformaldehyde, embedded in paraffin, and finally stained with hematoxylin and eosin (H&E).</p><!><p>Mouse MC3T3-E1 pre-osteoblasts ( 10 To investigate the proliferation of cells on the scaffolds, 10 4 MC3T3-E1 cells were seeded in 48-well plates containing scaffolds at 37 with 5% CO ℃ 2 . The cell proliferation on different scaffolds was assessed on day 1, 3, 5, and 7 by cell counting kit (CCK-8; Dojindo, Japan). Briefly, the culture medium was removed and 10% CCK-8-containing medium was added to each well in dark. After 2 h of incubation, 100 μL CCK-8 solution was transferred to a 96-well plate and examined by Multiscan Spectrum (Synergy Mx, Biotek, USA) at 450 nm. To study the scaffolds biocompatibility, MC3T3-E1 cells (1.0 ×10 4 cells/well) were seeded in a 48-well plate and the scaffolds were gently placed into the plate 24 h later. After an additional 24 h of incubation, the scaffolds were removed and the cells in the plate were stained with PI and Calcein-AM, respectively. Finally, cells were observed under a fluorescence microscope (Olympus IX83).</p><p>Mouse MC3T3-E1 pre-osteoblasts were seeded in 48-well plate at a density of 10 4 cells/well with α -MEM (Hyclone) supplemented with 10% FBS (Gibco) and 1% Penicillin-Streptomycin-L-glutamine (Hyclone). 48 h after incubation, the cells reached confluence and the growth medium was removed and replaced with osteogenic medium comprising growth media supplemented with dexamethasone (100 nM; Sigma), L-ascorbic acid (50 μg/mL; Sigma), and β-glycerophosphate (10 mM; Sigma). The scaffolds were gently placed into the plate and the medium was replaced every 2 days.</p><p>The expressions of osteogenesis-related genes were quantitatively analyzed by real-time reverse-transcriptase polymerase chain reaction (real-time PCR) on day 7.</p><p>The total RNA was extracted using Trizol reagent (Invitrogen, USA) and the complementary DNA (cDNA) was obtained by synthesizing DNA from 1 μg of total RNA via reverse-transcription using an iScript cDNA Synthesis Kit (Bio-Rad) following the manufacturer's instructions. Real-time PCR was performed with EvaGreen Dye (Bio-Rad) using RT-PCR instrument (CFX Connect, Bio-Rad). The forward and reverse primers for different genes were listed in Table S1. Cycle threshold (Ct) values were used to determine fold differences according to the ΔΔCt method. β-actin was used as an internal reference to normalize the data. On day 10, two representative osteogenic proteins (OPN and OCN) were evaluated by immunofluorescence. The cells co-cultured with different scaffolds were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.25% Triton X-100 for 5 min, and incubated with 1% bovine serum albumin (BSA) for 1h at room temperature. Then the cells were incubated with primary antibodies in 1% BSA overnight at 4</p><p>(mouse anti-OPN monoclonal antibody, 1:50, Novus; rabbit anti-℃ OCN polyclonal antibody, 1:100, Absin). The cells were then incubated in dark with 1:500 dilution of secondary antibodies in 1% BSA for 1h at room temperature (Alexa Fluor ® 488-conjugated anti-mouse IgG and Alexa Fluor ® 555-conjugated anti-rabbit IgG, Cell Signaling). Finally, the cytoskeleton and nuclei were stained with phalloidin (Phalloidin-iFluor™ 555 Conjugate, AAT Bioquest; AbFluor™ 488-Phalloidin, Abbkin) and DAPI (Beijing Solarbio Science and Technology, China), respectively. All staining were followed by rinsing with PBS three times. Representative images were obtained using a fluorescence microscope (Olympus IX83).</p><p>Alkaline phosphate (ALP) activity of MC3T3-E1 pre-osteoblasts with different scaffolds was quantified using ALP activity assay kit (Beijing Solarbio Science and Technology, China) on day 7. Briefly, cells were removed from the scaffold surfaces using Triton X-100 (1% v/v) and then centrifuged (12,000 rpm at 4 ) for 30 min to ℃ remove all cell debris. The supernatant was mixed with p-nitrophenyl phosphate (Songon) at 37 for 60 min. 4-Nitrophend NaOH was added to the cell supernatant ℃ and the ALP activity was examined by Multiscan Spectrum (Synergy Mx, Biotek, USA) at 510 nm. For standardization, the total protein content was calculated by a bicinchoninic acid (BCA) protein assay kit (Beyotime). The ALP activity was ultimately expressed as the total protein content (μM/mg). For ALP staining, the cells were fixed with 4% paraformaldehyde for 15 min, followed by staining using azo-coupling ALP color development kit (Beijing Solarbio Science and Technology, China) for 20 min in dark. Finally, cells were imaged using a fluorescence microscope (Olympus IX83).</p><p>The calcium nodules formed by the MC3T3-E1 cells co-cultured with different scaffolds were stained by Alizarin Red S (Al, Sigma, USA) on day 21. Specifically, cells were fixed in 4% paraformaldehyde for 15 min. The cells were washed thrice with distilled water and stained with Al for 20 min. Afterwards, the excess Al was thoroughly removed with distilled water and the deposited calcium was imaged. For quantitative analysis, the stained samples were dissolved with 10% cetylpyridinium chloride and analyzed by a Multiscan Spectrum (Synergy Mx, Biotek, USA) at 600 nm.</p><!><p>All surgical procedures were approved by the Animal Ethics Committee of West China Hospital of Sichuan University, China. Sixteen male Sprague-Dawley (SD) rats (8 weeks old, 200 ± 20 g) were chosen to construct distal femoral condyle defect models for the evaluation of bone osseointegration in vivo. The rats were randomly split into four groups: P, PG, PGH and PGHD, and were anaesthetized by isoflurane.</p><p>A hole (3 mm in diameter and 7 mm in depth) was made using a dental drill and the scaffold sample was press fit into the hole. 8 weeks post-surgery, the rats were sacrificed and their femurs were used for micro-CT scanning and histological staining. The harvested femoral specimen were fixed by 4% buffered paraformaldehyde, dehydrated using graded ethanol solution (70%, 85%, 95% and 100%), and embedded in polymethylmethacrylate without decalcification. The specimens were then microtomed into 5 μm thick slices along the cross-sectional surface, stained by methyl blue and basic fuchsin and observed under optical microscopy (Olympus IX83). The area of new bone tissue was calculated by the Image J software. Fluorescence observation was performed using CLSM (Nikon, Japan). The excitation/emission wavelengths used to observe the chelating fluorochromes were 543/620 nm and 488/520 nm for alizarin red S (AL, red) and calcein (CA, green), respectively.</p><!><p>Statistical analysis was performed using SPSS software (version 22.0; IBM Corp., Armonk, NY, USA), and the statistical significance was analyzed using oneway analysis of variance (ANOVA) followed by the LSD post hoc test. The relationships between different parameters were performed using Pearson's correlation test. Statistical significance was considered for p < 0.05, while high significance was p < 0.01.</p><!><p>Fig 1a show the compressive moduli data of P and PG scaffolds with different pore size. Both mechanical properties decrease with increasing scaffold pore size. The scaffolds with 500 µm pores demonstrated compressive moduli comparable to that of human cancellous bone (<3 GPa) [29], and are therefore selected for further investigation. The electrical conductivity of PG is 2.75×10 -3 S/cm (Fig 1b), which is twelve order of magnitude higher than that of pristine P (~10 -15 S/cm), and is comparable to semiconducting material.</p><!><p>The CLSM images (Fig 6a ) showed that without NIR irradiation, MG-63 cells on P, P+, PG, and PGH spread well with strong pseudopodia attachment on the surfaces in all directions. The cells also showed bright cell nuclei and actin in all groups. For PGHD, the presence of cancer drug has led to marked change of MG-63 cell morphology, i.e., round shape with no clear orientation, indicating inhibition of the cell growth. Upon NIR irradiation (0.30 W/cm 2 for 10 min), MG-63 cells on all samples displayed round shape with no pseudopodia and featured thinning of the actin layer around the cell nucleus.</p><p>The quantitative CCK-8 results of MG-63 (Fig 6b ) showed that in the absence of NIR irradiation (NIR-), only PGHD demonstrated significantly reduced MG-63 viability (59.8%) due to the presence of anti-cancer drug. All other groups (P, PG, PGH and control) showed no obvious anti-cancer effect (cell viability ~100%). With NIR irradiation, the number of live cells declined drastically in PG+, PGH+ and PGHD+, while the cell viabilities of P, P+, PG, and PGH were similar to that of the control group. Under 0.30 W/cm 2 NIR irradiation, the scaffolds (e.g. PG+, PGH+, and PGHD+) could reach the 45 within 100 s in immersed condition and remain ℃ stabilized for the remainder of the treatment time. Literature suggests that the temperature in the range of 45~50 can result in rapid necrosis of tumor cells ℃ (cancer ablation) because of their lower heat tolerance result can result in DNA and protein denaturation [30,31]. It is worth noting that the cell viability of PGHD+ further decreased to 24.9%, significantly lower than that of PG+ and PGH+ (45.7% and 45.4%, respectively).</p><p>The results of live/dead staining (Fig 6c ) were in accordance with the CCK-8 results., confirming PGHD+ is the most effective group in cancer ablation due to the combined effect of cancer drug release and hyperthermia introduced by PPT.</p><!><p>The bone regeneration is essential for bone defect repair. Osteogenic differentiation of the scaffolds was investigated both in vitro and in vivo and related markers were quantified by real-time PCR (Fig 10a). ALP, OPN and OCN are the corresponding makers representing different stages of osteogenic differentiation (ALP: early stage; OPN: secondary stage; OCN: late stage.) [34,35]. Col-I is one of the key components of extra cellular matrix deposition [36]. After culturing for 7 days, the gene expressions of ALP, OPN, OCN and Col1α1 in PG, PGH and PGHD were significantly upregulated as compared to P, where PGH and PGHD had similar performance. The osteo-related protein expressions of different scaffolds were also evaluated by immunofluorescence (Fig 10b and 10c). The highest OPN and OCN fluorescence intensity was observed in PGH and PGHD, followed by PG and then P.</p><p>The trend was consistent with what was observed from the real-time PCR results. The above in vitro osteogenic differentiation data revealed that the boneregeneration capability of different groups follows the order: PGH and PGHD>PG>P.</p><p>The best osteogenesis ability demonstrated by PGH and PGHD groups can be attributed to the presence of nanostructured HA coating, which promoted the serum protein adsorption, cell adhesion, attachment and and proliferation [37]. The calcium and phosphate ions released from HA can also upregulate the expression of osteogenic genes and activate secretion and extracellular matrix mineralization [38]. The presence of G in PEEK of PG group also demonstrated improved the scaffold osteogenic differentiation due to the osteoinductive capability of G [39,40]. Clinical translation of biomedical materials/products replies on rigorous in vitro and in vivo studies. Well-designed correlation analysis may help to identify the key in vitro parameters that can be used to predict the bone regeneration capacity in vivo.</p><p>This could subsequently reduce the economic and time-cost for in vivo studies [41]. In this study, Pearson's correlation test was used to analyze the relationship between the biocompatibility parameters (CCK8-1d, 3d, 5d and 7d), osteogenesis-related genes</p><!><p>In this study, we design and developed a 3D printed hybrid PEEK/graphene scaffolds with drug laden bioactive hydroxyl apatite coating. The scaffold has tailored porous structure / mechanical properties, and has combined drug release/photothermal therapeutic function. Results show that the scaffold with loaded antibiotic and/or anticancer drugs and on-demand photothermal conversion effect can achieve near total eradication of Escherichia coli (E. coli) and Methicillin-resistant Staphylococcus aureus (MRSA), as well as effective ablation of osteosarcoma cancer cells. The coated scaffold also exhibited strong bone regeneration ability, demonstrating its strong potential in applications such as bone defect repair as well as multimodal management of osteosarcoma and osteomyelitis.</p>

ChemRxiv

Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib

The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The co-crystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions \xe2\x88\x921 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs.

structure_of_the_human_protein_kinase_zak_in_complex_with_vemurafenib

INTRODUCTION<!>Cloning, protein expression and purification<!>Crystallisation<!>TM shift assay<!>Kinase activity assay<!>Isothermal titration calorimetry<!>Proteomic sample preparation<!>Tandem mass spectrometry and data analysis<!>Peptide library screening<!>Inhibitor screen<!>ZAK kinase substrate specificity<!>Validation of TM shift hits with a kinase activity assay<!>ZAK crystallisation<!>Structure of the ZAK-vemurafenib complex<!>Thermodynamics of vemurafenib binding to ZAK<!>Vemurafenib binding mode<!>Comparison of B-RAF and ZAK bound to vemurafenib<!>Impact on rational drug design<!>CONCLUSIONS

<p>The human leucine zipper- and sterile alpha motif-containing kinase (ZAK, also referred to as MLT, MLTK, HCCS-4, MRK and AZK) belongs to the mixed lineage kinase (MLK) family of protein kinases.(1) Its kinase domain shares about 40% sequence identity with other MLK family members such as MLK1 or DLK. Differential splicing leads to the expression of two ZAK isoforms.(2),(3) Besides the kinase domain, α-ZAK comprises a leucine zipper, a SAM domain and a C terminal portion of unknown function. In the much shorter isoform β-ZAK, this C terminal portion including the SAM domain is replaced by a most likely disordered tail (Figure 1A). The comparison of cancer tissue with the adjacent normal tissue by transcriptome sequencing revealed that the ZAK isoforms were differentially expressed in colorectal, bladder and breast cancers with α-ZAK being higher expressed in the cancer tissue.(2),(4) However, whether the changes in isoform usage are causative for or a result of cell transformation is not clear yet.</p><p>Physiologically, ZAK has been classified as a MAP3K.(5) Its activation is induced by ribosomal stress(6), osmotic shock(7) and ionizing radiation(8), with PKN1 being a molecular trigger of ZAK activation(9), resulting in trans-autophosphorylation of ZAK activation loop residues T161 and S165(8). Once activated, ZAK phosphorylates MAP2K7 thus leading to JNK activation.(5) Since the JNK proteins regulate cellular processes such as apoptosis and inflammation(10) ZAK can be regarded as a key switch determining cell fate.</p><p>This property may be exploited for the development of cardioprotective drugs. ZAK is highly expressed in adult human cardiomyocytes(11),(12) and has been associated with cardiomyocyte hypertrophy(13). In a recent study, increased cellular ZAK activity led to enlarged cardiomyocytes and increased expression of the hypertrophy marker brain natriuretic peptide (BNP).(14) In support of a crucial role in cardiomyocyte signalling ZAK has been shown to be required for the activation of the MAPKs JNK and p38 by doxorubicin(15), a pathway that has been linked to the significant cardiotoxic adverse effects of this commonly used cytostatic agent(16),(17). A range of clinically used anticancer drugs that potently inhibit ZAK effectively suppressed doxorubicin-mediated phosphorylation of JNK and p38 thus potentially protecting the heart from doxorubicin-induced heart failure.(15) These drugs including nilotinib(18), ponatinib(15) and sorafenib(19) also resulted in down-regulation of inflammatory cytokines such as IL-1β and IL-6 suggesting anti-inflammatory properties of ZAK inhibition(15). The findings have been confirmed in ZAK-deficient mice, with doxorubicin failing to induce elevated serum levels of inflammatory cytokines.(15)</p><p>However, the role of ZAK as a regulator of apoptosis and cell survival has also been associated with common adverse effects of drugs with strong ZAK off-target activity(20),(21). The administration of these drugs induces the formation of cutaneous squamous cell carcinoma (cSCC), as reported for sorafenib (in 5–10% of all patients)(22), dabrafenib (6–11%)(23) and vemurafenib (20–26%)(24). Since many of the cSCC occur on sun-exposed skin areas, UV light has been assumed to be the trigger of cSCC development.(20) Both the knockdown of ZAK and the administration of compounds with ZAK inhibitory activity prevented UV light from inducing apoptosis in cancer cells.(20),(21) Moreover, ZAK inhibition accelerated the UV light-driven development of cSCC in the hairless mouse model.(20),(21) A prominent alternative model to explain cSCC formation is the inhibitor-driven paradoxical B-RAF activation.(25) Which model may be more valid under physiological conditions or whether both mechanisms cooperate to promote cSCC formation is still under debate.(26)</p><p>In order to provide a structural model for the development of ZAK-specific tool compounds and the design of kinase inhibitors that seek to avoid ZAK as an off-target, we report here the first high resolution structure of the ZAK kinase including the leucine zipper domain in complex with the B-RAF inhibitor vemurafenib. The unique binding mode of the inhibitor vemurafenib provides a structural model for the design of B-RAF inhibitors with improved specificity.</p><!><p>cDNA coding for ZAK residues 5 to 309 (Mammalian Gene Collection, MGC) was PCR amplified using the forward primer TACTTCCAATCCATGGGTGCCTCCTTTGTGCA and the reverse primer TATCCACCTTTACTGTCATTTAAGCTCCTGCTCCTTAAAGC. The PCR product was then inserted into the vector pFB-LIC-Bse via ligation independent cloning.(27) After the transposition of the coding sequence into an engineered Baculovirus genome (Bac-to-Bac, Invitrogen), the viral DNA was transfected into TriEx cells (Novagen) cultivated in InsectXpress medium (Lonza). Protein expression was performed as previously described.(28)</p><p>Cells were resuspended in lysis buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol) and lysed by sonication. The lysate was cleared by centrifugation and loaded onto a Ni NTA column. After vigorous rinsing with lysis buffer the His6 tagged protein was eluted in lysis buffer containing 300 mM imidazole. While the protein was subjected to dialysis to get rid of the imidazole the N terminal tag was cleaved by TEV protease. Contaminating proteins, the cleaved tag and TEV protease were removed by another Ni NTA step. Finally, ZAK5–309 was concentrated and subjected to gel filtration using an AKTA Xpress system combined with an S200 gel filtration column. The elution volume 92.0 mL indicated the protein to be monomeric in solution. The final yield was 2.8 mg ZAK5–309/1 L insect cell medium.</p><!><p>50 nL of a solution containing 24 mg/mL ZAK5–309 and 500 µM vemurafenib (20 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol) were transferred to a 3 well crystallisation plate (Swissci), mixed with 100 nL precipitant solution (100 mM bis-tris-propane pH 6.5, 200 mM sodium malonate, 20% PEG3350, 10% ethylene glycol) and incubated at 4°C. The slowly growing crystal was detected after 14 days and mounted after 42 days in precipitant solution cryoprotected with additional 20% ethylene glycol. Data were collected at Diamond Light Source, analysed, scaled and merged with Xia2(29). The structure was solved by molecular replacement with Phaser(30) using a MLK1 model as a template (PDB ID 3DTC) and refined with Refmac5(31). The model was validated using MolProbity(32). A summary of data collection and refinement statistics is given in Table S3. The model and the structure factors have been deposited with the PDB ID 5HES.</p><!><p>The assay was performed according to a previously established protocol.(33) A solution of 2 µM ZAK5–309 in assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol) was mixed 1:1000 with SYPRO Orange (Sigma). The compounds to be tested were added to a final concentration of 10 µM. 20 µL of each sample were placed in a 96-well plate and heated from 25 to 96°C. Fluorescence was monitored using a Mx3005P real-time PCR instrument (Stratagene) with excitation and emission filters set to 465 and 590 nm, respectively. Data were analysed with the MxPro software.</p><!><p>ZAKtide ( ANHWHTVHLRA) was synthesised by Micheal Berne (Tufts Medical School). 20 µM peptide were phosphorylated by 4 nM ZAK5–309 in the presence of inhibitors (assay buffer 20 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol, 100 µM ATP, 5 mM MgCl2). The reaction mixtures were incubated at 37°C for 1 hour before being quenched by the addition of formic acid to a final concentration of 1% (v/v). Both the substrate and product peptides were monitored by RapidFire™ mass spectrometry. Samples were loaded onto a C4 solid phase extraction (SPE) cartridge. The cartridge was then washed to remove non-volatile buffer salts using deionised water containing 0.1% formic acid (solvent A) at a flow rate of 1.5 mL/min for 4.5 s. The sample was eluted with acetonitrile containing 15% deionised water and 0.1% formic acid (solvent B) at a flow rate of 1.25 ml/min for 4.5 s. The eluent was analysed on an Agilent 6530 Accurate-Mass quadrupole time-of-flight (Q-TOF) mass spectrometer operated in positive ionisation mode. Ion chromatograms were extracted for the +3 charge state of the substrate (MW 1340.69 Da) and product (MW 1420.65 Da) and integrated using the RapidFire™ Integrator software. The ratio of product area to product plus substrate area was used as the response, and the baseline signal from a sample inactivated with formic acid prior to addition of substrate was subtracted. The data were fit to the following equation using the nls function in the R programming language: normalised response=d+(a-d)/(1+10(log inhibitor concentration - log IC50)×HillSlope).Mean pIC50 was obtained by taking the geometric mean of pIC50s determined from duplicate experiments.</p><!><p>Measurements were performed at 37°C on a MicroCal VP-ITC (GE Healthcare). ZAK5–309 was dialysed overnight into assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol). The syringe was loaded with 120 µM ZAK5–309, the cell was filled with 15 µM vemurafenib. Every 10 minutes 10 µL of the protein solution were injected into the cell for a total of 28 injections. The heat flow data were analysed with the MicroCal ORIGIN software package employing a single binding site model.</p><!><p>The samples were prepared as previously described.(34) In brief, protein samples were reduced and alkylated in solution and subjected to chloroform/methanol precipitation. The precipitate was resuspended in 6 M urea in 100 mM Tris pH 7.4, digested with elastase at 37°C overnight and desalted using C18 Sep Pak column cartridges (Waters). After drying down in vacuo samples were resuspended in buffer A (98% H2O, 2% acetonitrile, 0.1% formic acid).</p><!><p>Samples were analysed using a nano-liquid chromatography tandem mass spectrometry (LC-MS/MS) system consisting of a Dionex Ultimate 3000 UPLC coupled to a hybrid quadrupole orbitrap instrument (Q Exactive, Thermo). Samples were loaded at a flow rate of 20 µL/min onto a PepMAP pre-column (C18, 300 µm×5mm, 5 µm particle size, Thermo) for one minute and separated at a flow rate of 250 nl/min on an nEASY column (C18, 75 µm×500 mm, 2 µm particle size, Thermo) for 60 minutes using a gradient of 2%–35% acetonitrile (v/v) in 5% DMSO (v/v) and 0.1% formic acid (v/v). All scans were performed at a resolution of 70,000 at 200 mass/charge and the 15 most abundant precursors were selected for HCD fragmentation. Raw MS data were de novo sequenced by PEAKS Version 7 (Bioinformatics Solutions) with search criteria at 10 ppm for MS1 and 0.05 Da for MS2. A database search (human SwissProt, 85,809 sequences) with subsequent posttranslational modification searches, where all modifications reported in UNIMOD were considered, was then applied to the de novo identified MS/MS spectra. False discovery rates of 1% threshold were applied. MS/MS spectra with phosphorylation modifications were inspected manually.</p><!><p>The library consisted of 182 peptide mixtures and was arrayed in a 1536-well plate at 50 µM concentration in 2 µL reaction buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EGTA, 1 mM DTT, 0.1% Tween 20) per well. Peptide mixtures had the general sequence Y-A-X-X-X-X-X-S/T-X-X-X-X-A-G-K-K(biotin).(35) In each well in the array, one of the X positions was fixed as a single amino acid at the indicated position, while the others were an equimolar mixture of the 17 amino acids excluding cysteine, serine and threonine. Two additional peptide mixtures were included that fixed either Ser or Thr at the phosphoacceptor position with all X positions left as mixtures. Purified ZAK was added to 10 – 30 µg/µL concentration along with ATP (to 50 µM including 0.03 µCi/µL γ-[33P]ATP), and plates were sealed and incubated at 30°C for 2 hours. Aliquots (200 nL) from each well were then spotted onto a streptavidin membrane (Promega), which was extensively washed, air-dried, and exposed to a phosphor screen to quantify radiolabel incorporation into each peptide. Quantified signals were normalized so that the average value of all amino acids at a given position were equal to 1. The heat map was generated in Microsoft Excel following log2 transformation of the normalized data.</p><!><p>Both α-ZAK and β-ZAK comprise the kinase domain and the leucine zipper (Figure 1A). This region of the protein was chosen for structural analysis. To aid crystallization trials, we tested a small kinase inhibitor library (~150 compounds that have been approved or have been in clinical trials before 2012, purchased from Selleckchem) using a melting temperature (TM) shift assay(36). In this assay inhibitor binding is assessed by a shift in melting temperature of the target protein that usually linearly correlates with inhibitor binding strength.(37) In agreement with earlier reports nilotinib(18), sorafenib(19), dabrafenib(21) and vemurafenib(21),(38) significantly increased TM for ZAK supporting strong interaction with the kinase (Figure 1B,E). With this simple assay we identified not only the inhibitors already reported to bind ZAK but also additional clinical inhibitors with significant activity for ZAK as judged by their TM increase (Table S1). Ponatinib induced with 15°C the highest TM shift. The phosphorylation of ZAK in the activation loop residues T161 and S165 had no impact on the stabilisation pattern. Interestingly, most of the identified ZAK stabilisers were type II inhibitors capable of binding kinases in the DFG-out conformation (type II inhibitors have been marked with an asterisk in Figure 1E).</p><!><p>ZAK is a catalytically active kinase.(8) Testing ZAK for its ability to phosphorylate a combinatorial peptide library(35) revealed a unique substrate specificity (Figure 1C and Table S2). Besides preferring threonine as the phosphoacceptor, ZAK was selective for hydrophobic residues at positions +1 (AVIFY) and +3 (VFYWQ) from the phosphoacceptor. The additional preferences (tryptophan at −2, histidines at −1 and +2) have not been previously reported for a kinase. While the sequence flanking the primary ZAK autophosphorylation site T161 ( RFHNHTTHMS) corresponded well to the determined consensus sequence (Figure 1C), the secondary ZAK autophosphorylation site S165 ( HTTHMSLVGT) was a more distant match to the consensus sequence. Interestingly, even the activation segments of the physiological ZAK substrates MAP2K4 ( GQLVDSIAKT) and MAP2K7 ( GRLVDSKAKT) are predicted to be poor ZAK substrates as isolated peptides. These findings suggest that selective and efficient substrate targeting by ZAK involves complex formation with downstream signalling factors, which has been generally observed for MAP3K interactions with MAP2Ks(39).</p><!><p>The optimal ZAK peptide substrate (ZAKtide, ANHWHTVHLRA) was synthesised (Michael Berne, Tufts Medical School) and served as the substrate in a kinase activity assay. ZAKtide was incubated with ZAK and ATP/Mg2+ at 37°C, and the relative levels of unphosphorylated and phosphorylated ZAKtide in the reaction mixture were quantified using mass spectrometry. Under the chosen conditions the apparent KM value for ATP was determined to be 140 µM which is in the range of KM values for previously investigated protein kinases(40). We measured IC50 values for compounds that stabilised ZAK by more than 5°C in TM shift assay (Figures 1D and S1). All of the tested compounds inhibited ZAK with IC50s ranging from 12 nM for rebastinib to 210 nM for apatinib confirming the TM shift data (Figure 1E). However, probably due to differences in binding modes TM shift values did not always correlate well with experimental IC50 values.</p><!><p>Our attempts to co-crystallise ZAK with ATP-γ-S and a subset of the TM shift hits (ponatinib, motesanib and vemurafenib) were not successful (summarised in Figure 2B). Though ZAK crystallised in complex with ATP-γ-S the diffraction was poor. Therefore, ZAK was subjected to autophoshorylation resulting in ZAK that was monophosphorylated mainly at the activation loop residue T161 (Figures 2C,D and S2) and ZAK diphosphorylated at T161 and S165. These differentially phosphorylated ZAK variants were co-crystallised with the inhibitors mentioned above. Only the combination of monophosphorylated ZAK and vemurafenib resulted in crystals with suitable diffraction properties. The structure was solved by molecular replacement using a MLK1 model (PDB ID 3DTC) as a template. A summary of data collection and refinement statistics is given in Table S3.</p><!><p>The ZAK kinase domain comprised the canonical kinase catalytic domain structure with an N lobe containing the β1-β5 sheets and the αC helix and the mainly helical C lobe. As expected, the inhibitor vemurafenib interacted with the ATP binding site (Figure 2A). Besides this conserved architecture common to all protein kinases the ZAK structure revealed some unique features.</p><p>The αC helix is predicted by PSIPRED to comprise residues 48–59. However, in the crystal structure it was moved away from the ATP binding site and comprised only residues 53–59. This SRC/CDK-like inactive conformation was probably due to the interaction with vemurafenib which pushed the αC helix outward. As a result the conserved salt bridge between K45 and E53 which is regarded a hallmark for the active kinase conformation was not formed (Figure S3).</p><p>The ZAK kinase and SAM domains are linked by 60 residues that are predicted to comprise a leucine zipper. The helix in the N terminus of the linker was attached to the kinase C lobe. W283 formed a π stacking interaction with F276, while L291, L294 and L297 formed a hydrophobic surface patch interacting with residues located in the αG helix (Figure 2F). The C terminus of the linker was not resolved in the structure with the electron density dropping sharply after residue 299. Based on the prediction of helical secondary structure for residues 301–322 we assume that the linker is composed of two helices interconnected by a flexible hinge. For Nek2, another protein kinase with a leucine zipper succeeding the kinase domain, the leucine zipper is required for homodimerisation and activation.(41) It remains to be investigated if a similar mechanism is also valid for ZAK. However, our construct ZAK5–309 did neither dimerise in size exclusion chromatography nor did the leucine zippers interact in the crystal.</p><p>The conserved P loop typically constitutes the most flexible part of the kinase N lobe. It bridges the β1 and β2 sheets and contributes to position the ATP β- and γ-phosphates for catalysis. In kinases bound to ATP and in 98% of the publicly available structures of kinase-inhibitor complexes, the P loop adopts an extended conformation.(42) However, in the ZAK-vemurafenib complex, the P loop was folded over the inhibitor thus forming a hydrophobic cage. This kinked conformation was mainly driven by the G23 backbone amide having polar contacts with the vemurafenib chlorine atom and by F27 forming a π stacking interaction with the chlorophenyl moiety.</p><p>As mentioned above ZAK tightly bound to a range of type II kinase inhibitors suggesting that the ZAK DFG motif at the base of the activation loop is capable of adopting the DFG-out conformation. However, the bound vemurafenib stabilised the DFG-in conformation with D151 pointing towards the ATP binding pocket and F152 pointing towards the αC β4 loop that anchors the C terminus of the αC helix to the top of the C lobe (Figure 2E). The protein used for crystallisation was phosphorylated in the activation loop residue T161 (Figures 2C,D and S2). In the crystal structure, the phosphate moiety of pT161 was in proximity of E51 N terminal of the αC helix, H158 in the activation loop and C285 of a neighbouring ZAK molecule interacting through crystal contacts. This interaction seems to be indispensable for crystallisation in space group P 41 since we did not obtain any such crystals of ZAK in a different phosphorylation state. The location of pT161 in the crystal structure is surprising as this phosphorylation site is located at the expected position in the sequence for activating phosphorylation sites (approximately 11 residues N terminal of the APE helix) and is expected to anchor the activation segment to the HRD arginine. It remains therefore to be investigated if the observed conformation is a consequence of inhibitor binding or may also be observed in the apoprotein. The region C terminal of pT161 was not resolved in the experimental electron density creating a 6 residue gap and indicating a disordered activation loop in solution despite its phosphorylated state. We speculate that T161 phosphorylation alone may not be sufficient to induce the attachment of the activation loop to the C lobe indicating additional S165 phosphorylation to be required to achieve full ZAK activity.</p><!><p>In order to determine the thermodynamic parameters for the binding of vemurafenib to ZAK in solution we performed isothermal titration calorimetry (ITC). Due to the poor vemurafenib solubility the experimental setup had to be optimised. Finally we carried out the binding experiment as a reverse titration in which a solution of 120 µM unphosphorylated ZAK was titrated into 15 µM vemurafenib at 37°C (Figure 3A). The binding was enthalpically driven (ΔH= −9.6 kcal·mol−1) with a minor entropic contribution to binding (−TΔS= −1.1 kcal·mol−1) resulting in a dissociation constant (KD) of 29 ± 4 nM. The slow equilibration following the injections likely reflected structural rearrangements within ZAK. Takeninto account the tight binding of type II inhibitors we suggest that in solution both the ZAK DFG-in and DFG-out conformations prevail in equilibrium, and that the interconversion rates are low. The measured KD was consistent with the measured IC50 value of 23 ± 4 nM (Figure 1D) and with KINOMEscan data of the close vemurafenib analogue PLX4720 to ZAK with a KD of 41 nM.(38) The insertion of the phenyl ring into PLX4720 was reported to increase the inhibitor's affinity for ZAK by factor 2.(21)</p><!><p>Our structural model suggests that vemurafenib does not inhibit ZAK by simply blocking the ATP binding site but by significantly distorting the kinase fold. Vemurafenib tightly interacted with five ZAK structural motifs (Figure 3B). The central 7-azaindole firmly anchored vemurafenib in the position generally occupied by the ATP purine forming two ATP mimetic hydrogen bonds with the backbone of E83 and A85 in the ZAK hinge region. The interaction was further strengthened by a π stacking interaction of Y84 with the condensed ring system.</p><p>The bifluorinated phenyl ring substituted with a propylsulfonamide group interacted with the DFG motif and penetrated with the propyl moiety into a pocket formed by the displaced αC helix and the DFG motif. Three hydrogen bonds were formed with all backbone nitrogens of the DFG motif thus trapping it in the DFG-in conformation. A water bridge interconnected the vemurafenib ketone linker and the D151 side chain contributing additional interactions with ZAK.</p><p>The second substituent of the 7-azaindole in vemurafenib is a chlorophenyl ring in 5 position. Even though the ring protruded from the ATP binding site it was entwined by the displaced ZAK P loop thus shielded from the surrounding solvent. The main driving force of the kinked P loop conformation seemed to be a π stacking interaction of F27 with the chlorophenyl ring.</p><!><p>The vemurafenib binding modes in B-RAF and ZAK share the same hinge interaction, but they differ considerably in the P loop conformation. While in B-RAF the P loop adopts an extended conformation not making contacts to vemurafenib(43), the ZAK P loop extensively interacts with vemurafenib (Figure 3D,E). The inhibitor PLX4720 differs from vemurafenib by the substitution of the chlorophenyl ring for a chlorine atom (Figure 3C).(43) The affinities of PLX4720 and vemurafenib for ZAK and B-RAF have been determined by a KINOMEscan competitive binding assay.(21) PLX4720 is a more potent inhibitor for B-RAF when compared with vemurafenib (IC50s 32 nM vs 65 nM), for ZAK this property is reversed (IC50s 9.5 nM vs 4.0 nM).(21) Our structural data suggest that this is due to the P loop interacting with the chlorophenyl ring. Folded and distorted P loop conformations have been associated with favourable inhibitor selectivity.(42) A structural consequence of a folded P loop, in particular when it is associated with an αC out movement, is the generation of a cavity between the P loop and αC. Such a cavity has for instance successfully targeted by the ERK inhibitor SCH772984.(44) The structure of the vemurafenib ZAK complex revealed a similar binding pocket extending from the carbonyl moiety of the inhibitor suggesting that this structural feature could be explored for the generation of selective ZAK inhibitors. Distortion of the P loop has also been linked to the specificity of imatinib for the tyrosine kinase ABL over c-SRC. The KD for imatinib binding ABL is by factor 2300 lower than for c-SRC(45) despite the high conservation of both binding sites. Besides the more favoured DFG-out conformation in ABL it is the P loop conformation (kinked in ABL(46) and extended in c-SRC(45)) that accounts for the difference.(47),(48),(49)</p><!><p>Vemurafenib has low nanomolar activity for a range of kinases, but its highest activity is displayed towards ZAK.(21) It is therefore likely that the vemurafenib scaffold can serve as a starting point for the development of more selective ZAK inhibitors. ZAK is the only human kinase bearing a cysteine residue in P loop −1 position.(50) In the ZAK/vemurafenib complex, this cysteine is in close proximity to the vemurafenib chlorophenyl moiety. Taken together, the replacement of the chlorophenyl moiety by an adequate electrophilic warhead could potentially lead to a specific covalent ZAK inhibitor. This strategy has been successfully employed for a number of kinases such as JAK3(51) and CDK7(52).</p><p>Likewise, the insights into the binding mode of vemurafenib to ZAK might help to generate inhibitors with increased B-RAF selectivity. For pharmacological reasons vemurafenib, and not PLX4720, was chosen to be tested in clinical trials(43), even though the introduction of the chlorophenyl moiety decreased the affinity of the inhibitor for B-RAF(21). It was previously not known that the improved pharmacological profile had to be paid by increased off-target activity towards ZAK. The chlorophenyl moiety interacts with ZAK F27 and D92. Replacing the chlorophenyl moiety by a substituent that retains the pharmacological profile but prevents the interaction with ZAK F27 and D92 might lead to a drug with reduced adverse effects.</p><!><p>Here we provided the first structural model for the common off-target of clinically approved kinase inhibitors ZAK. Inhibition of this kinase has been linked to significant adverse effects of currently used drugs, and the structural insight provided by the ZAK complex with the B-RAF inhibitor vemurafenib suggests new rational design strategies avoiding unintended ZAK inhibition by future drug candidates. The excellent druggability of ZAK demonstrated by our limited inhibitor screening campaign using a clinical kinase inhibitor set together with the presented unique structural features may also lead to highly selective ZAK inhibitors that may find applications as cardioprotective agents during chemotherapy or as new inhibitors treating heart failure and fibrosis as well as inflammatory conditions.</p>

PubMed Author Manuscript

Promysalin Elicits Species-Selective Inhibition of Pseudomonas aeruginosa by Targeting Succinate Dehydrogenase

Natural products have served as an inspiration to scientists both for their complex three-dimensional architecture and exquisite biological activity. Promysalin is one such Pseudomonad secondary metabolite that exhibits narrow-spectrum antibacterial activity, originally isolated from the rhizosphere. We herein utilize affinity-based protein profiling (AfBPP) to identify succinate dehydrogenase (Sdh) as the biological target of the natural product. The target was further validated in silico, in vitro, in vivo, and through the selection, and sequencing, of a resistant mutant. Succinate dehydrogenase plays an essential role in primary metabolism of Pseudomonas aeruginosa as the only enzyme that is involved both in the tricarboxylic acid cycle (TCA) and in respiration via the electron transport chain. These findings add credence to other studies that suggest that the TCA cycle is an understudied target in the development of novel therapeutics to combat P. aeruginosa, a significant pathogen in clinical settings.

promysalin_elicits_species-selective_inhibition_of_pseudomonas_aeruginosa_by_targeting_succinate_deh

INTRODUCTION<!>The Inhibitory Activity of Promysalin Is Not Affected by Iron Concentration<!>Affinity-Based Protein Profiling Identifies Succinate Dehydrogenase as the Biological Target of Promysalin<!>Computational Molecular Docking Supports Succinate Dehydrogenase as the Target of Promysalin<!>Whole Genome Sequencing of Promysalin-Resistant PA14 Identifies a Mutation in Succinate Dehydrogenase<!>Promysalin Leverages Differences in the Metabolic Flux of the Tricarboxylic Acid Cycle To Elicit Species-Selectivity<!>DISCUSSION<!>

<p>The continued rise of antibiotic resistant bacterial infections warrants the development of novel treatments with unique modes of action. To date, there is a general lack of diversity among cellular targets of approved antibiotics with recent reports estimating that fewer than 25 targets are represented.1 Most of these compounds are nondiscriminatory (broadspectrum), and they target essential pathways such as cell wall or protein synthesis.2 Although some "narrow-spectrum" therapies are available, they target large subsets of bacteria (anaerobes vs aerobes, Gram-positive vs Gram-negative) instead of focusing on particular pathogenic species. The latter method of treatment would be preferred in an effort to reduce adverse side effects to the host and microbiome communities and to minimize the development of resistance. However, both financial and technical limitations have thwarted such efforts to date.3 Furthermore, the identification of either (1) unique targets that would permit selective killing or (2) compounds that discriminate species is not trivial; this presents a clear unmet need that is ripe for discovery.</p><p>The combination of microbial diversity and evolutionary pressure has incentivized bacteria to create natural products with extraordinary selectivity and bioactivity. These scaffolds serve with distinction as antibacterial agents as an estimated 70% of marketed antibiotics are derived from natural products. One specific example exists within the rhizosphere where predominantly Gram-negative bacteria, particularly the Pseudomonads, utilize chemical warfare to both colonize the environment (quorum sensing) and defend themselves (antibiotics).4,5 Of particular health interest is the bacterial species Pseudomonas aeruginosa (Pa), an opportunistic environmental pathogen inherently resistant to many antibiotics yet rarely infective to healthy individuals.6 However, those with compromised immune systems (i.e., burn victims, chemotherapy patients, and the chronically hospitalized) or cystic fibrosis are especially susceptible to a fatal infection. In 2013, the Centers for Disease Control listed multidrug resistant Pa one of the top 15 urgent/serious microbial threats facing society, and just this year they increased its priority to the highest level, "critical", demonstrating a pressing need to develop new therapeutics which target this pathogen of interest.7</p><p>Recent efforts by both the De Mot8 and Muller9 laboratories have focused on this call by targeting untapped resources within the soil, which are rich in diversity. Thorough work by both groups has revealed natural products with complex chemical architecture and unique bioactivity providing inspiration for organic chemists as platforms for further discovery. One such example is the Pseudomonad secondary metabolite promysalin, which possesses species-selective inhibitory activity against Pa (initially reported IC50 = 0.83 µg/mL; MBC = 100 µg/mL against PA14), while inducing swarming and biofilm formation in a related species, P. putida (Pp).10 In 2015, our group completed the first total synthesis of the natural product, which enabled our elucidation of promysalin's relative and absolute stereochemistry and also confirmed the reported biological activity.11 We also showed for the first time that promysalin repressed fluorescence of P. putida KT2440, which is presumably attributed to the inhibition of pyoverdine production by the bacterium. We postulated that this phenotype might result from its ability to bind iron and therefore synthesized a concise library of analogs to test this hypothesis. Not only did this work provide initial structure–activity relationships that guided the findings provided herein, but it also confirmed that promysalin was capable of chelating iron, albeit weakly.</p><p>Despite the interesting and unique biological effects of promysalin, its mode of action remained unclear. Two potential mechanisms could explain the species-selective mode of action of this natural product. First, promysalin might act as a sideromycin, an antibiotic that hijacks siderophore transport through an iron-binding motif (Figure 1).12 Alternatively, promysalin could be targeting a difference in primary metabolism between species of Pseudomonads.13 The latter mechanism initially seemed counterintuitive: one would not expect that an essential enzyme, critical to nearly all life, could be the target of a narrow-spectrum agent. Nonetheless, here we report the surprising finding that the target of promysalin is succinate dehydrogenase (Sdh), a conserved enzyme that plays a key role in both in the tricarboxylic acid cycle (TCA) and in respiration.</p><!><p>Previous reports have demonstrated that the inhibitory activity of sideromycins is correlated to iron concentration as they rely on a chelation strategy to penetrate bacterial cells.14 In an elegant display of chemical creativity, the Miller group used these molecules as inspiration and developed a second-generation of synthetic sideromycins, whereby established antibiotics were covalently tethered to known siderophores, effectively creating a "Trojan Horse" strategy that was remarkably successful.15,16 This approach is best exemplified by BAL30072, a novel siderophore-sulfactam conjugate that entered Phase 1 clinical trials.17 Similarly, these molecules also rely on iron concentration and their activity can be enhanced by either the introduction of strong iron chelators or prior removal of iron from the media. Based on this knowledge, we sought to probe the bioactivity of promysalin against Pa strains PAO1 and PA14 over a range of iron concentrations. Under iron-limited conditions, we expected transcriptional upregulation of iron transport systems would facilitate the diffusion of promysalin into the cell and consequently increase its potency akin to sideromycins. However, our studies revealed that there was no identifiable effect of the available iron on efficacy as indicated by IC50 values, suggesting that iron chelation is coincidental and separate from antibiotic activity.</p><p>Previous work investigating the mechanism of action of BAL30072 identified the iron receptor PiuA as the active transporter responsible for the uptake of the molecule into Pa.18 PiuA is a member of the TonB dependent transporter (TBT) family, which are membrane-bound proteins responsible for the active transport of siderophores by means of the proton motive force.19 Transcription of such systems is up-regulated in response to stress20 and conversely down-regulated when an equilibrium is met, as excess iron is toxic. Previous findings have shown that TBTs regulate pyoverdine production and also vary widely between Pseudomonads, which could potentially explain our prior results. Initially, we investigated the ability of promysalin to form an Fe3+-bound complex with a variety of iron sources (Fe(acac)3, (NH4)5[Fe(C6H4O7)2], and FeCl3) by UV–visible spectroscopy. In all instances, we did not observe the characteristic Fe3+–siderophore complex at ~500 nm seen in other systems like enterobactin (Figure S1). Recently, one of our laboratories solved the crystal structure of PiuA and revealed the putative binding site of BAL30072.19 To further confirm that promysalin was not interacting via the PiuA system we sought to use isothermal microcalorimetry titration experiments to determine the extent at which the natural product binds. However, these studies again refuted our earlier hypothesis as no appreciable interaction was observed (Figure S2). Taken in sum, these findings demonstrate that although promysalin is capable of binding iron, it does not appear to be acting as a viable siderophore and/or using siderophore transport channels to elicit its response.</p><!><p>With our initial siderophore-based hypothesis disproven, we next planned to implement affinity-based protein profiling (AfBPP) to identify likely candidates. At the onset of this project, it was unclear if promysalin covalently modified its target; we therefore decided to install a photoaffinity probe to ensure capture of the biological moiety. The Yao group previously developed a concise route to a stand-alone diazirine photoprobe flanked on one end with an alkyne for AfBPP and a primary amine on the other primed for amidation chemistry.21 Initially, we envisioned installing the diazirine photoprobe to the side chain hydroxyl group, as that position was reactive based on our protecting group scheme from our previous total synthesis; however, our previous analog studies discouraged this modification.22 Instead, we focused our efforts on the alkylation of the amide nitrogen, which can be easily accessed from our earlier routes. For preliminary screening, we appended a propargyl moiety to the amide nitrogen, S4, (Scheme S1), which was approximately 3-fold less active than the natural product (218 vs 67 nM in PA14), thus permitting our strategy to synthesize the amide probe (full synthetic details are provided in the Supporting Information). Antibacterial activity of the probe was confirmed, with an IC50 value of 1.7 µM (in PA14), supporting its use for proteomic studies.</p><p>With the probe in hand we turned to AfBPP to elucidate the protein targets of promysalin. For each experiment, three different sample types were prepared for gel-free in situ proteomic analysis (Figure 2A). Cultures of P. aeruginosa PAO1 and PA14 were grown to log phase and incubated with either (1) promysalin photoprobe, (−)-2, (2) inactive promysalin photoprobe, (−)-3, or (3) promysalin (−)-1 followed by promysalin photoprobe (−)-2 (competitive inhibitor); experiments (2) and (3) serve to identify and eliminate any false positives. After UV irradiation, cells were lysed, reacted in situ with biotin-azide, and enriched on avidin beads. Enriched proteins were subjected to a trypsin-digest and labeled with light, medium or heavy isotopes via dimethyl-isotope labeling.23 Isotope labels were switched throughout biological replicates and samples with corresponding labels were pooled prior to LC-MS/MS measurement. Statistical analysis revealed only a small number of significantly (p-value ≤0.05; log2-ratio ≥1) enriched proteins (Figures 2B,C and S3). The most prominent hit in both Pa strains (PAO1 and PA14) as visualized by the volcano plot in Figure 2 was the succinate dehydrogenase Csubunit (SdhC); furthermore, the enrichment could be outcompeted by promysalin thereby providing preliminary validation of SdhC as the biological target.</p><p>In efforts to initially confirm our proteomic studies we sought to determine an in vitro IC50 against Sdh itself. While previous reports mention isolating Sdh from Pa membranes we instead leveraged a commercially available colorimetric mitochondrial Sdh assay.24 This would both give us confidence in Sdh as the target (as bacterial and mammalian enzymes are homologous) and provide information regarding selectivity between the two kingdoms. We observe complete inhibition of complex II at 200 µM and an IC50 of 2.5 µM. It should be noted that the ~50-fold difference in activity between the in vitro mammalian assay and the in vivo bacterial studies suggests that promysalin preferentially targets bacteria, which warrants future studies.</p><!><p>With consistent proteomic data and in vitro inhibitory activity in hand we sought to identify a putative binding site with computational modeling. Ideally, we would have preferred to cocrystallize promysalin with Sdh; however, the Pseudomonas protein has not yet been crystallized. The homologous enzyme in E. coli has been structurally characterized,25 and thus this served as a starting point for modeling the Pseudomonas enzyme. Previous work also identified several small molecules that inhibit E. coli Sdh at the ubiquinone-binding site (Figure 3A), and based on shared structural features of these compounds we anticipated that promysalin would bind at the analogous site (Figure 3B).26</p><p>We aligned each of these known inhibitors onto the Pseudomonas enzyme then used these compounds as the basis for pharmacophoric matching using a broad range of possible promysalin three-dimensional conformations. Upon energy minimization, these yielded many bound poses with comparable predicted energetics; thus, we leveraged our existing structure–activity relationship (SAR) data to narrow down the possible models.22</p><p>To our satisfaction, we found that the SAR was fully consistent with only one of these very diverse models: this points to the stringency of the constraints that arise from our thorough SAR characterization and provides confidence in the final model. There are three key observations that allowed us to reject all possible models but this one. First, we previously reported that the replacement of the ester linker with an amide abolished activity: in the docked model the ester adopts a conformation where the oxygen is engaged in intramolecular hydrogen bonding, whereas an amide substitution here would abolish this favorable interaction (Figure 3C). Second, replacing the salicylate hydroxyl group with a methoxy group greatly reduced activity: in the docked model this hydroxyl group engages in hydrogen bonding with a nearby aspartate and tyrosine; alkylating the oxygen would render these interactions impossible (Figure 3D and F). Finally, adding a methyl group to the dehydroproline heterocycle resulted in a compound with moderate activity; in the docked model this position points outward from the binding pocket, explaining how incorporation of an extra substituent is tolerated (Figure 3E).</p><p>This model is additionally consistent with information that was not part of the SAR used in its selection (Figure 3F). The model includes a hydrogen bond between a backbone carbonyl of SdhC and the alcohol side chain on the myristate region: the modeled position and orientation of the side chain alcohol explains why its stereochemistry was important for activity (changing this stereochemistry would lead to a steric clash), and yet its removal was also tolerated. Separately, we note that promysalin must bind in a manner that can accommodate the diazirine photoprobe with only minimal effects on bioactivity (~10× less active): the terminal amide in this model engages in two hydrogen bonds with the enzyme, and still would allow the alkyne moiety of the photoprobe to project toward the hydrophobic groove occupied by the fatty acid side chain.</p><p>Another key consequence of this model relates to the strainspecific activity of promysalin. We mapped the sequences for each Sdh subunit for PAO1, PA14, and KT2440 back onto this model of binding: notably, there was not a single sequence difference among the three at this site. The model therefore implies that the observed differential activity is not based on binding preferences of promysalin for Sdh, but rather upon some other factor that distinguishes these strains.</p><!><p>There is precedence that bacteria and fungi can generate resistance to Sdh inhibitors. For example, carboxin resistance is of major concern in the agriculture industry and a number of Sdh mutations have been disclosed which render the compound inactive.27,28 In an effort to validate both our proteomic results and our proposed docking model we sought to select for a promysalin-resistant mutant in PA14. Toward this end, bacteria were subjected to a range of concentrations (sub-lethal to lethal) of promysalin daily for a 24-day period. After the course of treatment, two morphologically distinct mutant strains were obtained (Figure 4A). Strain O5, which had a similar morphology to that of the parent strain was >60-fold more resistant to promysalin. The mutant contained a nonsynonymous single nucleotide polymorphism (SNP) in SdhB, which resulted in an I206V mutation within the ubiquinone binding site at the interface of SdhB and SdhC (Figure 4B). This subtle mutation is unique to promysalin as it has not been identified in carboxin-resistant strains and is likely attributed to a greater loss of hydrophobic contact with promysalin when compared to ubiquinone.</p><p>A second resistant strain (N5) was also identified and based on its "abnormal" morphology was suspected of having distinct mutations. Whole genome sequencing (WGS) of this strain revealed the same SNP in Sdh and a second mutation in YfiR, a regulator of intracellular c-di-GMP levels.29,30 YfiR mutants have been shown to form small colony variants (SCVs) thereby explaining the altered morphology.30 SCVs due to YfiR mutation, which were initially isolated from the lung of a cystic fibrosis patient, are known to possess higher levels of persistence and biofilm formation.30 Strain N5 displayed a 10-fold increase in resistance to promysalin (IC50 = 646 nM) and a significant increase in biofilm formation, in line with previous studies of this mutation (Figure 4A). Taken in sum, the WGS data further validates our proteomic, in vitro, and docking model confirming succinate dehydrogenase as the biological target of promysalin in PA14.</p><!><p>The tricarboxylic acid (TCA) cycle is an essential pathway in primary metabolism and facilitates the release of stored energy through a series of eight reactions.31 Succinate dehydrogenase is an enzyme that is part of both the TCA cycle and the electron transport chain (housed in membrane).32 Its specific function within the process is to catalyze the oxidation of succinate to fumarate with simultaneous reduction of the cofactor ubiquinone (CoQ10) to ubiquinol (Figure 5A). Under stress, however, alternative pathways can be employed. The glyoxylate shunt pathway is one such alternative which circumvents four of the eight steps in the TCA cycle, one of which involves Sdh, for specific metabolic uses.33 In the glyoxylate pathway, isocitrate is converted to glyoxylate and sequentially converted to malate, thereby bypassing several transformations including the oxidation of succinate. Alternatively, isocitrate can also be directly converted to succinate. However, in this pathway, the succinate produced is often released for energy production and biosynthesis, suggesting metabolism and subsequent cellular function can persist without Sdh.</p><p>Based on this understanding of the TCA cycle, we postulated that we could rationalize the species-selectivity of promysalin on differences in metabolism which would become clear through microbiological growth assays in defined media. Toward this end, we first attempted, albeit unsuccessfully, to rescue growth of Pa through media supplementation of fumarate. These results were not surprising based on the dual modality of Sdh, as this enzyme not only converts succinate to fumarate but also facilitates electron transport. Although the supplementation assay would rescue the former deficiency, it would not address the latter. We next hypothesized that through purposefully selected feeding studies we could potentially override any inherent species-specific preferences in primary metabolism. To begin, we grew each strain (PA14, PAO1, PP KT2440, and PP RW10S1) in either TSB or M9 minimal media supplemented with either succinate or glucose. As expected, promysalin was active only against Pa and not Pp in TSB and M9 media supplemented with glucose as these carbon sources allowed the bacteria to utilize either the full TCA cycle or the shunt pathway in a fully aerobic process. Conversely, a clear zone of inhibition is present in both Pp strains (gentamicin shown as a control) as can be seen in Figure 5B, including the producing organism, demonstrating that promysalin is capable of inhibiting the growth of Pp in presumably nonenvironmental circumstances. While inhibition of the producing organism is surprising, it is not unprecedented as bacteria can develop modes of self-resistance to their own antibiotics. Toward this end, we sought to explain this finding by revisiting the isolation paper where the genome of the producing strain was fully sequenced.10 In that report, no transporter or resistance genes were disclosed; however, the gene cluster encoding the biosynthesis of the natural product is found immediately adjacent to the TCA genes and presumably under the control of the same promoter. This would allow the bacteria to modify its metabolism accordingly whenever antibiotic production was activated. Taken in sum, these results shed light on how promysalin can elicit is species-selectivity through the inhibition of Sdh.</p><!><p>We report herein a multidisciplinary approach to identify the biological target of the Pseudomonad secondary metabolite promysalin. Based on the narrow-spectrum activity of the natural product, we expected to either identify a target unique to Pa or a transporter specific to the natural product. Instead, we uncovered succinate dehydrogenase, an enzyme involved in primary metabolism, as the biological target; computational modeling, in vitro assays, and whole genome sequencing of resistant mutants further validated these findings. Previous studies have shown that other rhizosphere natural products, like siccanin, a fungal natural product, also target Sdh. This small molecule was "rediscovered" through an initial screen for Pa membrane inhibitors but was later shown to be species-selective preferentially targeting Pa, but not E. coli or Corynebacterium glutamicum.34 When considering promysalin and siccanin, recent studies investigating the effect of growth conditions on essential functions of Pa confirm SdhABCD as essential, regardless of growth media. These findings complement the siccanin data, as SdhABCD has been found to be nonessential in corresponding E. coli investigations.35 This difference in activity can be understood via the dual roles that Sdh serves both in metabolism by means of the TCA cycle and in respiration through the electron transport chain (ETC). While Pa is able, under specific conditions, to grow and survive via fermentation, respiration is almost solely responsible for ATP production (via oxidative phosphorylation following the ETC); consequently, unless in the proper environment, Pa requires the ETC to generate ATP and survive.35,36 This facultative anaerobic behavior is a critical difference between Pa and Pp as Pp possesses a highly versatile aerobic metabolism, often favoring the Entner–Doudoroff pathway.37 Furthermore, recent work by the Collins lab has demonstrated that metabolic flux in Pa greatly varies between growth conditions (i.e., carbon sources), and that by targeting specific enzymes within the TCA cycle, one can potentiate antibiotic activity. These findings may help to explain the differential activity between PAO1 and PA14, though they cannot fully rationalize the inactivity in Pp.13 In a separate study looking at systems-level metabolic pathways, it has been postulated that Pp may be able to interchangeably utilize the glyoxylate shunt pathway in lieu of the TCA cycle without sacrificing overall growth.38 Future work in our laboratory will seek to confirm these computational findings via transcriptomic studies.</p><p>Growth of Pa in sublethal concentrations of promysalin over a 24 day period led to the identification of a single, consistent mutation in SdhB, which was conserved across all replicates. This mutation, I206V, presumably results in a reduction of hydrophobic contacts with promysalin, reducing its affinity, while only having a minor effect on ubiquinone binding as demonstrated by its similar growth profile. In addition to the SdhB mutation, a portion of the resistant population also displayed the SCV phenotype, which WGS revealed to be a double mutant of both SdhB and YfiR. The abnormal morphology of the single colonies, containing the YfiR mutation, is consistent with YfiR knockouts, which were first discovered in Pa CF sputum isolates.30 YfiR acts as the regulator in the YfiBNR system closely regulating YfiN, which functions as a diguanylate cyclase, producing c-di-GMP. In wild-type strains, YfiN is repressed by YfiR; however, in YfiR mutants, the derepression of YfiN leads to an increased production of c-di-GMP.30 Adaptations of this mutation increase the number of persister cells and also form more robust biofilms (Figure 4A).39 Presumably, the initial YfiR mutation in Pa arose in a similar manner, that is, from the sublethal treatment of antibiotic. It will be interesting to see if the mutation of YfiR is a common defense mechanism utilized by Pa to resist antibiotic treatment.</p><p>Taken together, we initially identified the target of a species-selective antibiotic via proteomic studies. The success of these studies hinged on our previous analog findings thereby allowing for the chemical synthesis of a diazirine photoprobe which retained activity. Succinate dehydrogenase was identified using AfBPP and was further validated with in vitro assays, feeding studies, and whole genome sequencing of resistant mutants. Computational molecular docking was used to predict the putative binding pose within the ubiquinone pocket, and additionally provided insight into the basis for the observed I206V resistance mutation. Furthermore, we show that, under specific media conditions, the species-selective nature of promysalin is abolished to the extent that it is capable of inhibiting growth of its producing strain. Our findings add to the emerging discoveries focusing on the targeting of the TCA cycle both to potentiate existing antibiotics and also to develop narrow-spectrum therapies, which will undoubtedly find utility both in drug discovery and in deconvoluting multispecies microbiomes.</p><!><p> ASSOCIATED CONTENT </p><p>*Supporting Information</p><p>Experimental details, synthesis details, NMR spectra (PDF)</p><p>The authors declare no competing financial interest.</p>

PubMed Author Manuscript

Replacement of a Thiourea-S with an Amidine-NH Donor Group in a Platinum\xe2\x80\x93Acridine Antitumor Compound Reduces the Metal\'s Reactivity with Cysteine Sulfur

The reactivity of two DNA-targeted platinum\xe2\x80\x93acridine conjugates with cysteine sulfur was studied. The conjugate containing an amidine-NH donor group cis to the chloride leaving group showed considerably reduced reactivity with N-acetylcysteine compared to the prototypical derivative containing a thiourea-S linkage. The opposite scenario has been observed previously in reactions with nucleobase nitrogen. Possible consequences of the unique target-selective tuning of the substitution chemistry for the pharmacodynamic properties and biological activity of these agents are discussed.

replacement_of_a_thiourea-s_with_an_amidine-nh_donor_group_in_a_platinum\xe2\x80\x93acridine_antitum

Introduction<!>Results and Discussion<!>Conclusions<!>Materials<!>NMR Spectroscopy<!>Incubations and Chromatographic Separations<!>Mass Spectrometry

<p>The high reactivity of the anticancer drug cisplatin (cis-[PtCl2(NH3)2]) with the intracellular sulfur-containing nucleophile glutathione (γ-l-glutamyl-l-cysteinylglycine, GSHa) causes adverse effects, such as severe systemic toxicities and tumor resistance.1, 2 One major goal in platinum drug development, therefore, is to design complex geometries that react less avidly with cysteine sulfur. This has been achieved, for instance, in picoplatin (cis-[PtCl2(NH3)(2-methylpyridine)]), by replacing one ammine ligand in cisplatin with a sterically hindered pyridine derivative,3, 4 and in carboplatin (cis-[Pt(CBDCA)(NH3)2], CBDCA = cyclobutane-1,1-dicarboxylate), by introducing a bidentate leaving group in place of the chlorido ligands. While carboplatin is remarkably inert in reactions with GSH,5 the stability of the dicarboxylate chelate compromises the metal's ability to bind to its pharmacological target, DNA.6 This dilemma is manifest in the fact that the second-generation drug, while less toxic than cisplatin, has to be administered at significantly higher doses to achieve the same therapeutic effect as the parent drug.7</p><p>Here we report a unique case of a chemical modification to a DNA-targeted platinum agent that reduces the reactivity of the metal with cysteine sulfur while it was previously found to accelerate the reaction with nucleobase nitrogen. In a recent study,8 we investigated the biological effect of replacing a thiourea (sp2-S) with an amidine (sp2-NH) donor cis to a chloride leaving group. The new platinum–acridine agent reported, [PtCl(en)(L)](NO3)2 (en = ethane-1,2-diamine; L = N-(2-(acridin-9-ylamino)ethyl)-N-methylpropionamidine, acridinium cation) (compound 1, Figure 1) proved to be a significantly more efficient DNA binder and more cytotoxic agent than the prototypical agent, PT-ACRAMTU9, 10 (ACRAMTU = 1-(2-(acridin-9-ylamino)ethyl)-1,3-dimethylthiourea, acridinium cation) (compound 2, Figure 1). Moreover, this work has lead to the first member of this class of compounds endowed with antitumor activity in aggressive non-small cell lung cancer in a mouse xenograft model.8 To test the effect of this simple structural modification on the metal's reactivity with protein sulfur, we incubated both analogues with N-acetylcysteine (N-AcCys), a modified amino acid mimicking GSH, and monitored the progress of the reactions by 1H NMR spectroscopy. In addition, we analyzed the mixtures after completion of the reactions by in-line high-performance liquid chromatography electrospray mass spectrometry (LC–MS).</p><!><p>In arrayed one-dimensional 1H NMR experiments, the reaction of complex 1 with one equivalent of N-AcCys (25 °C, 10 mM phosphate buffer, D2O, pH* 6.8) was found to proceed considerably slower than the reaction of PT-ACRAMTU (2) under the same conditions. The relative rates at which the amino acid reacted with 1 and 2 were deduced from the change in integral intensities of 1H NMR signals assigned to the platinum complexes (see Supporting Information for spectral data and detailed procedures). Under the conditions chosen, the reaction between 1 and N-AcCys follows a second-order rate law, typical of a mechanism that is dominated by direct substitution of the chlorido ligand by cysteine sulfur and is not limited by the rate of aquation.2 In contrast, the more rapid reaction of PT-ACRAMTU (2) with N-AcCys followed neither second-order nor first-order kinetics, suggesting that the reaction of this derivative proceeds via a more complicated mechanism (Figure 2). In both cases, the 1H NMR spectra of the mixtures indicated formation of multiple products. Attempts to assign the species formed by 1 and 2 using two-dimensional [1H-15N] NMR spectroscopy, a technique applied previously in nucleotide binding studies of [15N]-en labeled conjugates,8, 11 were unsuccessful due to the complexity of the reaction mixtures.</p><p>To overcome this difficulty, LC–MS was used to analyze the product distribution in mixtures of platinum complex incubated with one equivalent of sulfur nucleophile (37 °C, 6 h, 10 mM phosphate buffer, pH 7.2). The two most abundant adducts formed by compound 1 were unambiguously identified as the mononuclear species [Pt(en)(L)(N-AcCys*)]+ ([I]+, 14%) and the dinuclear complex [{Pt(en)(L)}2(μ-N-AcCys*)]4+ ([II]4+, 72%) in electrospray mass spectra recorded in positive-ion mode (Figure 3). (The asterisk indicates the dianionic form of metal-bound N-AcCys at neutral pH.) These findings suggest that adduct I, formed by substitution of chloride by cysteine sulfur, rapidly reacts with unreacted complex 1 to form the dinuclear species, II (Scheme 1). The tendency of cysteine sulfur to readily induce bridged complexes with platinum(II) drugs has been amply demonstrated.2 The platinum–amidine linkage and the en chelate in complex 1 appear to be resistant to nucleophilic attack by cysteine based on the presence of only a minor amount of free acridine L (III, 3%) and the absence of ring-opened adducts. In contrast, the LC–MS data acquired for PT-ACRAMTU (2) reveal a more complicated reaction pattern. In analogy to complex 1, the chloride substitution pathway leads to the dinuclear adduct, [{Pt(en)(ACRAMTU)}2(μ-N-AcCys*)]4+ (II′, 32%), most likely via intermediate I′, which could not be detected in this case (Figure 3). In addition, significant amounts of ACRAMTU (III′, 33%) and the bisintercalator complex, [Pt(en)(ACRAMTU)2]4+ (IV, 26%) (Scheme 1), were observed in the HPLC profiles (Supporting Information). Formation of the latter complex can be explained with chloride substitution in compound 2 by free ACRAMTU in the reaction mixture. The release of ACRAMTU by N-AcCys was unexpected and shows that thiourea, while a typical nonleaving group in reactions of compound 2 with DNA nitrogen,12, 13 becomes substitution-labile in the presence of cysteine sulfur.</p><p>The reactivity of the Pt–Sthiourea bond and its cis labilizing effect on chloride are potential contributors to the increased cysteine reactivity of PT-ACRAMTU (2) compared to analogue 1. The rationale for incorporating a thiourea-based nonleaving group in the original design was to enhance the reactivity of the metal with DNA nucleobases by exploiting the cis activating effect of sulfur. In their pioneering studies of the inorganic kinetic cis-effect, Tobe and coworkers demonstrated for the complexes [PtCl(en)(dmso-S)]+ and [PtCl(en)(dms)]+ (dmso = dimethylsulfoxide, dms = dimethylsulfide) that the sulfur donors greatly accelerate chloride substitution by an incoming nucleophile relative to analogous complexes with [N3Cl] donor sets.14, 15 In the same studies, the authors demonstrated that the relative rate enhancement (kS/kN) is large for strong incoming nucleophiles, such as sulfur ligands, but negligible for weak ligands like water. The comparative kinetic study of amidine-modified complex 1 and thiourea-based PT-ACRAMTU (2) shows that replacement of the sulfur with an imino donor group reduces, as expected, the metal's reactivity with cysteine sulfur but enhances its binding with DNA nitrogen. These findings strongly suggest that, unlike the reaction with cysteine, the reaction with DNA is not controlled by the electronic cis-effect but, as concluded previously, most likely by steric factors and/or H-bonding that favor complex aquation and facilitate nucleophilic attack of nucleobase nitrogen on the metal center.8</p><p>In addition to the unique monofunctional–intercalative adducts formed by PT-ACRAMTU and its second-generation analogues, one critical feature that sets these hybrid agents apart from cisplatin-type cross linkers is their inherent DNA-targeted character. While intercalation plays an important role in the mechanism of action of these agents, rapid platination of DNA nucleobases appears to be crucial for potent inhibition of cancer cell proliferation. Characteristically, derivatives that react with DNA sluggishly or not at all have proven only marginally cytotoxic.16, 17 Since the Pt–Scysteine bond is resistant to nucleophilic attack by DNA nitrogen,2 the GSH metabolites formed by compounds 1 and 2 (analogous to I/I′ and II/II′, Scheme 1), even if they reach the nucleus, should be relatively nontoxic because of their inability to induce cytotoxic DNA adducts. Likewise, ACRAMTU (III′) and the bisintercalator IV (a reversible DNA binder18, 19), which are potential metabolites formed in the reaction between 2 and GSH, are considerably less cytotoxic than the hybrid agents.9, 19</p><!><p>In summary, we report the first case of a simple structural modification within a DNA-targeted platinum antitumor agent that increases the target affinity of the metal while concomitantly reducing unwanted reactivity with protein sulfur. Based on these findings, the amidine analogue, 1, should have a major pharmacological advantage over PT-ACRAMTU (2), which may contribute to the superior potency of the new analogue in vitro and in vivo. To test this hypothesis, future studies will delineate relationships between the cytotoxicity and intracellular distribution of 1, PT-ACRAMTU, and related complexes in cancer cells characterized by elevated cytosolic GSH levels. Another important issue to be addressed is the relatively high toxicity of our new hybrid agent in treated animals. The results of a necropsy performed on the test animals treated with at sublethal doses of the hybrid agent (unreported data) revealed mild to yellow discoloration of the kidneys. We speculate that the adverse effects on the kidneys are the result of platinum binding to sulfur of glutathione (GSH). Cisplatin–GSH adducts are metabolized to strong nephrotoxins that cause damage to the renal proximal tubules.2 Thus, additional structural modifications in compound 2 may be needed to reduce this toxicity to levels as low as those observed for picoplatin and carboplatin.</p><!><p>The platinum-acridine complexes 1 and 2 were synthesized according to published procedures.8, 9 For the preparation of biological buffers, biochemical grade chemicals (Fisher/Acros) were used. HPLC grade solvent were used in all chromatographic separations. All other chemicals and reagents were purchased from common vendors and used without further purification. Stock solutions of the platinum compounds were prepared immediately before use.</p><!><p>NMR spectra in arrayed experiments were collected at room temperature on a Bruker 500 DRX spectrometer equipped with a triple-resonance broadband inverse probe and a variable temperature unit. Reactions were performed in 5-mm NMR tubes containing 2 mM complex 1 or 2 and 2 mM N-acetylcysteine (10 mM phosphate buffer, D2O, pH* 6.8). The 1-D 1H kinetics experiments were carried out as a standard arrayed 2-D experiments using a variable-delay list. Incremented 1-D spectra were processed exactly the same, and suitable signals were integrated. Data were processed with XWINNMR 3.6 (Bruker, Ettlingen, Germany). The concentrations of platinum complex at each time point were deduced from relative peak intensities, averaged over multiple signals to account for differences in proton relaxation.</p><!><p>Reactions of 1 and 2 with N-acetylcysteine were performed at a 1:1 drug-to-amino acid ratio in 10 mM sodium phosphate buffer (pH 7.1). Incubations were performed at 37 °C for 6 h. The mixtures were separated by reverse-phase HPLC using the LC module of an Agilent Technologies 1100 LC/MSD Trap system equipped with a multi-wavelength diode-array detector and an autosampler. A 4.6 × 150 mm reverse-phase Agilent ZORBAX SB-C18 (5 μm) analytical column was used in all of the assays, which was maintained at 25 °C during separations. A monitoring wavelength of 413 nm was used to detect acridine-containing fragments. The following eluent systems were used for the separations: solvent A, degassed water/0.1% formic acid, and solvent B, methanol/0.1% formic acid. The gradient used in the separation was 98% → 30% solvent A over 30 min at a flow rate of 0.5 mL/min. Peak integration was done using the LC/MSD Trap Control 4.0 data analysis software.</p><!><p>Mass spectra were recorded on an Agilent 1100LC/MSD ion trap mass spectrometer. After separation by in-line HPLC, adducts were directly infused into the atmospheric-pressure electrospray source. Ion evaporation was assisted by a flow of N2 drying gas (350 °C) at a pressure of 40 psi and a flow rate of 10 L/min. Positive-ion mass spectra were recorded with a capillary voltage of 2800 V and a mass-to-charge scan range of 150 to 2200 m/z.</p>

PubMed Author Manuscript

Targeting MDM2-p53 Interaction for Cancer Therapy: Are We There Yet?

Inactivation of the tumor suppressor p53 and/or overexpression of the oncogene MDM2 frequently occur in human cancers, and are associated with poor prognosis, advanced forms of the disease, and chemoresistance. MDM2, the major negative regulator of p53, induces p53 degradation and inactivates its tumor suppressing activity. In turn, p53 regulates MDM2 expression. This MDM2-p53 negative feedback loop has been widely studied and presents an attractive target for cancer therapy, with a few of the inhibitors of this interaction already having advanced into clinical trials. Additionally, there is an increasing interest in understanding MDM2\xe2\x80\x99s p53-independent activities in carcinogenesis and cancer progression, which may also have implications for cancer therapy. This review aims to highlight the various roles that the MDM2-p53 interaction plays in cancer, the p53 independent oncogenic activities of MDM2 and the various strategies that may be used to target MDM2 and the MDM2-p53 interaction. We will summarize the major preclinical and clinical evidences of MDM2 inhibitors for human cancer treatment and make suggestions to further improve efficacy and safety of this interesting class of cancer therapeutics.

targeting_mdm2-p53_interaction_for_cancer_therapy:_are_we_there_yet?

INTRODUCTION<!>MDM2 As A Negative Regulator of p53<!>MDM2 As An Oncogene, Independent of p53<!>Clinical Relevance of Targeting MDM2 for Cancer Therapy and Prevention<!>STRATEGIES TO TARGET MDM2 FOR CANCER THERAPY<!>Inhibiting MDM2 Expression<!>Inhibiting MDM2-p53 Binding<!>Modulating E3 Ubiquitin Ligase Activity of MDM2<!>SMIs Disrupting MDM2-p53 Interaction<!>SMIs Binding to MDM2<!>SMIs Binding to p53<!>SMIs InhibitingMDM2 E3 Ubiquitin Ligase Activity<!>SMIs Inducing MDM2 Post-Translational Modifications<!>SMIs Inhibiting MDM2 Expression<!>Nutlins (Cis-Imidazolines)<!>Spiroxindoles<!>Tryptamine Derivative (JNJ-26854165/Serdemetan)<!>Other Compounds in Clinical Trials<!>DISCUSSION AND FUTURE DIRECTIONS

<p>Human malignancies remain a leading cause of death in both women and men worldwide; there are still major challenges in understanding cancer etiology, pathogenesis, treatment and prevention. It is widely accepted that carcinogenesis and cancer progression are complex and multi-factorial processes with the emergence of cells possessing features such as sustained proliferation, evasion of growth suppressors, resistance to cell death and replicative mortality, increased angiogenesis, and activation of invasion and metastasis [1]. The cellular changes at genetic and epigenetic levels during carcinogenesis and cancer progression have been the center of cancer research for several decades. These genetic changes involve deletions, mutations, and rearrangements in a set of specific genes (typically oncogenes and tumor suppressors) that may affect their protein products. Oncogenes generally encode cell proliferation and apoptosis-controlling proteins; and their amplification or activation leads to development of the malignant phenotype [2–4]. In contrast, tumor suppressor genes activate anti-proliferative and pro-apoptotic pathways, thus halting cell cycle progression; and inactivation or loss of tumor suppressor genes also leads to the malignant phenotype.</p><p>Since its discovery more than thirty years ago, p53 has become the most widely studied tumor suppressor gene [5]. This short-lived protein has multiple functions in normal cells and is essential in preventing cancer onset and development. There are several outstanding reviews which document the functions and regulations of p53 [6–17]. As a transcription factor, p53 is responsible for maintaining genomic integrity and is activated in response to diverse stress signals, leading to DNA repair, cell cycle arrest, apoptosis, and senescence [9, 16]. The importance of p53 in cancer biology and etiology can be gauged by the fact that this protein is deleted or mutated in more than half of all human malignancies [15]; with the loss of its wild-type function negatively impacting prognosis and response to cancer therapy. These observations render the restoration of p53 functions in the cancerous cell as an attractive anticancer approach. However, after more than two-decades of intensive preclinical and clinical research, activation of p53, including p53 gene therapy, has not been proven to be a practical clinical approach to cancer therapy. One of the reasons for ineffective consequence of p53 therapy may be the inactivation of endogenous and exogenous p53 by its negative regulators such as MDM2 (murine double minute 2) [18].</p><p>MDM2 is a cellular phosphoprotein that forms a complex with p53 [18–22]. MDM2 has E3 ubiquitin ligase activity which plays a critical role in the degradation of p53 [23]. MDM2 is overexpressed in many human malignancies, indicating that it is a major mechanism utilized by cancer cells to escape p53 surveillance [24]. Indeed, the MDM2 gene is frequently amplified in most cancers; and alterations in the p53 and MDM2 genes and/or their protein products, separately or concomitantly, lead to poor prognosis and treatment failure in cancer patients [25, 26]. Therefore, considering the obstacles faced in p53-based cancer therapy [27–31], we [32, 33] and others [34] have proposed that MDM2 is a valuable molecular target for cancer therapy. In the last 15 years or so, the MDM2-p53 interaction has become a focal point of research in both academia and the industry to develop better targeted cancer therapeutics [28, 30, 33, 34]. In this review, we aim to critically evaluate the rationale and status of targeting MDM2 and advances made in the development of MDM2 inhibitors as novel cancer therapeutics and what the future holds for this class of novel cancer therapeutics.</p><!><p>The p53 protein was first discovered in 1979 as a cellular partner of simian virus 40 large T-antigen; it was found to migrate as a 53-kD band in gel electrophoresis, thus imparting its name [6]. Subsequent studies showed that p53 maintains genomic integrity of the cell, prevents malignant transformation, induces cell cycle arrest and apoptosis in response to stress signals [16]. Disruption of this gene facilitates the oncogenic process leading to increased cancer risk [12, 13]. Indeed, p53 mutations are seen in more than 50% of all human cancers, being highly prevalent in both solid cancers as well as in leukemias and lymphomas; thus providing an insight into the genetic machinery that controls the carcinogenic process [15]. With the discovery of MDM2, a more complete picture of the p53 pathway emerged a decade after p53 discovery.</p><p>The mdm2 gene is located on chromosome 12q13–14 and encodes for a 491 amino acid protein [19]. Under normal conditions, MDM2 is expressed in the nucleus, but it trans-locates to the cytoplasm to mediate the degradation of its substrates. The first indication that MDM2 acts as an oncogene came from the observation that it could cause spontaneous transformation of an immortalized murine cell line, BALB/c 3T3 and that mdm2 overexpression rendered rodent fibroblasts tumorigenic in nude mice [20]. The direct evidence for the crucial role of MDM2 in negatively regulating p53 comes from the fact that targeted deletion of the mdm2 gene in mice is embryonic lethal due to p53-mediated apoptosis, whereas simultaneous deletion of the TP53 gene rescues the lethality [35, 36].</p><p>The auto-regulatory feedback loop between MDM2 and p53 may be the most important finding in this field. As illustrated in (Fig. 1), the MDM2 protein binds to the N-terminal transactivation domain of p53, inhibiting its transcriptional activity [37–39], promotes p53 export out of the nucleus [40, 41], prevents p53 from interacting with transcriptional co-activators [42], and targets p53 for ubiquitination and degradation by the proteasome [41, 43, 44]. On the other hand, p53 regulates the transcription of the mdm2 gene, thus forming a negative feedback loop that maintains cellular p53 at low level [45]. The MDM2-p53 interaction was initially thought to result from the mutual binding of MDM2 and p53 via their N-terminal domains, but recently the p53 C-terminus has also been shown to be involved in the MDM2-p53 interaction [46, 47]. The mechanisms responsible for the MDM2-induced p53 degradation have been intensively investigated. MDM2 serves as an E3 ubiquitin ligase via its C-terminal RING finger domain and ubiquitinates p53 at several lysine residues [23]. While low levels of MDM2 induce p53 mono ubiquitination and nuclear export, high levels of MDM2 cause polyubiquitination and degradation of p53 in the nucleus [48, 49]. MDM2 can also auto ubiquitinate itself, leading to self-degradation [23].</p><p>Several studies demonstrate that the MDM2-p53 interaction is more complicated than previously thought. There have now been increasing evidences indicating that the MDM2-p53 feedback loop is regulated or modulated by a host of factors (as summarized in Table 1), including ribosomal proteins [50–63], proteasome activator (PA28γ) [64], polycomb protein RNF2 [65, 66] and RYBP [67], the ARF tumor suppressor [68–71], DNA damage response elements (14-3-3-σ, PML, and JMY) [72–74], and MDMX [75–77], among others. These molecules may bind to MDM2 and/or p53, altering their conformation, modification, interaction, and modulating the E3 ligase activity of MDM2 towards itself and p53. This complex interplay of cellular players adds multiple layers of regulation to the MDM2-p53 loop. The MDMX-MDM2-p53 interaction can serve as an excellent example of the complexity of p53 signaling pathways. MDMX (also referred as MDM4) possesses a high degree of homology to MDM2, especially in its N-terminal p53 binding domain [75, 77]. Similar to MDM2, it possesses, at its N-terminus, a p53 binding domain and at its C-terminus, a RING finger domain through which it forms hetero dimers with MDM2. MDMX is highly and/or abnormally expressed in human tumors and seems to promote carcinogenesis [77–79]. MDM2 and MDMX perform non-redundant functions to inactivate p53 during embryogenesis and throughout development [79–81]. MDM2 and MDMX are proposed to work independently to inhibit p53 activity, or alternatively, MDM2 and MDMX may form a complex that is more effective at inhibiting p53 transactivation or enhancing p53 turnover. MDM2 and MDMX are also postulated to form heterooligomers through their RING domains, whereby MDMX increases MDM2's E3 ligase activity (as shown in Fig. 1). MDM2 can also directly ubiquitinate and degrade MDMX in response to DNA-damage stimuli [82–85].</p><!><p>In addition to its role in the MDM2-p53 loop, recent findings indicate that MDM2 also has critical roles in carcinogenesis, independent of p53. For example, bladder cancer and sarcoma patients with increased MDM2 levels and mutant p53 present a worse prognosis than those with a single defect [26]. In animal studies, 33% lymphomas that arise in Eμ-myc transgenic mice with deleted or mutated p53 show overexpression of MDM2 [86, 118]. Studies with genetically engineered mice support the hypothesis that MDM2 has p53-independent functions that contribute to carcinogenesis. p53−/−mdm2+/−mice and p53−/−mice expressing an MDM2 trans-gene develop more sarcomas than p53−/−mice [119]. Many investigations have now identified numerous MDM2 interacting molecules that are involved in cell proliferation, apoptosis, and tumor invasion and metastasis [120–123].</p><p>The representative p53-indpendent activities of MDM2 in carcinogenesis and cancer progression are depicted in (Fig. 2). In addition to the inhibition of p53, MDM2 overexpression in cancer cells influences various cellular signaling pathways and contributes to tumor development and progression. MDM2 binds and regulates multiple proteins that are involved in cell cycle control, apoptosis, DNA repair, cell migration and invasion, angiogenesis, and chemo resistance. These signaling pathways work together to ensure the integrity of genetic information, and as such MDM2 may be considered as a central meeting point regulating genome stability, cell transformation and ultimately tumor formation and progression.</p><p>The MDM2's p53-independent activities have been intensively investigated, but remain not fully understood. MDM2 overexpression may contribute to genomic instability seen in cancer cells. For instance, MDM2 contributes to chromosomal breakage and failure of genomic integrity by direct interaction with proteins such as dihydrofolate reductase (DHFR), a key enzyme in folate metabolism [124]. MDM2 also plays an important role in DNA repair mechanisms by binding directly to the DNA repair protein Nbs1 [125]. MDM2 overexpression inhibits p21, the cyclin-dependent kinase inhibitor [126]. MDM2 directly interacts with p21, inducing its conformational change and ubiquitin-independent proteasomal degradation [126–128]. This may at least, in part, explain the MDM2's p53-independent activity in promoting cell proliferation.</p><p>Furthermore, MDM2 overexpression drives cell cycle progression in the S-phase by binding with retinoblastoma (Rb) protein and E2F-1, members of the Rb-E2F tumor suppressor pathway [129–131]. MDM2 stabilizes E2F-1, inducing E2F-1 transcriptional activation and ultimately cell cycle progression, while inhibiting the ability of Rb to induce G1 arrest. MDM2 regulates Rb protein stability through both its ubiquitin ligase activity as well as by directly interacting with the C8 subunit of the 20S proteasome, thereby facilitating its destruction [129]. Interestingly, MDM2-dependent degradation of Rb increases DNA methyltransferase DNMT3A activity which is associated with silencing of tumor suppressor genes [132]. MDM2 overexpression also inhibits apoptosis induced by co-overexpression of E2F-1 and DP-1(E2F dimerization partner 1), in osteosarcoma cells lacking p53 and Rb [133].</p><p>Other evidence supporting MDM2's anti-apoptotic roles includes its interaction with the pro-apoptotic proteins p73 and FOXO3a [134–136]. MDM2 mediates the NED Dylation of the pro-apoptotic form of p73-TAp73, promoting its cytoplasmic translocation and mitigating its p53-transactivational activity [134]. MDM2 negatively regulates the stability of FOXO3a by mediating its ubiquitination and degradation [135]. FOXO3a is also responsible for regulating p27, which leads to MDM2-mediated control of cell cycle progression via oncogenic growth factor or Ras activation [137]. In addition, MDM2 positively regulates XIAP (X-linked inhibitor of apoptosis protein) by enhancing its translation and subsequent expression [138].</p><p>Apart from its roles in cell cycle and apoptosis control, MDM2 also regulates angiogenesis by increasing expression levels of transcription factors such as hypoxia inducible factor (HIF-1α), subsequently increasing the expression of vascular endothelial growth factor (VEGF) [139–141]. In addition, MDM2 has been identified in the epithelial-tomesenchymal transition process via its capability to target E-cadherin for proteasomal degradation [142], implicating its role in cancer metastasis. MDM2 also interacts with MTBP (MDM2 binding protein), a metastasis suppressor, and over-expression of MDM2 can reverse MTBP's function, indicating a potential role for MDM2 in cancer metastasis [143]. MDM2 binds to and stabilizes Slug mRNA, increasing its expression and cellular invasiveness in p53-null cells [144]. MDM2 contributes to chemo resistance by degrading HUWE-1 which ubiquitinates the antiapoptotic protein Mcl-1 [145]. Further, MDM2 enhances p65-mediated transcriptional activity of NFκB in ALL cells, leading to resistance towards doxorubicin-induced apoptosis [146].</p><p>In brief, as discussed above, the p53-independent activities of MDM2 are important because more than half of all human cancers exhibit mutated and non-functional forms of p53. If MDM2 is to be considered as a valid and viable therapeutic target, the oncogenic activity of MDM2, independent of its role as a negative regulator of p53, must be understood in greater detail [147].</p><!><p>Multiple lines of preclinical and clinical evidences suggest the existence of a direct relationship between MDM2 and cancer development and progression [26] (Table 2). MDM2 overexpression has been observed in many cancer types [148, 151, 156, 165, 179], with the overall frequency of gene amplification being 7% and the highest frequency being observed in soft tissue tumors [24]. These tumors often show a higher incidence of MDM2 amplification than p53 mutation. Overall, a negative correlation is seen between occurrence of p53 mutations and MDM2 amplification, thus supporting the hypothesis that MDM2 negatively regulates p53. In many cases, a higher frequency of MDM2 protein overexpression is observed in tumors with wild-type p53.</p><p>A well-documented mechanism for MDM2 overexpression is the single nucleotide polymorphism at nucleotide 309 (SNP309) in its promoter [180, 181]. The clinical significance of SNP309 remains to be clarified. Although the MDM2 SNP309 variant increases MDM2 expression and is associated with tumor formation [182–184], the SNP is not associated with increased risk or a prognostic factor in certain cancers such as glioblastoma [185]. MDM2 overexpression induced by TGF-β is observed in late stage metastatic breast cancer and correlates with poor prognosis [186]. Other mechanisms apart from gene amplification, such as increased transcription and translation also contribute to MDM2 over-expression [109]. High levels of both MDM2 and MDMX proteins without increased copy number have been observed in some cancer types, such as melanoma, Ewing's sarcoma, colon carcinoma, and retinoblastoma [109].</p><p>In breast cancer cell lines, it appears that MDM2 expression is linked to the ER (estrogen receptor) status of the cells [187]. Higher MDM2 expression is observed in ER+ cells. Interestingly, estrogen-mediated increase in cell proliferation correlates with increased MDM2 levels, without a concomitant decrease in the p53 protein level [188]. These findings may have an implication in breast cancer diagnosis and treatment.</p><p>MDM2 is also linked to chemo resistance in several cancer types. Overexpression of MDM2 is an important event in regulating sensitivity to chemotherapy in childhood acute lymphoblastic leukemia (ALL) [189]. Functional inactivation of p53 by mutation or other mechanisms is common in relapsed neuroblastoma and associated with MDM2 gene amplification, contributing to chemo resistance [190]. Several studies have reported that either increased MDM2 activity or the SNP309G allele is associated with cellular resistance to topoisomerase II inhibitors [191].</p><!><p>As aforementioned, in most situations, MDM2 overexpression is oncogenic and is associated with late stage disease, resistance to chemotherapy and radiotherapy, and poor prognosis, making it a valid and valuable target for developing cancer therapeutics. We and others first tested the value of MDM2 as a cancer target in preclinical models using antisense oligonucleotides (ASOs) and RNA interference (siRNA) to inhibit MDM2 expression. The results have validated that MDM2 inhibition in cells and mouse models of human cancer is indeed a viable approach to suppress tumor cell growth in vitro and in vivo [33, 192–197]. To date, investigations on the MDM2-p53 interaction has provided a basis for the design of novel small molecule therapeutics aiming at inhibiting MDM2 activity and eventually reactivating the wild-type p53 function [198–202]. In vivo studies have shown restoration of wild-type p53 function can lead to the suppression of soft tissue sarcoma, lymphomas, and liver cancers [202].</p><p>Studies on the crystal structure of the N-terminal domain of MDM2 reveal a deep hydrophobic cleft on which the p53 peptide binds as an amphipathic alpha helix, a structure also present in other pro-apoptotic proteins such as Bax [203]. In fact, three amino acids in p53 (Phe19, Trp23 and Leu26) are essential for the binding between p53 and MDM2, and they are inserted into the deep hydrophobic depression on the surface of the MDM2 molecule [203]. Various approaches followed in the discovery of novel small molecule inhibitors of MDM2-p53interaction have been recently developed. The major strategies that can be used to target MDM2 includes: (i) blocking MDM2 expression; (ii) inhibiting the E3 ubiquitin ligase activity of MDM2; (iii) inhibiting the MDM2-p53 interaction; and (iv) targeting the protein-protein complex in relation to MDM2 interactive proteins.</p><p>Cutting-edge technologies in structure biology, bioinformatics, computer-aided drug design, and high throughput screening, have led to the identification of numerous molecules belonging to different classes of chemical structures that have significant MDM2 inhibitory effects. The list of natural compounds and synthetic small molecules capable of selectively inhibiting MDM2 and/or the MDM2-p53 interaction keeps increasing rapidly. In the following sections, we will focus primarily on specific and selected molecules that have (or might have) a strong association at the clinical level.</p><!><p>Several early studies by our group [32, 192–194, 197] and others [204, 205] using antisense oligonucleotides to inhibit MDM2 expression have established the proof of principle for this approach in cell and mouse models of human cancer. MDM2 down-regulation in various cancer cells results in p53 stabilization and activation of the p53 pathway in cancer cells in vitro, as well as in tumor xenografts in nude mice. Interestingly, not only p53 wild-type cells, but also cells that express mutant p53, respond to MDM2 inhibition [32, 197]. It has been suggested that the p53-independent stabilization of the cyclin-dependent kinase inhibitor, p21, due to MDM2 down-regulation might contribute to the antitumor activity of MDM2 antisense oligonucleotides and MDM2 inhibitors in these mutant cell lines [200]. MDM2 inhibition also results in chemosensitization and radiosensitization of cancer cells in various in vivo and in vitro models [33, 206].</p><p>Other gene targeting strategies include the use of ribozymes and RNA interference techniques. MDM2 ribozymes induce cell growth arrest and increase apoptosis in vitro [207]. RNAi-mediated MDM2 knockdown leads to similar effects on proliferation, survival, apoptosis, and cell cycle progression [208]. Also, aptamers targeting MDM2 provide another line of attack [209]. More recently, several groups have used novel approaches to deliver MDM2-siRNA for cancer therapy [210, 211].</p><p>In the past few years, we and others have investigated the value of MDM2 as a molecular target for natural product chemotherapeutic and chemopreventive agents [212–214]. Several well-known chemopreventive agents such as curcumin, genistein, and ginsenosides have been demonstrated to down-regulate MDM2 oncoprotein expression. These compounds were able to influence MDM2 levels in tumors with wild-type p53 as well as those with non-functional or mutant p53. For a comprehensive discussion on natural product MDM2 inhibitors, readers are directed towards a recent review [215].</p><!><p>MDM2 will be unable to down-regulate p53 if it is prevented from interacting with p53 protein. Therefore, inhibition of MDM2-p53 binding appears to be a desirable strategy for p53 stabilization and activation. However, targeting protein-protein interactions by small molecules is challenging. Generally, protein-protein interactions are difficult to interrupt by low molecular weight chemical entities as they involve large, flat interfaces with buried surfaces [216]. However, in the case of the MDM2-p53 interaction, it has been demonstrated that a limited number of amino acid residues (namely, Phe19, Trp23 and Leu26 in the N-terminal domain of p53) are crucial for the binding of the two proteins [203]. There exists a narrow, continuous, hydrophobic pocket on the MDM2 protein for p53 binding with the aforementioned amino acids critically regulating the interaction. Therefore, it is reasonable to expect that a synthetic molecule with three hydrophobic groups projecting into this pocket would essentially mimic the key amino acid residues of p53, thus competitively inhibiting the MDM2-p53 interaction. The nutlin class of MDM2 inhibitors developed by Roche Company from the screening of a large combinatorial library is based on this principle [198–200]. These inhibitors possess the capability to displace p53 from MDM2 in vitro with nanomolar potency (IC50 = 90 nM for nutlin-3a). Crystallographic studies demonstrate that nutlins bind to the p53-interacting domain of MDM2 in a way that closely resembles the molecular interactions of the crucial amino acid residues from p53 [198].</p><!><p>The ubiquitin ligase activity of MDM2 is crucial for its negative regulation on p53 protein stability, indicating that this may be a valid drug target. Recently, small molecule inhibitors have been identified that specifically target the E3 ligase activity of MDM2. These compounds inhibit the ubiquitination of p53 in vitro, with IC50 values in the micromolar range [217–221]. They are able to activate p53 signaling, inducing apoptosis in a p53-dependent manner. The first report on MDM2 E3 ligase inhibitors dates back to 2002 with the arylsulfonamide, bisarylurea, and acylimidazolone compounds being discovered in an MDM2-mediated p53 ubiquitination chemical library screen [218]. They do not affect the physical interaction between MDM2 and p53, probably inhibiting MDM2 in an allosteric fashion by blocking a structural rearrangement of MDM2 necessary for p53 ubiquitination [218]. Selective inhibition of p53 ubiquitination is therapeutically desirable as opposed to total loss of MDM2 ubiquitin ligase activity (which will also inhibit MDM2 autoubiquitination, leading to enhanced MDM2 levels). In addition, several natural product MDM2 inhibitors have been shown to induce MDM2 autoubiquitination and degradation, although the exact mechanism of action remains to be revealed [215].</p><!><p>Advances in the understanding of the conformation and structure of MDM2 have sparked the rational design of small molecule MDM2 inhibitors. Selective binding of another molecule to the surface of MDM2 can prevent the MDM2-p53 interaction, leading to the accumulation of p53 in the nucleus, and the subsequent induction of pro-apoptotic and anti-proliferative p53 functions. Most new chemical entities developed as MDM2 inhibitors are based on this principle [222]. High throughput screening methods, combined with combinatorial library synthesis, have led to the development of a number of small molecule MDM2 inhibitors with diverse chemical structures. These include chalcones, piperazine-4-phenyl derivatives, nutlins (cis-imidazolines), spiro-oxindoles, sulfonamides, benzodiazepinediones, isoindolinones, and terphenyls among others [221, 223–225]. Their corresponding binding domains on MDM2 and/or p53 proteins are shown in (Fig. 3).</p><!><p>The imidazoline derivatives (better known as Nutlins) are one of the first small molecules for selective inhibition of MDM2-p53 binding [198]. They act by mimicking the three critical amino acid residues (Phe19, Trp23, Leu26) within the hydrophobic pocket of MDM2. Crystallographic studies demonstrate that the chlorophenyl moieties perfectly fit deeply into the Leu26 and Trp23 pockets while the isopropylphenyloxy moiety mimics Phe19 with the phenyl groups serving as a connector to place the phenyloxy group into the Phe19 pocket. Nutlins do not induce p53 phosphorylation on Ser15, suggesting that they are non-genotoxic, selective inhibitors. Studies also suggest that the 4-methoxy functionality in nutlin3 mimics p53 Leu22. Nutlin-3, the most potent analog of the series, induces p53 levels and enhances p53 transcriptional activity. It has been shown to be effective in various cancer models with wild-type p53, such as neuroblastoma, colon, mantle cell lymphoma, breast and osteosarcoma [202, 226–228]. Nutlin-3 also activates p53 and induces apoptosis and cellular senescence in myeloid and lymphoid leukemic cells [229, 230]. An interesting observation has been that androgen deprivation followed by two weeks of Nutlin-3 administration in LNCaP bearing nude mice leads to a greater tumor regression and dramatically increases survival, indicating cross talk between p53, MDM2, and the androgen receptor [231].</p><p>This class of compounds have been shown to have effects on other proteins as well. In the absence of functional p53, Nutlin-3 disrupts p73-MDM2 interaction and increases p73 transcriptional activity, leading to greater apoptosis and growth inhibition in hepatocellular carcinomas [232]. Interestingly, Nutlin-3a, a compound that was designed to inhibit MDM2-p53 binding, has been shown to interfere with the MDM2-E2F-1 interaction [233]. Simultaneous treatment of a p53 mutant peripheral nerve sheath tumor cell line with Nutlin-3a and cisplatin leads to an increase in E2F-1 levels and E2F-1-mediated apoptosis [234]. Furthermore, E2F-1 knockdown inhibits Nutlin-3-mediated apoptosis [234]. Nutlin-3 also inhibits VEGF via inhibition of HIF-MDM2 interaction [235]. Nutlin-3 has also been shown to inhibit the protein expression of NFκB target genes ICAM-1 and MCP-1 in a p53-dependent manner [236]. ICAM-1 and MCP-1 are involved in cell migration and metastasis, suggesting a role for Nutlin-3 in the treatment of advanced diseases. The nutlin series of compounds have been shown to cause cell death via interaction with proteins such as TRAIL, PUMA, and others [229]. Nutlin therapy has also been shown to sensitize different cell lines such as laryngeal, lung, and prostate cancer cells to ionizing radiation [237]. Nutlin-resistant cell lines have wild-type MDM2 but mutations in the p53 DNA binding and dimerization domains. This chemoresistance can be overcome by the p53 targeting agent RITA [238]. Interestingly, the nutlins do not bind the MDM2 homolog MDMX. Furthermore, despite the similarity between MDM2 and MDMX, MDM2 inhibitors such as Nutlin-3 are far less effective against MDM4. However, MDMX inhibitors not only can activate p53 and induce apoptosis in breast cancer cells, but also synergize with MDM2 inhibitors for p53 activation and induction of apoptosis [239]. Nutlins-3a and −3b (inactive enantiomer) have been demonstrated to be substrates of multi-drug resistance protein-1, and act to reverse multi-drug resistance by saturating the efflux capabilities of these transporters [240]. However, several nutlins developed in the early phases failed to proceed to the trials due to poor pharmacokinetic properties [241].</p><p>Derivatives structurally related to nutlins include pyrrolidine-2-carboxamide derivatives, and imidazothiazole derivatives (with a proline moiety) [223]. A series of compounds with 3-imidazolyl-indole scaffolds as dual inhibitors of MDM2/MDMX-p53 interaction have been identified [242]. Spiroxindole derivatives represent another class of MDM2-p53 inhibitors designed to mimic the Trp23 residue that is the most critical for binding of p53 to MDM2. MI-63, MI-219, MI-319, and MI-147 belong to this class. Oxindoles were synthesized using a de novo approach to mimic the indole ring and also the side chain of Trp23. Shangary and others then used a substructure search technique to identify natural products with an oxindole substructure [202], such as spirotryprostatin A and alstonisine. MI-219, the clinical grade compound, is shown to alter the functional activity of MDM2 through enhancing its auto ubiquitination and degradation [243]. MI-219 causes p53 activation by blocking the MDM2-p53 interaction in wild-type p53 cells but exhibits much lower activity in cells with mutant p53, which is consistent with its mechanism of action as a specific inhibitor of the MDM2-p53 protein-protein interaction [202]. However, in the studies, MI-219 failed to achieve tumor regression in various xenograft models of human cancer though it was able to completely inhibit tumor growth [244]. Recently, the group has reported the synthesis of a series of highly potent diastereomeric spiro-oxindoles, in which stereochemistry affects binding affinity to MDM2 greatly [245]. Optimization of the pharmacokinetic properties of one of the analogs reported therein has yielded MI-888 (Ki = 0.44 nM) with highly selective activity in inhibiting the growth of wild-type p53 cancer cell lines [296, 297]. MI-888 exhibits very good oral bioavailability in rats along with complete tumor regression in mice bearing osteosarcoma (SJSA-1) and acute leukemia (RS4;11) xenografts [297]. An analogue of MI-888 has been advanced into Phase I clinical development [246].</p><p>Other MDM2 binding SMIs include chalcone and the NAcylpolyamine derivatives. The latter class represents cyclic peptides in β-sheet confirmation which inhibit the association of both MDM2 and MDMX with p53 [247]. The chal-cone derivatives have been shown to have anticancer activities in a number of cancer cells. Several derivatives prepared from Licochalcone-A, obtained from licorice root, inhibit MDM2-p53 interaction by binding near the Trp23 pocket of MDM2 [248]. A second generation of chalcone derivatives bearing a boronic fragment has also been synthesized and it is believed that boronic acid analogs may form a stronger salt bridge with Lys51 than corresponding chalcone analogs of caxboxylic acid moiety [248–250]. Surprisingly, the DNA damaging agent 5-fluorouracil has been demonstrated to stabilize and activate p53 by blocking MDM2 feedback inhibition through ribosomal proteins [251]. This finding warrants further investigation into the mechanism of current chemotherapeutic agents to unravel if they activate p53 by inhibiting MDM2.</p><p>Recently, Amgen has developed a series of piperidinone based compounds [252]. These compounds also interact with the Trp23, Leu26, and Phe19 residues in MDM2 apart from ordering the N-terminal residues of MDM2, thus resulting in very high affinity for MDM2 binding (MDM2 IC50= 1.1 nM) [252]. In a mouse SJSA-1 tumor xenograft model, oral administration of AM-8553 at 200 mg/kg once daily resulted in partial tumor regression, demonstrating its excellent antitumor activity and clinical translational potential [252].</p><!><p>The molecule RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis) acts by binding to the N-terminal of p53 and reactivating p53 function [249]. It is shown that RITA acts not only on wild-type p53, but also on mutant p53. RITA may bind directly with mutant p53 or by disrupting the inhibitory complex that mutant p53 forms with p63 and smad2, or the complex with p73. However, some investigators have demonstrated that RITA does not block MDM2-p53 binding [253, 254]. Therefore, a better understanding of the binding mode of this class of compounds is required for further development of them as effective anti-cancer agents.</p><!><p>The 5-deazaflavin compounds inhibit the E3 ligase activity of MDM2 by targeting the RING finger domain of the oncoprotein. These compounds are also known as HDM2 ligase inhibitor (HLI series). Treatment of these compounds results in p53 activation, despite causing stabilization of both p53 and MDM2. This activation is speculated to result from the direct binding and inhibition of the RING finger domain, the functional domain necessary for MDM2's E3 ligase activity. These compounds also affect the E2 ligase activity. However, at high concentrations, HLIs also cause cell death in p53 mutant cells [255].</p><p>Selective inhibitors of E3 ligase include three chemically distinct classes of compounds (namely, benzsulfonamides, ureas, and imidazolones). These are selective inhibitors of MDM2 E3 ligase, with little or no effect on auto ubiquitination of MDM2. They comprise of simple reversible inhibitors of MDM2 that bind to it in a manner noncompetitive with respect to the substrates, Ub-Ubc4 (E2) and p53. The lead candidate of tryptamine derivatives, JNJ26854165, blocks the degradation of p53 by inhibiting the binding of MDM2-p53 complex to proteasome. Treatment with this compound induced p53-mediated apoptosis in wild-type p53 cells, while in mutant p53 cells, JNJ26854165 induced S-phase cell cycle arrest and E2F1-mediated apoptosis. Notably, the compound is active against Nutlin-3a resistant samples, indicating involvement of a pathway other than MDM2-p53 inhibition [109, 220, 256].</p><p>Sempervirine, a natural indole alkaloid identified by a high throughput electrochemiluminescent screen that screened more than 100,000 natural compounds, has also been identified as an inhibitor of MDM2's E3 ligase activity [257]. Sempervirine inhibits the MDM2-mediated ubiquiti-nation and degradation of p53, resulting in the accumulation of p53. This compound also preferentially induces apoptosis in wild-type p53 expressing cancer cells, meriting further investigation of its anticancer activity [257].</p><!><p>Based on the reported chemopreventive activities in several human cancers, we have demonstrated that the soy-derived isoflavone genistein directly down-regulates MDM2 at both the transcriptional and post-translational levels, independent of the p53 status [212]. Our studies have also shown that genistein down-regulates MDM2 and increases p21 levels, independent of tyrosine kinase inhibitory activity of the compound. Another common flavonoid, apigenin, has been shown to induce p53 via MDM2 attenuation and to inhibit the phosphorylation of MDM2 by Akt (which, in turn, increases MDM2 stability and p53 degradation) in ovarian cancer cells [258]. Similarly, the sesquiterpene lactone parthenolide, isolated from the European feverfew herb, Tanacetum parthenium, induces MDM2 ubiquitination and proteasomal degradation in an ATM-dependent manner, subsequently activating p53 and other MDM2-regulated tumor suppressor [259, 260]. Berberine, a natural isoquinoline alkaloid obtained from the herb Rhizoma coptidis and used in Traditional Chinese Medicine, has been widely investigated due to its myriad pharmacological effects, including anticancer, anti-inflammatory, and antibacterial activities. Berberine induces apoptosis in acute lymphoblastic leukemia (ALL) cells through downregulation of the MDM2 oncoprotein in wild-type p53 ALL cell lines [261]. Berberine also decreases DAXX transcription, and subsequently prevents the formation of the MDM2-DAXX-HAUSP complex, resulting in the persistent self-ubiquitination of MDM2 in ALL cells [261].</p><!><p>Several natural products have shown inhibitory effects on MDM2 gene expression [215]. Ginsenoside saponins has been known to regulate multiple steps in cell proliferation, modulate the expression of tumor suppressors, oncogenes, growth factors, cell death mediators, pro-inflammatory molecules, and protein kinases [262]. These anticancer activities follow a well-defined structure-activity relationship. Over the past few years, our group has identified two new ginsenoside products, 20(S)-25-hydroxy-dammarane-3β,12β, 20-triol and its methoxyl derivative (25-OCH-PPD and 25-OCH3-PPD) which exhibit excellent anticancer activity against various human cancers, including prostate, pancreatic, lung, and breast cancers by decreasing MDM2 protein levels both in vitro and in vivo. 25-OCH3-PPD also inhibits the transcriptional activity of MDM2 in cancer cells [263–267].</p><p>Curcumin, a dietary polyphenol, also down-regulates MDM2 transcription through the PI3K/mTOR/ETS2 pathway and curcumin exposure sensitizes human cancer cells to chemotherapy and radiation via MDM2 [213]. Semisynthetic derivatives of the marine alkaloids such as the makaluvamines have been shown to decrease the levels of MDM2 oncoprotein, to cause apoptosis by the caspase-mediated pathway, and to inhibit the PI3k-Akt pathway [268–274].</p><!><p>A cis-imidazoline derivative belonging to the Nutlin family entered clinical trials in late 2007/early 2008. This compound codenamed RG-7112 molecule has just completed phase I clinical trials in patients with solid tumors, and hematologic neoplasia (ClinicalTrials.gov; identifiers: and ). This multicenter, open trial was conducted in the United States and France and designed to determine the maximum tolerated dose and the optimal dosing schedule of RG-7112, administered as monotherapy in patients with advanced solid tumors. A first cohort of patients received the starting dose of 20mg/m2/day, once daily for 10 days in each 28-day cycle. RG7112 treatment was seen to stabilize the p53 protein and induce p53 target genes such as CDKN1A, NOXA, PUMA, FAS and BAX. The compound exhibited good dose tolerance and positive effects in the treatment of acute or chronic leukemia and patients responded well to an escalation in dosage. It is reported that a single patient with AML who has been leukemia-free for 9 months subsequent to RG7112 treatment [275]. Reductions in lymph node and spleen size, as well as in circulating leukemia cells, were noticed in chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) conditions [276–278].</p><p>RG-7112 has also been tested in patients with well-differentiated liposarcoma prior to debulking surgery (ClinicalTrials.gov; identifiers: ) [277]. The study conducted in France sought to study biomarker evidence of MDM2 and p53 pathway alterations in a clinical setup. The results of these initial proof of mechanism studies are preliminary since a very small number of patients (20 patients) were enrolled. Although, the data indicate that RG7112 does reach its target in a solid tumor and correlates with increased p53 and p21 levels, increased MIC-1 levels (induced by p53), and decreased proliferation, the data did not reach statistical significance. Also, as the tumors were not microdissected, it may be possible that the heterogeneous tumor tissue contained only a small fraction of cells responsive to RG7112 treatment. The most frequent adverse event seen was hematological toxicities. Thus, RG7112 may be a part of neoadjuvant therapy in combination with existing clinically approved non-genotoxic chemotherapeutics. This will help prevent aggravating the hematological toxicities that are a hallmark of standard DNA damaging drugs. Further information on the molecule's pharmacokinetic profile and adverse effects are awaited. Currently, an ongoing clinical trial is recruiting participants for a multicenter, open-label, Phase Ib study for RO5045337 (the oral formulation of RG7112) that will evaluate the safety, pharmacokinetics and efficacy of this drug in combination with doxorubicin in patients with soft tissue sarcoma. Another small molecule MDM2 inhibitor, RO5503781, presumably with a similar structure, has been launched into clinical trials, presently recruiting participants for a multicenter, open label, dose-escalating study to evaluate the safety, pharmacokinetics, pharmacodynamics and efficacy in patients with advanced malignancies except leukemia. In the clinical trials, this drug is being tested as a single agent or in combination with cytarabine (ClinicalTrials.gov; identifiers: and ).</p><!><p>SAR405838 (MI-888), an analogue of the MI series (MI-219: clinical prototype), discovered by Shangary et al. at the University of Michigan, was launched into phase I clinical trials by Sanofi S.A in 2012 (ClinicalTrials.gov; identifier: ). This study, as of date, is still recruiting participants and no results are available. The study aims to assess the drug's efficacy in patients with dedifferentiated liposarcoma and determine safety and the maximum tolerated doses (MTD).</p><!><p>Another MDM2 inhibitor in clinical trials is the E3 ubiquitin ligase inhibitor JNJ-26854165 (ClinicalTrials.gov; identifier: ) [220, 279, 280]. The first observations from the clinical trials were presented in 2009. This orally bioavailable drug has just completed phase I clinical trials for the treatment of advanced stage or refractory solid tumors in USA [276]. The starting dose of JNJ-26854165 in the trial was 4 mg/day as a single oral dose. Side effects reported with the treatment of JNJ-26854165 included nausea, vomiting, fatigue, anorexia, insomnia, electrolyte imbalance, creatinine elevations, and asymptomatic QTc prolongation. No hematological or cardiovascular toxicities were observed. A single patient with higher dose exhibited a grade 3 QTcF prolongation which was reversed after discontinuation of the treatment. Linear pharmacokinetics was seen in 20 to 400 mg dose range, with the preclinically determined therapeutic concentration being achieved at doses above 300 mg [276]. The levels of p53 were upregulated in skin while MDM2 levels were enhanced in tumors (probably due to loss of auto ubiquitination function) in a dose dependent manner. Similarly, the levels of MIC-1, a plasma macrophage inhibitory cytokine, a member of the TGF-β super family and induced by p53, were also increased in dose-dependent manner. A dose of 350 mg was used on expanded cohort of patients to confirm maximum tolerated dose, and a separate trial was started with alternate dosing schedule (150 mg twice a day) to minimize QTc prolongation.</p><!><p>A novel chemical moiety thioureidobutyronitrile (Kevetrin) is also being launched into Phase I dose escalation and safety clinical trials this year. In preclinical studies, Kevetr in activates p53, induces p21 and PUMA (p53 up-regulated modulator of apoptosis), a p53 activated proapoptotic protein (ClinicalTrials.gov; identifier: NCT 01664000). Recently, Novartis has launched an orally active MDM2-p53 interaction inhibitor, codenamed CGM097, into clinical trials for treatment of p53 wild-type tumors (ClinicalTrials.gov; identifier: ).</p><p>In addition to these MDM2 small molecule inhibitors, other compounds in clinical development include MK-8242 (or SCH 900242), which is being tested in two phase I clinical trials (ClinicalTrials.gov; identifier: and ). This compound is being tested as a single agent in patients with advanced solid tumors and in combination with cytarabine in acute myelogenous leukemia (AML) subjects. Lately, Daiichi Sankyo Inc. has commenced phase I clinical trial of DS-3032b (ClinicalTrials.gov; identifier: ) in patients with advanced solid tumors or lymphomas.</p><p>Of note, several of the naturally occurring compounds such as ginseng saponins and curcumin have been extensively studied for efficacy and safety in clinical setups, although they were not initially investigated as MDM2 inhibitors. They present a veritable treasure house of MDM2 inhibitors for future development. In (Fig. 4), we present the structures of several prototype MDM2 inhibitors both synthetic and natural origin, while in (Table 3) we summarize the mechanisms of action of these prototype synthetic and natural SMIs along with their clinical development statuses.</p><!><p>Disruption of the MDM2-p53 interaction with small molecule inhibitors is an attractive cancer therapeutic strategy. However, drug discovery efforts in this area have been primarily focused on strategies to inhibit the binding of p53 to the N-terminal domain of MDM2 and several novel scaffolds (both from rational drug synthesis and natural sources) have been developed [286–293]. Some of these compounds such as the dihydroimidazothiazole derivatives synthesized by scientists at Daiichi employ structural modifications of the nutlins followed by subsequent optimization of potency and pharmacokinetic behavior [289]. We now know that other domains in MDM2 are also involved in the MDM2-p53 interaction and mutations in these domains are associated with cancer [294]. In fact, it has been reported that the ligands binding to the MDM2 acid domain cause p53-mediated inhibition of cell growth and induce apoptosis [295]. This highlights the importance of the acidic domain in addition to the N terminus as a potential target for small molecular MDM2 inhibitors. Directly inhibiting the MDM2-p53 interaction, as a means of restoring p53 wild-type functions, is potentially useful in the treatment of cancers which harbor wild-type p53, but there still exist concerns as to how viable this concept would be clinically: how efficient will a chemical moiety in inhibiting a highly specific protein–protein interaction? What will be the adverse effects of unleashing a potent pro-apoptotic molecule like p53 on healthy cells? And since p53 transcriptionally activates MDM2, will increased p53 lead to increased MDM2 levels due to the MDM2-p53 feedback loop? Present evidence is in favor of these inhibitors; with a number of them entering clinical trials, the ultimate proof of concept may be just around the corner. More than twenty different chemical classes have been claimed to inhibit the MDM2-p53 interaction, but the majority of studies have been directed toward three classes: benzodiazepinediones, spiro-oxindoles (the MI series of compounds), and cis-imidazolines (Nutlins). Further preclinical and clinical studies on other compounds may provide more information on the value of targeting MDM2 and MDM2-p53 interaction.</p><p>In order to critically evaluate the mechanism of action and therapeutic potential of a MDM2 inhibitor, it should have the following desirable "drug-like" properties: (i) high binding affinity and specificity towards MDM2, (ii) high cytotoxicity in cancer cells with wild-type p53, and (iii) a highly desirable pharmacokinetic (PK) profile [202]. The p53-binding site of MDM2 is highly hydrophobic, and therefore all the non-peptide drugs that have been developed are, necessarily, lipophilic, and, thus lack aqueous solubility. It is true that several potent MDM2 inhibitors have been tested in animal models of human cancer for their anticancer activity. However, some of these compounds such as Nutlin 3A and MI-219 were not able to achieve complete tumor regression and also showed variable activity in different cancer types (Nutlin-3 and MI-219 inhibit tumor growth completely in SJSA-1 derived xenografts but show minimal activity against HCT-116 colon cancer xenografts) [244]. These results were consistent with data obtained from in vitro cell experiments [244, 296]. It is noteworthy that the antitumor activity of these compounds (nutlins as well as the MI series) was achieved at doses that caused no visible toxicity to the animals, as evidenced by body weight and gross organ morphology at necropsy [198–200, 244, 246, 275, 276, 296, 297]. Since derivatives of nutlins (such as RG7112) with optimized pharmacological parameters (with respect to bioavailability) are able to achieve tumor regression (either partial or complete), it is evident that potent and highly optimized MDM2 inhibitors can achieve impressive anticancer activity in animal models of human cancers [275]. Indeed, researchers at University of Michigan have carried out structural modifications in their MI-series based on the stereo-chemical properties of these compounds alongwith optimization of the pharmacokinetic parameters (by adding more "biological friendly" side chains that increase bioavailability) [244–246]. These efforts have yielded MI-888 which exhibits excellent oral bioavailability alongwith complete tumor regression in two animal models of human cancer [246, 296, 297]. An analogue of MI-888 has also advanced into phase-I clinical trials [297]. An interesting study by Azmi et al. details the use of the essential trace element zinc along with the MDM2 antagonist, MI-219 [298]. Zinc is an important part of the p53 biochemistry, with p53 binding to DNA through a structurally complex domain stabilized by zinc atom [298]. The MDM2 protein also carries a C-terminal RING domain that coordinates two zinc atoms, which are responsible for p53 nuclear export and proteasomal degradation [298]. Zinc chloride supplemented MI-219 regimen suppresses the p53 feedback MDM2 activation, thus increasing its efficacy [298].</p><p>However, till date, two MDM2 inhibitors (compounds inhibiting the MDM2-p53 interaction), Nutlin-3 and MI-219, appear to meet enough "drug-like" criteria. In addition, the benzodiazepinedione compound TDP665759, with an IC50 value of 704 nM appears to be another suitable compound. On the contrary, other small molecule inhibitors that target the MDM2-p53 interaction have either exhibited modest cytotoxic activities in cells or do not possess high binding affinity and/or specificity towards MDM2. Moreover, information on their cellular mechanisms of action, in vivo anti-cancer activity in preclinical set-up, cellular specificity for cancerous cells versus normal cells has not yet been reported.</p><p>Inhibitors of the MDM2-p53 interaction are unique in the aspect that, unlike, several traditional chemotherapeutics, these compounds induce p53 accumulation and activation in a non-genotoxic manner (without inducing DNA damage or requiring ATM/ATR-dependent p53 phosphorylation). Thus, these compounds do not technically affect p53 post-translational modification pathways such as phosphorylation, acetylation and sumoylation that cause p53 activation in response to genotoxic stress.</p><p>Although these inhibitors are highly effective against tumors containing wild-type p53, tumors with mutated or nonfunctional p53 may not be responsive. In fact, mutant p53 is known to stabilize MDM2. Considering that MDM2 has multiple p53-independent oncogenic activities, compounds such as nutlins that have been developed to specifically inhibit MDM2-p53 interaction would display poor effects on p53-inactivated tumors. The development of resistance following MDM2 inhibitor treatment still needs to be further investigated. Moreover, the pharmacokinetic properties and bioavailability of synthetic MDM2 inhibitors need major improvements before clinical use can be permitted. For instance, several initial analogs in the nutlin series have inadequate bioavailability in vivo [202].</p><p>As aforementioned, MDM2 is one of the most important oncogenes in the entire process of cancer. In addition to being a negative regulator of p53, it interacts with numerous other proteins involved in diverse cellular functions ranging from cell proliferation, apoptosis, to metastasis and angiogenesis, indicating that these signaling pathways have a potential to be explored as therapeutic targets. Targeting these interactions may provide alternative or complementary approaches to targeting the MDM2-p53 interaction, especially for the treatment of cancers with mutant p53 or loss of p53 function. Given the p53-independent activities of MDM2 in cancer, it becomes necessary to focus on MDM2 itself as a drug target. The oncogenic activities of MDM2 that have potential to be translated into possible molecular targets for cancer therapy include: (i) binding and destabilization of p21, (ii) binding and inhibition of the p53 homologues, p63 and p73, (iii) positive modulation of HIF-1α transcription factor/VEGF [299], (iv) interaction with ribosomal proteins, (vi) inhibition of MDM2 by the tumor suppressor ARF, (vii) inhibition of pRb, (viii) its interaction with hormone receptors such as androgen receptor and estrogen receptor, and (iv) its role in epithelial-mesenchymal transition and metastasis. However, more studies are required to elucidate the role(s) of these interactions, and to define the circumstances under which these interaction(s) can be successfully targeted. The use of system biology and modern high throughput drug screening techniques, combined with an increasingly in-depth understanding of the biochemistry and molecular biology of MDM2 will help us to develop novel MDM2 inhibitors.</p><p>MDM2 expression is regulated at multiple levels through various cellular mechanisms. Therefore, it is imperative to understand these mechanisms to better develop therapeutic strategies for MDM2 inhibition. In addition to p53, the transcription of MDM2 is regulated by other transcription factors such as NF-κB [300], Fli-1 [301], ETS [213], AP1 [302], and NFAT1 [303]. Inhibition of these transcription factors may provide another strategy to overcome MDM2 deregulation in cancer cells. Indeed, we have seen that curcumin suppresses MDM2 though the inhibition of ETS transcription factor. Thus, we believe a therapeutic regimen combining an MDM2 transcriptional inhibitor may benefit those harboring high level of MDM2 and/or inactivated p53.</p><p>Another mechanism by which MDM2 can be prevented from destabilizing p53 is by inhibiting its E3 ubiquitin ligase activity. However, not much progress (in terms of number of new chemical entities generated) has been made on MDM2 E3 ubiquitin ligase inhibitors on account of the biological complexity of the ubiquitination process. It is hoped that a better understanding at the molecular level of how exactly MDM2 functions as an E3 ubiquitin ligase will facilitate structure-based drug design and discovery. Conceptually, E3 ligases are very attractive drug targets as they mediate a majority of protein destruction mechanisms. Moreover, targeting MDM2 E3 ubiquitin ligase may have a broader spectrum of activity in cancers exhibiting mutant and non-functional p53. In fact, a study employing a combinatorial regimen of bortezomib (a proteasome inhibitor) along with nutlin shows excellent activity against myeloma [304, 305].</p><p>At present, most of the available MDM2 inhibitors lack activity against MDMX. Although Nutlin 3a and MI-219 activate p53 in cancer cells with overexpressed MDM2, they do not have similar effects in cells with overexpressed MDMX. MDM2 inhibitors are not able to bind to MDMX although MDMX has similarities in the N-terminal p53-binding domains to that of MDM2. Subtle structural differences in the MDMX-p53 interaction pocket drastically reduce the binding affinity of both Nutlin 3a and MI-219 for MDMX. This observation is supported by in vitro binding studies which demonstrate that Nutlin 3a is 500-fold less potent against MDMX compared with MDM2 [306]. MDM2 and MDMX possess non-redundant roles in modulating p53 activity and are the major antagonists of p53 in vivo. Therefore, dual antagonists for both MDM2 and MDMX may effectively reactivate p53 in cancer. RO-5963 has been identified as a dual inhibitor of MDM2-p53 and MDMX-p53 interactions. Structural analyses suggest that the compound induces p53 activity via formation of MDM2-MDMX homodimers and heterodimers [306].</p><p>Finally, natural compounds present an attractive area for the development of MDM2 inhibitors as they have been shown to target both the p53-dependent and -independent activities of MDM2, exhibiting impressive activity even in p53 mutant or non-functional conditions, thus eliminating the need for p53-dependent mechanisms to exert their effects. On the other hand, most synthetic small molecule inhibitors of MDM2 are known to restore the pro-apoptotic and anti-proliferative functions of p53 by disrupting the MDM2-p53 interaction and, therefore, are less effective in p53 mutant tumors. Further research on the reasons for the effectiveness of naturally occurring compounds in a p53 mutant scenario may provide clues about the structural requirements of such p53-independent MDM2 inhibitory activity. The unique frameworks of the natural compounds can inspire to develop novel synthetic and semi-synthetic derivatives with improved efficacy, pharmacokinetic, and bioavail-ability profiles, thus opening new avenues in MDM2-p53 research.</p>

PubMed Author Manuscript

Time-resolved proteomic analysis of quorum sensing in Vibrio harveyi

Bacteria use a process of chemical communication called quorum sensing to assess their population density and to change their behavior in response to fluctuations in the cell number and species composition of the community. In this work, we identified the quorum-sensing-regulated proteome in the model organism Vibrio harveyi by bio-orthogonal non-canonical amino acid tagging (BONCAT). BONCAT enables measurement of proteome dynamics with temporal resolution on the order of minutes. We deployed BONCAT to characterize the time-dependent transition of V. harveyi from individual-to group-behaviors.We identified 176 quorum-sensing-regulated proteins at early, intermediate, and late stages of the transition, and we mapped the temporal changes in quorum-sensing proteins controlled by both transcriptional and post-transcriptional mechanisms. Analysis of the identified proteins revealed 86 known and 90 new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

time-resolved_proteomic_analysis_of_quorum_sensing_in_vibrio_harveyi

Introduction<!>Results<!>Detection of quorum-sensing regulators<!>Quorum sensing causes global changes in protein synthesis<!>Bioinformatic analysis reveals regulation of functionally related protein groups<!>Dening the temporal order of protein regulation in response to quorum sensing<!>Quorum sensing regulates type VI secretion proteins in V. harveyi<!>Discussion and conclusions<!>Cell culture<!>Molecular methods<!>In-gel digestion<!>Protein quantication and ratio statistics<!>Bioinformatic analysis

<p>Bacteria assess their cell numbers and the species complexity of the community of neighboring cells using a chemical communication process called quorum sensing. Quorum sensing relies on the production, release, accumulation and group-wide detection of signal molecules called autoinducers. Quorum sensing controls genes underpinning collective behaviors including bioluminescence, secretion of virulence factors, and biolm formation. [1][2][3] The model quorum-sensing bacterium Vibrio harveyi integrates population-density information encoded in three autoinducers AI-1, CAI-1, and AI-2, which function as intraspecies, intragenus, and interspecies communication signals, respectively. [4][5][6] V. harveyi detects the three autoinducers using the cognate membrane-bound receptors LuxN, CqsS, and LuxPQ, respectively. [7][8][9] At low cell density (LCD), autoinducer concentrations are low, and the unliganded receptors act as kinases, funneling phosphate to the phosphorelay protein LuxU. 10 LuxU transfers the phosphoryl group to the response regulator protein LuxO, which activates transcription of genes encoding ve homologous quorum regulatory small RNAs (qrr sRNAs). 11,12 The Qrr sRNAs post-transcriptionally activate production of the transcription factor AphA and repress production of the transcription factor LuxR. AphA and LuxR are the two master quorum-sensing regulators that promote global changes in gene expression in response to population density changes. [12][13][14][15] At high cell density (HCD), autoinducer binding to the cognate receptors switches the receptors from kinases to phosphatases, removing phosphate from LuxU and, indirectly, from LuxO. Dephosphorylated LuxO is inactive so transcription of the qrr sRNA genes ceases. This event results in production of LuxR and repression of AphA. 12 Thus, the circuitry ensures that AphA is made at LCD, and it controls the regulon required for life as an individual, whereas LuxR is made at HCD, and it directs the program underpinning collective behaviors.</p><p>Previous microarray studies examined the transcriptomic response during quorum-sensing transitions. That work showed that AphA and LuxR control over 150 and 600 genes, respectively and $70 of these genes are regulated by both transcription factors. 15 Both AphA and LuxR act as activators and as repressors, and thus the precise pattern of quorumsensing target gene expression is exquisitely sensitive to uctuating levels of AphA and LuxR as cells transition between LCD and HCD modes. Developing a comparable understanding of the quorum-sensing-controlled proteome requires measurement of dynamic changes in protein abundance throughout the transition from individual to collective behavior.</p><p>In this work, we used the bio-orthogonal non-canonical amino acid tagging (BONCAT) method to track the proteomewide quorum-sensing response in V. harveyi with temporal precision. BONCAT enabled us to identify 176 proteins that are regulated during the transition from individual to collective behavior; 90 of these proteins are in addition to those identied in earlier studies. We show that a broad range of protein functional groups, including those involved in metabolism, transport, and virulence, change during the transition to group behavior. We demonstrate how particular temporal patterns of protein production are linked to particular tiers of the regulatory cascade by comparing the proteomic proles of the regulon controlled by the post-transcriptional Qrr sRNAs to the regulon controlled by the transcriptional regulator LuxR. Using this approach, we, for example, determined that the V. harveyi type VI secretion system is LuxR-regulated.</p><!><p>The BONCAT method was developed to provide time-resolved analyses of the cellular proteome. 16,17 In a BONCAT experiment, the non-canonical amino acid L-azidohomoalanine (Aha; Fig. S1a †) is provided to cells and, subsequently, incorporated into proteins in competition with methionine. 18 Aha-labeled proteins are chemically distinct from the remainder of the protein pool and thus, labeled proteins can be selectively conjugated to affinity tags for enrichment and mass spectrometry analysis (Fig. 1a). Because Aha can be introduced into cells in a well-dened pulse, BONCAT offers excellent temporal resolution and high sensitivity to changes in protein synthesis in response to biological stimuli. 19 Our goal was to identify time-dependent changes in protein production associated with quorum sensing. We chose to monitor the transition from individual to group behavior in V. harveyi because the core transcriptional regulon is wellestablished, providing a solid foundation for comparisons between transcriptional and translational outputs. 15 To experimentally manipulate the transition from LCD to HCD, we used V. harveyi strain TL25 in which the genes encoding the autoinducer receptors for CAI-1 (cqsS) and AI-2 (luxPQ) and the AI-1 synthase (luxM) have been deleted. 15 Thus, V. harveyi TL25 responds exclusively to exogenously supplied AI-1, which enables precise control over the activation of quorum sensing.</p><p>The hallmark phenotypic response controlled by quorum sensing in V. harveyi is bioluminescence, which is activated by LuxR during the transition from LCD to HCD. 20 Thus, we reasoned that light production could serve as a proxy for activation of quorum sensing. 20 Upon treatment of a culture of V. harveyi TL25 with AI-1, bioluminescence increases sharply aer 30 min and plateaus at a level 400-fold higher than the preaddition level aer approximately 90 min (Fig. 1b). Detection of Aha incorporation in V. harveyi cultures by in-gel uorescence showed that BONCAT experiments could be performed in this system with a temporal resolution of ten minutes (Fig. 1c). Using the bioluminescence prole as a guide, we combined two techniques, BONCAT and stable isotope labeling with amino acids in cell culture (SILAC), to monitor both increases and decreases in protein synthesis in ten-minute intervals between 0 and 90 min following addition of AI-1 (Fig. 1b and S1c and d †). 19,21 V. harveyi cultures that were not treated with AI-1 served as references for relative quantication. As expected, the production of the luciferase subunits LuxA and LuxB tracked with the bioluminescence prole in cultures treated with AI-1 (Fig. 1b). We detect LuxB at 30 min, slightly before we can detect LuxA. The LuxB measurement is coincident with the rst increase in bioluminescence. Between 40 and 50 min, bioluminescence and LuxA and LuxB levels exhibited sharp increases, aer which, both continued to climb at slower rates. Between 60 and 90 min, the production rates of LuxA and LuxB remained nearly constant while bioluminescence continued to increase. LuxA and LuxB increased about 8-fold total in response to autoinducer supplementation. This result highlights the fact that BONCAT measures protein synthesis rates during individual time intervals (not total protein abundance), whereas bioluminescence output reports on the total accumulated LuxAB activity.</p><p>LuxA and LuxB are encoded by the lux operon, which also encodes LuxC, an acyl-CoA reductase, LuxD, an acyl transferase, and LuxE, a long-chain fatty-acid ligase. LuxCDE synthesize the substrate required by the LuxAB luciferase enzyme. All ve proteins exhibited large, concurrent increases in translation at 50 min (Fig. 1d). The increase in bioluminescence precedes production of LuxCDE, which suggests some basal level of luciferase substrate is present. The coincidence of the production of LuxA and LuxB with the onset of bioluminescence, and the simultaneous up-regulation of all of the proteins in the lux operon validate the BONCAT technique as a reliable method for time-resolved analysis of the quorum-sensing response.</p><!><p>At the core of the quorum-sensing circuit are the transcriptional regulators LuxO, AphA, and LuxR, which drive quorum-sensing transitions. Expression of luxO, aphA, and luxR are themselves controlled by multiple regulatory feedback loops. 13,15,[22][23][24] To assess the consequences of addition of AI-1 to V. harveyi TL25 on these core regulators, we monitored both mRNA and protein synthesis using qRT-PCR and BONCAT, respectively. LuxO, AphA, and LuxR all showed rapid changes in protein production within 20 min of AI-1 treatment (Fig. 2). AphA and LuxR reached near-maximal differences in translation at the 30 min point; AphA protein production decreased 4-fold and LuxR protein production increased 16-fold. The mRNA levels of aphA and luxR tracked with those of AphA and LuxR protein changes, with the exception that luxR mRNA decreased in abundance between 60 and 90 min while the protein level remained constant. LuxO protein exhibited a consistent 2-fold increase in abundance throughout the time-course, whereas the corresponding mRNA levels slightly decreased. This pattern is consistent with the recent nding that the Qrr sRNAs control luxO mRNA through a sequestration mechanism such that the Qrr sRNAs repress LuxO protein production while not signicantly altering mRNA abundance. 25</p><!><p>Using the above protocol for induction of quorum sensing in V. harveyi TL25, we next examined the quorum-sensingcontrolled proteome using BONCAT to monitor protein synthesis in ten-minute time intervals immediately following addition of AI-1. We collected a total of 700 174 MS/MS spectra and identied 9238 peptides and 1564 unique protein groups (Fig. S3a and b, dataset S1 †). Proteins were identied with an average of 6 peptides (median ¼ 4); 88% of proteins were iden-tied by 2 or more peptides (Fig. S3c †). Relative protein abundances at each time point were calculated with an average of 49 unique quantications (median ¼ 17) (Fig. S3d †). By comparing evidence counts, MS-MS counts, and MS intensities of Met and Aha-containing peptides, we estimated the extent of replacement of Met by Aha to be roughly 15% (Table S1 †). Proteins with differences greater than 1.5-fold with false discovery rateadjusted p-values less than 0.05 were considered signicant.</p><p>Induction of quorum sensing altered production of 176 proteins (Fig. 3a). Unsupervised hierarchical clustering partitioned the regulated proteins into 10 groups based on their temporal production proles (Fig. 3a and b). Proteins from the lux operon clustered closely (group F), and LuxR and AphA, which exhibited distinct production proles, were assigned to very small clusters. Several clusters showed differences in protein production at early time points (groups D, E, I), whereas other clusters changed more abruptly at the 50 min time point (groups B, D, F, H) (Fig. 3b). Differences in protein production between AI-1-treated and control cultures were modest within the rst 20 min, with only 7 and 19 signicant protein changes at 0-10 min and 10-20 min, respectively. The number of autoinducer-regulated proteins increased with time aer induction, with 42-119 proteins altered between 40-90 min aer AI-1 treatment (Fig. 3c and d). 90 of the AI-1-regulated proteins are newly associated with quorum sensing in V. harveyi (Fig. 3e, Table 2). In total, our analysis identied 278 proteins that are members of the previously established aphA, luxR, or quorumsensing regulons. 15 Interestingly, only 86 of these proteins exhibited signicant up-or down-regulation by BONCAT (Fig. S4 †).</p><!><p>To identify major shis in protein production in response to induction of quorum sensing, we used principal component analysis (PCA) to simplify the dataset by reducing the dimensionality from 9 time points to 2 principal components. Weighting vectors showing the contribution of each time point to the principal components highlighted three distinct proteomic states: (1) an early period in which few proteins changed (10-30 min), (2) a transitional period that included rapid changes in protein production (40-50 min), and (3) a late period in which many proteins exhibited large differences in translation (60-90 min) (Fig. 4a, Table S2 †). As conrmation of these states, proteins with principal component coordinates near the 1 st , 2 nd , and 3 rd sets of vectors exhibited time-course production proles with punctuated changes at early, middle, and late stages (Fig. 4b). Gene ontology analysis identied 13 protein groups regulated by quorum sensing (Fig. 4c and S5 †). Several of these groups were involved in transport, including iron, oligopeptide, and dicarboxylic acid transport. A set of 50 proteins with functional annotations for transporter activity was the largest of enriched ontology groups. Other groups of biological processes included bioluminescence, type VI secretion, siderophore synthesis, thiamine metabolism, and chemotaxis.</p><p>To identify groups of functionally related proteins with similar patterns of protein production, we mapped protein interactions from the STRING database onto the PCA plot and scanned for protein networks that localized via their principal components (Fig. 4d). Consistent with our gene ontology analysis, we identied interacting protein groups associated with regulation of bioluminescence, type VI secretion, chemotaxis, iron homeostasis, oligopeptide transport, and thiamine metabolism in the quorum-sensing response (Fig. 4d). For example, regarding peptide transport, synthesis of the substrate binding protein of the oligopeptide permease complex, OppA, decreased two-fold between 50-90 min. 26 Also, a large group of proteins ( 16) involved in iron transport exhibited decreased production proles late in the experiment, and a group of ironregulatory proteins (6) increased in levels. With respect to chemotaxis, we observed both increases and decreases in protein levels: homologs of methyl-accepting chemotaxis proteins and the CheA and CheY signaling proteins decreased, whereas putative methyl-accepting chemotaxis proteins increased in abundance. Taken together, these results suggest an overall quorum-sensing-driven remodeling of iron homeostasis and chemotactic behavior.</p><!><p>The Qrr sRNAs play a central role in dictating the transition between LCD and HCD states by controlling expression of the quorum-sensing transcriptional regulators, AphA, LuxR, and LuxO (Fig. 5a). 22,23 The Qrr sRNAs directly regulate 16 additional targets outside of the quorum-sensing cascade with functions in virulence, metabolism, polysaccharide export, and chemotaxis. 27 The direct Qrr targets constitute the set of "rstresponse" genes and also trigger the later, broader changes in downstream gene expression. With respect to the second wave of quorum-sensing gene expression changes, LuxR plays the major role. Therefore, we compared the temporal patterns of regulation of proteins known to be direct targets of either the Qrr sRNAs or LuxR. 27,28 We detected regulation of production of seven proteins known to be encoded by Qrr-regulated genes, all of which exhibited signicant differences in expression within 20 minutes of AI-1 treatment (Fig. 5b, Table S3 †). Conversely, 20 of the 21 LuxR-regulated proteins identied by BONCAT showed differences in production only aer at least 30 minutes of AI-1 induction. Thus, the differences in timing between Qrr-and LuxR-regulated genes reect the underlying structure of the quorum-sensing circuitry. We investigated the protein production proles of the newly identied proteins to pinpoint additional candidates for regulation by the Qrr sRNAs. We found 19 additional proteins that are regulated within 20 minutes of AI-1 treatment, suggesting that the corresponding mRNAs may be targeted by the Qrr sRNAs (Table 1). The candidates include two putative chemotaxis proteins, the serine protease inhibitor ecotin, the type III secretion protein chaperone SycT, a chitinase, and several other proteins involved in metabolism. Strikingly, the mRNA and protein production of VIBHAR_02788 (a predicted chemotaxis protein) increased 4-and 12-fold, respectively, within the rst 10 minutes aer AI-1 treatment, suggesting that VIBHAR_02788 is a good candidate for posttranscriptional regulation by the Qrr sRNAs (Fig. 5c).</p><p>The mechanisms that control production of quorum-sensingregulated proteins undoubtedly become more complex as the response progresses. We identied proteins that were regulated at all stages (early (0-20 min), intermediate (20-60 min), and late (60-90 min)) following AI-1 treatment (Fig. 5d, dataset S1 †). Differences in the timing of quorum-sensing-regulated proteins suggest that additional regulatory components or mechanisms orchestrate the transition from individual to group behavior. For example, direct LuxR targets were regulated in both the intermediate and late phases, despite the fact that LuxR reaches its peak production at 30 min (Fig. 2a). This result suggests that accumulation of LuxR or additional transcriptional regulators contribute to control of LuxR-regulated genes.</p><!><p>Components of the type VI secretion system (TSSS) were among the proteins most strongly up-regulated in response to AI-1 treatment (Fig. 6a). Identied TSSS proteins included the haemolysin co-regulated effector protein (Hcp; VIBHAR_05871), and two additional proteins whose homologs have been implicated in TSSS regulation and Hcp secretion (VIBHAR_05854 and VIB-HAR_05858). 29,30 TSSS proteins exhibited a coordinated increase in production at 50 min, a prole similar to that of LuxCDABE. In V. harveyi, the TSSS homologs are encoded by ve putative operons: VIBHAR_05855-05851, VIBHAR_05856-05858, VIBHAR_05865-05859, VIBHAR_05871-05866, and VIB-HAR_05872-05873 (Fig. S6a †). Analysis of the mRNA levels of the operons conrmed the increase in expression of TSSS components between 50 to 60 min aer AI-induction; timing consistent with second-tier regulation (Fig. 6b). Previous microarray data comparing wild-type, DluxR, DaphA, and DluxR DaphA V. harveyi strains showed that TSSS gene expression was reduced in DluxR strains, but expression was not altered in the DaphA strain, providing evidence that expression of TSSS genes is LuxR-dependent and AphA-independent (Fig. S6b †). 13 Consistent with this notion, ChIP-seq data identied a LuxR binding site in the bi-directional promoter region of VIB-HAR_05855-05856. 28 Using electrophoretic mobility shi assays, we conrmed the presence of this LuxR binding site and determined that LuxR binds to two additional promoter regions in the TSSS locus (Fig. S6c †). This result shows that, unlike Vibrio cholerae which deploys the Qrr sRNAs to posttranscriptionally regulate TSSS, V. harveyi uses LuxR to control TSSS production. 31 This nding suggests that although both organisms have TSSS under quorum-sensing control, they employ different regulatory strategies to achieve distinct timing of TSSS protein production.</p><!><p>Global transcriptomic studies of V. harveyi have uncovered a continuum of changes in gene expression during the transition from LCD to HCD. As V. harveyi responds to changes in concentrations of autoinducers, shis in the levels of the regulatory components AphA, LuxR, and the Qrr sRNAs occur, which in turn alter the expression of the downstream genes in the quorumsensing regulon. Here we used the BONCAT method to measure changes in the quorum-sensing-regulated proteome during the transition from LCD to HCD, with a time-resolution of 10 min. We found correlated changes in production of the LuxCDABE enzymes and in the intensity of bioluminescence produced by the culture, and we observed regulation of the core regulatory components AphA, LuxR, and LuxO. Notably, the increase in LuxO upon induction of quorum sensing occurred at the level of the protein, but not the mRNA, consistent with the hypothesis that the luxO mRNA is regulated by sequestration by the Qrr sRNAs. 25 The time resolution of the BONCAT method allowed us to identify proteins whose rates of synthesis were altered during the early, intermediate, and late stages of the LCD to HCD transition. The proteins found to be regulated within the rst 20 min of autoinducer treatment included seven of the 20 known Qrr sRNA targets along with 19 other proteins not previously associated with Qrr regulation. No known Qrr targets were regulated at later times. In contrast, changes in the known LuxR targets occurred between 30 and 90 min following induction. Notably, proteins in the TSSS were up-regulated between 40 and 50 min following autoinducer treatment, suggesting LuxR regulation of type VI secretion in V. harveyi; this conclusion was conrmed by electrophoretic mobility shi assays. Several LuxR-regulated genes exhibited changes in protein production only very late in the BONCAT experiment, which suggests either that they are responsive to accumulating LuxR levels, that they are regulated by another transcription factor downstream of LuxR, or that they are co-regulated by other factors.</p><p>We found quorum-sensing-dependent changes in 176 proteins that span a broad range of functional groups, including those related to iron homeostasis, molecular transport, metabolism, and chemotaxis. Ninety of these proteins are newly associated with quorum sensing in V. harveyi, and expand what is known about the roles that quorum sensing plays in these processes. 13,32 The remaining 86 proteins are members of the previously established quorum-sensing, AphA, and/or LuxR regulons. Interestingly, nearly 200 other proteins from these regulons were identied by BONCAT but were not signicantly up-or down-regulated. For example, the quorum-sensing regulon, which was dened by differences in gene expression between a mutant V. harveyi strain locked at LCD and a strain locked at HCD, contains 365 regulated genes as determined by microarray analysis. 15 We quantied protein expression levels of 127 (35%) of these genes, 45 (35%) of which were signicantly regulated. The differences between the genetic and proteomic results may arise, at least in part, from differences in regulation at the levels of mRNA and protein, or from differences in the growth media used in the two experiments (rich (LM) medium in the genetic study vs. minimal (AB) medium here). 13,15 Furthermore, we would not expect the rapid addition of saturating amounts of AI-1 to a V. harveyi culture to reproduce precisely the effects of genetically locking the strain into either the LCD or the HCD state. Determining how environmental conditions affect the quorum-sensing response will be important to the development of a full understanding of bacterial communication in complex natural environments.</p><p>The BONCAT method has allowed us to identify a diverse set of proteins that respond to the induction of quorum sensing in V. harveyi. The method facilitates monitoring of changes in protein synthesis on a time scale of minutes, and enables correlation of those changes with the underlying temporal pattern of regulation of the quorum-sensing response. The approach described here should prove useful in studies of a wide variety of time-dependent cellular processes.</p><!><p>For each set of experiments, overnight cultures of V. harveyi strain TL25 (DluxM DluxPQ DcqsS) was used to inoculate 625 mL of AB minimal medium containing 18 amino acids (-Met, -Lys) at an OD 600 of 0.003. 15 The culture was divided into six 100 mL aliquots. Three aliquots were supplemented with "light" Lys and three were supplemented with "heavy" Lys (U-13 C 6 U-15 N 2 L-lysine, Cambridge Isotope Laboratories). When the aliquoted cultures reached an OD 600 of 0.1 ($5 doublings), two "heavy" cultures (replicates 1 and 2) and one "light" culture (replicate 3) were treated with AI-1 at a nal concentration of 10 mM ('AI-1 added'); the other three cultures were le untreated ('no AI-1 added'). At the specied time intervals, Aha was pulsed into all six cultures at a nal concentration of 1 mM. Aer 10 min of Aha treatment, protein synthesis was halted by the addition of 100 mg mL À1 chloramphenicol (Sigma). Cells were pelleted, frozen at À80 C, and stored for downstream processing. Aha was synthesized as described previously. 33 Cultures were grown at 30 C in a shaking incubator at 250 rpm.</p><!><p>To measure changes in gene expression following induction of quorum sensing in V. harveyi TL25, cultures were grown as described above, divided in half, and AI-1 was added to one of the aliquots. Samples were collected every 10 min and RNA was isolated as described previously. 13 cDNA synthesis and qRT-PCR were performed as described previously. 22 The levels of gene expression were normalized to the internal standard hfq using either the DDC T method or the standard curve method. At least two replicates were collected for each sample ('AI-1 added' or 'no AI-1 added'). The graphs show the average of those measurements and are calculated as 'AI-1 added' divided by 'no AI-1 added'. Electrophoretic mobility shi assays were performed as previously described. 15 PCR products were generated using oligonucleotides (Integrated DNA Technologies) listed in Table S4. † BONCAT Cells were lysed by heating in 1% SDS in PBS at 90 C for 10 min and lysates were cleared by centrifugation. Protein concentrations were determined with the BCA protein quantitation kit (Thermo Scientic), and paired 'light' and 'heavy' cultures were mixed at equal quantities of total protein. Azide-alkyne click chemistry was performed as described in Hong et al. with a 0.1 mM alkyne-DADPS tag and allowed to proceed for 4 h at room temperature (Fig. S1e †). 34 The DADPS tag was synthesized as described previously. 35 Proteins were concentrated by acetone precipitation and solubilized in 2% SDS in PBS. Solutions were diluted to 0.15% SDS in PBS, and tagged proteins were captured by incubating with streptavidin UltraLink resin (Thermo Scien-tic) for 30 min at room temperature. Resin was washed with 35 column volumes of 1% SDS in PBS and 10 column volumes of 0.1% SDS in ddH 2 O. The DADPS tag was cleaved by incubating the resin in 5% formic acid in 0.1% SDS in ddH 2 O for 1 h. Columns were washed with 5 column volumes of 0.1% SDS in H 2 O, during which proteins remained bound, and proteins were subsequently eluted in 15 column volumes of 1% SDS in PBS. Protein enrichment was conrmed by SDS-PAGE, and eluted proteins were concentrated on 3 kDa MWCO spin lters (Amicon).</p><!><p>Concentrated proteins were separated on precast 4-12% polyacrylamide gels (Life Technologies) and visualized with colloidal blue stain (Life Technologies). Lanes were cut into 8 slices and proteins were destained, reduced, alkylated, digested with LysC (Mako), and extracted as described in Bagert et al. 19 Extracted peptides were desalted with custom-packed C 18 columns as described in Rappsilber et al., lyophilized, and resuspended in 0.1% formic acid (Sigma). 36 Liquid chromatography-mass spectrometric analyses Liquid chromatography-mass spectrometry and data analyses were carried out on an EASY-nLC-orbitrap mass spectrometer (Thermo Fisher Scientic, Bremen, Germany) as previously described with the following modications. 37 For the EASY-nLC II system, solvent A consisted of 97.8% H 2 O, 2% ACN, and 0.2% formic acid and solvent B consisted of 19.8% H 2 O, 80% ACN, and 0.2% formic acid. For the LC-MS/MS experiments, samples were loaded at a ow rate of 500 nL min À1 onto a 16 cm analytical HPLC column (75 mm ID) packed in-house with ReproSil-Pur C 18 AQ 3 mm resin (120 Å pore size, Dr Maisch, Ammerbuch, Germany). The column was enclosed in a column heater operating at 30 C. Aer ca. 20 min of loading time, the peptides were separated with a 60 min gradient at a ow rate of 350 nL min À1 . The gradient was as follows: 0-30% solvent B (50 min), 30-100% B (1 min), and 100% B (8 min). The orbitrap was operated in data-dependent acquisition mode to alternate automatically between a full scan (m/z ¼ 300-1700) in the orbitrap and subsequent 10 CID MS/MS scans in the linear ion trap. CID was performed with helium as collision gas at a normalized collision energy of 35% and 30 ms of activation time.</p><!><p>Thermo RAW les were processed with MaxQuant (v. 1.4.1.2) using default parameters and LysC/P as the enzyme. Peptide and protein false discovery rates were xed at 1% using a targetdecoy approach. Additional variable modications for Met were Aha (À4.9863), L-2,4-diaminobutanoate (À30.9768), a product of Aha reduction, alkyne-DADPS (+835.4300), and 5-hexyn-1-ol (+93.0868), a product of alkyne-DADPS cleavage. Multiplicity was set to 2, and light and heavy (+8.0142) lysine labels were specied for all experiments. Aha and 5-hexyn-1-ol modications were included in protein quantication. We required protein quantications to be calculated with at least two evidences for each set of experiments.</p><p>Both pooled variances and bootstrap statistical methods were employed as previously described to estimate the individual protein ratio standard errors. 19,38 First, pooled estimates of peptide variation were calculated separately for peptides with well-characterized ratios and those based on requantication in MaxQuant. Second, standard errors of the overall protein ratios were calculated by generating 1000 bootstrap iterations. These iterations were generated by resampling the replicates and peptides and adding a small amount of random variation to each measurement based on the pooled variance estimates. Once the bootstrapped samples were generated for each protein, the standard error of the protein ratio was calculated from the standard deviation of the bootstrapped iterations. Using the standard error, proteins with ratios signicantly different from 1 : 1 were identied using a Z-test and p-values were adjusted to account for multiple hypothesis testing using the Benjamini and Hochberg method. 39</p><!><p>Hierarchical clustering was performed with R (v. 3.1.1) using Ward's method. 40 Condence intervals (95 th percentile) for cluster time-series data were calculated by a bootstrapping approach using the tsplot function from the Python (v. 2.7) module seaborn (v. 0.4.0). Singular value decomposition was computed for PCA with the Python module matplotlib.mlab (v. 1.4.0). Gene ontology analysis was performed using a combination of GO terms and KEGG orthology and module terms. Group scores were dened as the mean of protein distances from the origin of the PCA biplot (PC1 vs. PC2). Statistical cutoffs (p-value < 0.05) were calculated using a bootstrapping approach that calculates scores for 100 000 groups randomly selected from the total pool of quantied proteins. Cutoffs were calculated individually for each group size (n ¼ 4, 5, etc.) and groups with fewer than 4 members were excluded. Version 9.1 of the STRING database was used for identifying protein interactions, and interacting networks were identied by manual inspection. 41</p>

Royal Society of Chemistry (RSC)

A novel application of generation model in foreseeing 'future' reactions

Deep learning is widely used in chemistry and can rival human chemists in certain scenarios. Inspired by molecule generation in new drug discovery, we present a deep learning-based reaction generation approach to perform reaction generation with the Trans-VAE model in this study. To comprehend how exploratory and innovative the model is in reaction generation, we constructed the dataset by time-split. We applied the Michael addition reaction as the generation vehicle and took the reactions reported before a certain date as the training set and explored whether the model could generate reactions that were reported after the date. We took 2010 and 2015 as the time points for the splitting of the Michael addition reaction respectively. Among the generated reactions, 911 and 487 reactions were applied in the experiments after the respective split time points, accounting for 12.75% and 16.29% of all reported reactions after each time point. The generated results were in line with expectations and additionally generated a large quantity of new chemically feasible Michael addition reactions, which also demonstrated the learnability of the Trans-VAE model for reaction rules. Our research provides a reference for future novel reaction discovery using deep learning.

a_novel_application_of_generation_model_in_foreseeing_'future'_reactions

Introduction<!>Results and discussion<!>Conclusion<!>Methods<!>/ 10

<p>Organic synthesis is one of the challenging processes in drug discovery, and the exploration of new organic reactions has always been a key stumbling block in the development of synthetic organic chemistry. 1,2 New reactions enrich synthetic routes in the chemistry and materials field. Conventionally, the majority of new reactions have been discovered by the "chemical intuition" of scientists, which was a complex task requiring sufficient luck. For instance, the product of the Diels-Alder reaction was known to chemists as early as 1906, but it was not until 1950 that the reaction was applied to total synthesis experiments. 3,4 The long and intricate progress of discovering new reactions intervenes the promotion of drug discovery. 5,6 Over the past few years, artificial intelligence (AI) technology has provided a number of important applications in various aspects of chemistry and has brought disruptive effects. [7][8][9][10][11][12][13][14][15] Reaching or even surpassing human-level capability at combining chemical reactions with AI remains a new challenge with broad feasible applications. The exploration of AI in chemical reactions primarily involved reaction prediction, 16,17 retrosynthesis analysis, 18,19 reaction condition optimization, 20 and reaction classification, 21 etc. In principle, reaction prediction can be realized by extracting the rules of various chemical reactions, and then directly deriving the products related to reactants. The current mainstream methods usually treat reaction prediction tasks as similarity transformation of molecular graphs or text translation, and the corresponding models are graph convolutional neural network (GCN) and sequence-to-sequence models. 22,23 The performance of text-based reaction prediction has been significantly improved since the release of google company's transformer model, which is entirely based on the attention mechanism. The Molecular Transformer proposed by Schwaller et al, in which the molecules involved in a reaction are all represented as the Simplified Molecular Input Line Entry System (SMILES), was a state-of-the-art SMILES-based sequenceto-sequence model that could reach a 90.4% top-1 accuracy on the USPTO_MIT Data Set with separated reagents. 24 In addition to innovations in the model structure, many strategies can assist AI in better comprehending chemical reactions, including data augmentation 25 and transfer learning, 26 which have shown satisfactory functions in tackling low chemical data regimes. However, it is arduous to discover new reactions by automatically extracting rules from known chemical reactions.</p><p>Inspired by molecular generation, which refers to the generation of undiscovered active or target molecules by extracting the characteristics from a set of molecules known to have specific biological activities, recent studies have turned their attention to generation models and put forward de novo reaction generation. In the task of molecular generation, many active molecules aiming at a specific target have been successfully generated, and some molecules are already in the clinical research stage, which provides a great reference for finding new reactions through generation models. The reactions generated by a reliable generation model can not only guide future chemical research, but also provide a wealth of reaction data to drive deep learning 2 / 10 models. However, a chemical reaction that implies the chemical transformations between reactants and products is a more intricate object for a computer than a pure chemical compound that contains only SMILES rules and information on structural properties. The first attempt at reaction generation was presented by Bort et al 27 . They constructed Bidirectional Long Short-Term Memory (LSTM) layers and trained on the database called USPTO. All reactions were modified from the original SMILES in the form of corresponding Condensed Graph Reaction (SMILES/CGR). Via visualizing latent variables as Generative Topographic Mapping (GTM), they located the position of Suzuki reaction, and found some reactions have particular structural motifs that were not present in the training data. But the new reactions generated in their work have not been proved by follow-up experiments. In subsequent studies by Wang et al, 28 the type of data set used for reaction generation was restricted to Heck reactions. And the Transformer XL, which is a fully attention-based model and more suitable for long sequences, was applied in their study. The result analysis proved that the generated reaction conforms to the Heck reaction rule, and the model also had a favorable grasp of deeper chemical knowledge such as site selectivity. They further selected some reactions for laboratory synthesis to verify the reliability of the generated reactions. Is there a simple and efficient way to test the reliability of the generation model and the novelty of generated reactions? We devised a scheme where the model is trained with chemical reactions published in journals prior to a certain time point to test if the model could produce reactions after that point in time. The schematic diagram of the method is shown in Figure 1.</p><p>In this study, we used the classical Michael addition, which is a representative reaction for carbon chain growth, carbon ring formation, and heteroatom introduction in organic synthesis, as a reaction generation vehicle. And the Trans-VAE 29 model where both encoder and decoder are built with transformer is applied to accommodate the long sequence generation of the reaction. We imported the Michael addition reactions before a certain date into the model as the training input, and part of the reactions generated by our model have been verified by chemists in the literature after the date. The result proved the superiority of the model in certain aspects of reaction generation. More importantly, some generated reactions were brand-new Michael addition reactions that have never shown up in the literature and are valuable for confirming chemical feasibility. With the Michael addition reaction being successfully generated and supported by literature, it not only provides us with a simple and effective way to verify the chemical level generation model, but also sets the stage for generating new types of future reactions in our next work.</p><!><p>The primary purpose of reaction generation is to generate reactions that can be used in future research. And secondly, it can also be used to expand the data volume of small data set reactions to break through the data volume bottleneck of deep learning technology in the field of chemistry. Obviously, it is more difficult to generate new reactions that meet the demand of researchers in the process of model generation. Take 2010 as the split time point, we utilized a total of 3,218 reactions to training the model and then generated 32,979 new Michael addition reactions, 911 of which were reported in the literature after 2010 and validated experimentally. Similarly, we divided the data with 2015 as the split date, and fed the 6,962 training reaction data before 2015 into the model, and finally generated 81,377 new reactions, 487 of which were applied in the literature after 2015. As listed in Table 1, we observe that generated reactions reported after 2010 accounted for 12.75% of all reactions reported after that date, and when 2015 is the split time point, the ratio is 16.29%. We also depict the variation of the ratio with the progress of reaction generation in supplementary Fig. S2, indicating that the model-generated reactions are very reliable and also provides a guarantee for the application of the rest of the new reactions to chemical research in the future.</p><p>We randomly selected some model-generated Michael addition reactions, which were reported after 2015 in Figure 2, as well as some brand-new examples of Michael addition reactions. As shown in Figure 2, (a)-(c) are model-generated reactions that have been applied in practical studies, (d)-(f) are completely new reactions. These examples are consistent with the reaction characteristic rule of Michael addition reaction. On basis of the reaction generated from the dataset with 2015 as split-date, we would evaluate the quality of the model generation in terms of the distribution and similarity of the generated reactions to the training reactions and the chemical properties.</p><p>Because a complete reaction includes reactants and products and the chemical rule between them, it is necessary to compare the component relationships between the training set and the generated set. As listed in Table 2, we counted the types of Michael acceptors and donors and products in the training set and generated set. To visually represent the distribution between the generated set and the training set, we used the t-SNE 30 (t-distributed stochastic neighbor embedding) method to visualize the molecular Morgan fingerprints 31 and further verified the validity of the generated molecules. t-SNE is a dimensionality reduction visualization technique that creates a dimensionality-reduced feature space where similar data points 3 / 10 in a high-dimensional space map to similar distances in a low-dimensional space, and their distributions also remain similar. Morgan molecular fingerprints is a circular topological fingerprint, which obtained by adapting the standard Morgan algorithm. In contrast to MACCS which depends on predefined molecular features to be matched, Morgan molecular fingerprints is a systematic exploration of atomic types and molecular connectivity by searching all the substructures in the compound for a given step by a search algorithm.</p><p>Figure 3A, 3B, and 3C show the t-SNE plots of the Michael donor, acceptor, and product in the generated set with the Morgan fingerprints of the corresponding reactants in the training set, respectively. It can be seen from the plots that the training set molecules overlap well with the corresponding generated set, which indicates that both the reactant and product molecules generated by the model varied around the training set with a certain novelty and also fit the distribution of the training set.</p><p>Shifting the gaze to the overall reaction level, the process of combining the corresponding reactants and product molecules into a reaction means that the model must learn the Michael addition reaction rule. Despite that the Michael addition reaction is one of the most widely used catalytic carbon-carbon bond forming tools in organic synthesis, its rule is complicated for the Trans-VAE model. To further demonstrate that the reactions generated by the model belong to Michael addition reactions, we utilized TMAP to visualize the reaction fingerprint (rxnfp) of the reactions. The reaction fingerprint is derived from the reaction representation learned by the Bidirectional Encoder Representations from Transformers (BERT) model, which used unsupervised learning to construct a reaction space in a large database consisting of unannotated chemical reaction SMILES, and fine-tuned on limited labeled data to construct an accurate reaction classifier. 32 TMAP is a method to visualize the highdimensional space as a tree diagram. 33 As shown in Figure 3E, TMAP connects reactions in the generated reactions(10,000 reactions randomly selected from the generated set) and training dataset based on rxnfp similarity, with each reaction represented as a point in the tree diagram. In addition, the USPTO-50K which contains ten major classes of chemical reactions was downloaded and curated by Liu et al 34 were used to form the backbone of chemical space. Furthermore, we used UMAP 35 to reduce the dimensionality of the rxnfp to validate the distribution of the training set and generated set (Figure 3D). It turns out that the model grasps and reproduces the reaction rules in the training set relatively satisfactorily.</p><p>Michael addition reaction, which can effectively build various carbon-carbon or carbon-hetero bonds, has good compatibility with various functional groups. It can also be applied to the preparation of complex compounds and has very important practical value. To explore in detail whether our model fully understand the Michael addition reaction, we perform an in-depth analysis of the generated Michael addition reaction set. Firstly, we divide the Michael addition reaction into intermolecular and intramolecular reactions. If a molecule contains both donor and acceptor functional groups, intramolecular reactions may occur to constitute carbon rings or heterocycles. As listed in Table3, there are 6,707 intermolecular reactions and 255 intramolecular reactions in training dataset. As for generated reactions, intermolecular reaction accounts for 99.6%, which is consistent with the distribution of intermolecular reactions in training set. As shown in supplementary Fig. S3, we listed several representative examples of intermolecular reactions and intramolecular reactions from training and generated datasets. Because the Michael addition reaction is reversible and the thermodynamically most stable product usually predominates. Five-and six-membered rings are usually more stable due to the lower ring tension Our model accurately captures this feature and the intramolecular reaction of Michael addition is mainly used for the synthesis of more stable fiveor six-membered rings.</p><p>Besides alkene Michael acceptors, electron-deficient alkynes conjugated with electron-withdrawing groups can also be used as Michael acceptors, although alkynes are less reactive than alkene Michael acceptors. Table 4 shows the distribution of Michael acceptors types, mainly divided into alkene acceptors and alkyne acceptors. We select several alkyne Michael addition reactions from the training and generated sets and displayed them in supplementary Fig. S4.</p><p>In the Michael addition reaction, when the enolate as the Michael donor is formed from a simple carbonyl compound under the action of a base, an important feature of the reaction is that the stereo structure of the product is closely related to that of the enolate. As listed in supplementary Fig. S5A, there are two examples from training dataset, where the Z-enolate creates anti-product and E-enolate creates syn-product. It is confirmed that the model has perceived this rule, and the generated reactions follow it satisfactorily as depicted in supplementary Fig. S5A. In the case of simple carbonyl compounds with asymmetric Michael donors, the acceptor reacts mainly with α-carbon atoms having more substituents, depending on the stability of the intermediate enol. In general, the more electron-donating substituents on the double bond, the more stable the enol is and the more Michael addition reaction is promoted. A typical instance is shown in supplementary Fig. S5B.</p><p>For stable carbanion conjugated to multiple heteroatoms, reactions with the acceptor typically yield 1-4 addition products. Most of these heteroatom-containing stable groups are easy to leave and can be considered as conjugated auxiliary groups. We also list some instances in supplementary Fig. S6 where we can see that the carbon atom in the middle of the two carbonyl 4 / 10 groups is more acidic than the carbon atom on the other side of the carbonyl group, so it is more likely to be deprotonated by a base to form a carbanion. The reactions generated by our model also fit this signature.</p><p>The molecular structure of Michael acceptor includes an electron-withdrawing group and an unsaturated system. Almost all alkene compounds substituted with electron-withdrawing group can be utilized as Michael acceptors as exhibited in supplementary Fig. S7. However, if the acceptor molecule contains two or more electron-withdrawing groups at the same time, the regioselectivity of the reaction is usually controlled by the relatively active group. supplementary Fig. S7A2 is a generated reaction example that conforms to this rule. We can see that the Michael donor in supplementary Fig. S7A2 has two electronabsorbing groups, nitro, and cyano. Since nitro is more electron-absorbing, the carbon atom to which nitro is attached is more likely to lose a proton to form a carbanion. The model learns this principle during training and reflects it in the generated reaction.</p><p>It is worth mentioning that, in addition to carbon nucleophiles, some heteroatom groups can also be used as donors for the Michael addition reaction due to their nucleophilic properties. For example, alkylamines or arylamines are widely used as Michael donors. The reaction has promising chemical selectivity and generally does not generate imine by-products. We have further added reaction data for the heteroatom Michael addition reaction to the data with 2015 as the split date. After retraining the model on the data, we observe whether the model still grasps the reaction Michael addition under the obfuscation of heteroatoms. In this study, we mainly consider heteroatoms such as N, S, O. Table 5 is the classification and proportion of heteroatom nucleophiles. It could be seen that the generated carbon nucleophiles, nitrogen nucleophiles, oxygen nucleophiles, and sulfur nucleophiles occupied most of the generated reactants, which is similar to the distribution of these four reactants in the training dataset. We present examples of Michael additions involving heteroatoms in the training and generated sets in supplementary Fig. S8, respectively. These results are exciting as it proves that our Trans-VAE model is sufficiently expressive to produce the correct reactions.</p><!><p>In this work, we applied the Trans-VAE model for the reaction generation task. To explore whether the model can 'break the time limit' and generate Michael addition reactions that have been applied in reports after a certain date, we simulate this scenario by dividing the dataset by 'time-split'. Thanks to the transformer-based encoder and decoder architecture, the model can capture both SMILES rules and Michael addition reaction feature information in the long sequence of reactions. We used 2010 as the split time point, and trained the model on the reactions before 2010. The results showed that the model generated reactions that were applied after 2010, accounting for 12.75% of all published reactions after that date. providing initial evidence of the reliability of the Trans-VAE model in reactions generation. To demonstrate the effectiveness rather than haphazardness of our model, we conducted another experiment with 2015 as the split time point, and the rate was 16.29%. We then further inspected whether the model mastered the rules of Michael addition reactions by analyzing the generated Michael addition reactions in terms of their chemical characteristics. The final analysis shows that the model captures reaction characteristics consistent with the now discovered chemical laws of Michael addition reactions, indicating the reliability of applying deep learning models to reaction generation, and laying the foundation for our subsequent exploration of the vast chemical space using deep learning models and the discovery of the generation of completely new types of chemical reactions.</p><!><p>Dataset. The reaction generation model was trained on SMILES files containing only Michael addition reactions that were extracted from the "Reaxys" database based on a search of reaction templates and/or reaction names (all entries using the "Michael addition reaction" phrase). The extracted Excel files were subjected to a pre-processing process with a series of python scripts to obtain a high-quality dataset that met the requirements for new reactions generation. In this step, reactions where the SMILES string was invalid or the reactants and products were identical were removed from the file, and the remaining reactions were canonized using RDkit 36 so that the same compound was represented by the same SMILES. Finally, the non-compliant reactions were filtered based on the Michael addition reaction template using RDkit's Python script. As for the time point of the reaction, it was considered that the same reaction may be reported in the literature at different times, we took the time when it was first reported and deleted the rest of the same reactions to obtain a dataset containing 12,322 Michael addition reactions. Taking 2015 as the split line, the reactions before 2015 were divided into training and validation sets (9:1), while those after this time were used as a reference for whether the model could generate 'future' reactions.</p><!><p>Model. With the rapid development of natural language processing (NLP) models, the text-based format has been widely used in previous works, such as chemical reaction prediction and ADMET prediction. We represented the reactions in the form of Simplified Molecular Input Line Entry System (SMILES) strings 37 , in which every character corresponds to an atom or chemical bond. The regularized expression which is arbitrarily extensible with reaction information is adopted as the rule for SMILES word segmentation. Based on this rule, a chemical atom with multiple characters and special environments (e.g 'Cl', '[O-]') is treated as one token instead of being divided into multiple tokens, which is more in line with chemical specifications. 38 The fundamental architecture of Trans-VAE consists of an encoder and a decoder, and the workflow of the model is displayed in Figure 1B. The encoder maps the discrete SMILES to a dense latent representation and transforms it into a continuous fixed-dimensional vector, while the decoder attempts to convert the vector in the latent space back into input with the smallest possible error. By adding noise to the encoded SMILES, molecules would have corresponding probability distribution in the latent space rather than individual points, and the decoder also learns to discover more robust representations from latent points. The training process intends to minimize the reconstruction loss between original SMILES and generative SMILES, while satisfying the probability distribution of the generated data is similar to that of the training data.</p><p>Consider the fact that the SMILES representation of the reaction has increased by two to three times compared to the molecule, which calls for the model to have salient performance for long sequences. Therefore, we applied the VAE model proposed by Dollar et al 30 which implements the transformer as the encoder and decoder. Compared with the recent commonly built Long Short-Term Memory (LSTM) and Gated Recurrent Unit (GRU) as encoder and decoder, it replaces the recurrence with the attention head entirely, which reduces the spreading path of information in the model and helps to grasp the longrange syntactic dependence in reaction SMILES. However, the highly parallelizable transformer also brings a lot of trainable parameters. To avoid parameter explosion, a convolution bottleneck is employed to stochastically compressed the encoder output and then feed it to the decoder.</p><p>The purpose of our work is to explore whether the Trans-VAE model can generate reactions reported afterward based on reactions before 2015, that is pay more attention to the exploration. Therefore, we give priority to sampling within the highentropy dimension, where all of the meaningful structural information is involved. Different from random sampling, highentropy sampling is better able to explore chemical phase space and obtain novel reactions, albeit may reduce valid SMILES.</p>

ChemRxiv

Spin Diffusion Driven by R-Symmetry Sequences: Applications to Homonuclear Correlation Spectroscopy in MAS NMR of Biological and Organic Solids

We present a family of homonuclear 13C-13C magic angle spinning spin diffusion experiments, based on R2nv (n = 1 and 2, v = 1 and 2) symmetry sequences. These experiments are well suited for 13C-13C correlation spectroscopy in biological and organic systems, and are especially advantageous at very fast MAS conditions, where conventional PDSD and DARR experiments fail. At very fast MAS frequencies the R211, R221, and R222 sequences result in excellent quality correlation spectra both in model compounds and in proteins. Under these conditions, individual R2nv display different polarization transfer efficiency-dependencies on isotropic chemical shift differences: R221 recouples efficiently both small and large chemical shift differences (in proteins these correspond to aliphatic-to-aliphatic and carbonyl-to-aliphatic correlations, respectively), while R211 and R222 exhibit the maximum recoupling efficiency for the aliphatic-to-aliphatic or carbonyl-to-aliphatic correlations, respectively. At moderate MAS frequencies (10\xe2\x80\x9320 kHz), all R2nv sequences introduced in this work display similar transfer efficiencies, and their performance is very similar to that of PDSD and DARR. Polarization transfer dynamics and chemical shift dependencies of these R2-driven spin diffusion (RDSD) schemes are experimentally evaluated and investigated by numerical simulations for [U-13C,15N]-alanine and the [U-13C,15N] N-formyl-Met-Leu-Phe (MLF) tripeptide. Further applications of this approach are illustrated for several proteins: spherical assemblies of HIV-1 U-13C,15N CA protein, U-13C,15N enriched dynein light chain DLC8, and sparsely 13C/uniformly 15N enriched CAP-Gly domain of dynactin. Due to the excellent performance and ease of implementation, the presented R2nv symmetry sequences are expected to be of wide applicability in studies of proteins and protein assemblies as well as other organic solids by MAS NMR spectroscopy.

spin_diffusion_driven_by_r-symmetry_sequences:_applications_to_homonuclear_correlation_spectroscopy_

Introduction<!>Samples<!>Solid-State NMR Spectroscopy<!>Numerical Simulations<!>Symmetry-Based Spin Diffusion Experiments at Moderate MAS Frequencies<!>Symmetry-Based Spin Diffusion Experiments at Very Fast MAS Frequencies: Alanine<!>Symmetry-Based Spin Diffusion Experiments at Very Fast MAS Frequencies: Applications to Proteins and Protein Assemblies<!>Conclusions

<p>With the recent developments in decoupling and recoupling techniques, solid-state NMR has become a powerful tool for determining molecular structure and dynamics in biological solids, ranging from amyloid fibrils to membrane proteins to intact viruses to protein assemblies.1–10 Magic angle spinning (MAS) recoupling methods, both dipolar- and scalar-based, have emerged as an essential tool for structural and dynamics characterization of uniformly and extensively enriched biopolymers.11–24 With the recent breakthroughs in probe technology, very fast MAS frequencies of the order of 40–70 kHz are now accessible to the experimentalist,25–27 and these fast MAS conditions result in greatly enhanced spectral resolution due to efficient averaging of the homonuclear 1H-1H dipolar interactions at spinning rates above 30 kHz, and due to efficient heteronuclear 1H-X decoupling that can be achieved at high rotation frequencies. The narrow 1H lines under very fast MAS also permit proton detection, and indeed, several investigators have recently demonstrated the benefits of proton-detected experiments in proteins, both in terms of greatly enhanced sensitivity and the additional information that can be gained by incorporating the proton dimension into the spectra.28–31 In the heteronucleus-detected experiments, the narrow lines attained at very fast MAS frequencies also give rise to significant sensitivity enhancements, and therefore, very fast MAS conditions are in principle advantageous across the board for structural and dynamics studies of proteins and protein assemblies. However, implementation of fast-MAS protocols for protein structure determination requires the development of modified homo- and heteronuclear recoupling methods,26,32–36 because many of the recoupling techniques that are commonly employed at MAS frequencies below 20 kHz do not work above 30 kHz.</p><p>Types of experiments that do not work in their original implementation under very fast MAS conditions comprise homonuclear spin-diffusion-based recoupling methods, such as proton-driven spin diffusion (PDSD)37,38 and dipolar-assisted rotational resonance (DARR)20,39 or RF-assisted diffusion (RAD).40 These techniques are ubiquitously used in structural analysis of proteins and protein assemblies at moderate MAS frequencies (10–20 kHz), both for resonance assignments and for deriving long-range distance constraints as well as for quantitative CP/MAS measurements.1,6,7,11,12,22,23,41–57 These sequences are some of the very few homonuclear recoupling methods (along with PAR34,58,59 and CHHC60,61) that do not suffer from dipolar truncation36,44,62–64 and are thus particularly advantageous for extracting distance information in uniformly and extensively enriched proteins.</p><p>In PDSD experiments, the recoupling efficiency depends strongly on the line broadening due to the residual 1H-X dipolar couplings and on the spinning frequency, and in some cases it is hard to achieve broadband homonuclear correlation spectra. This limitation is alleviated when a weak radio frequency (rf) field irradiation is applied on the protons during the mixing time, such as in DARR (RAD) experiments. For DARR (RAD) irradiation, the 1H rf field strength is ω1H=nωr, satisfying the rotary resonance matching condition and resulting in broadband rotational resonance recoupling. During the mixing time, 13C-13C polarization transfer is driven by the reintroduced 1H-13C and 1H-1H dipolar couplings, and the transfer efficiency for the coupled 13C spins is somewhat less dependent on MAS frequency than in the conventional PDSD experiment. While dependence of polarization transfer efficiency on the MAS frequency in DARR (RAD) experiments is not as dramatic as in the PDSD sequence, the dependence on the chemical shift difference between the coupled spins is very strong.65 This chemical shift difference dependence is particularly severe at very fast MAS rates (>30 kHz), resulting in lower and non-uniform polarization transfer efficiencies across the spectrum, and at frequencies of 40 kHz or above DARR/RAD would not be a method of choice for 13C correlation spectroscopy.</p><p>Recently, several amplitude- and phase-modulated DARR/RAD experiments were developed to improve the spin diffusion efficiency under very fast MAS conditions.36,65,66 Second-order rotational resonance conditions, ω1 = nωr ± Δωiso, were introduced by Ernst et al.36 Broadband homonuclear dipolar recoupling at very fast MAS frequencies can be performed by various amplitude-modulated 1H rf field irradiation schemes, referred to as mixed rotational and rotary-resonance (MIRROR) technique. Tekely et al. proposed a phase-alternated recoupling irradiation (PARIS)66,67 scheme to obtain more efficient polarization transfer at very fast MAS, and the phase alternated DARR irradiation compensates efficiently for rf field inhomogeneity and improves the magnetization transfer rate. Nevertheless, the chemical shift offset sensitivity still remains an issue in these sequences, and it is difficult to achieve uniform broadband polarization transfer.</p><p>In this work, we present a family of spin diffusion experiments in which the magnetization transfer is driven by rotor-synchronized R2nv symmetry-based recoupling. Such symmetry-based approaches in MAS recoupling pulse sequences were originally described by Levitt in a series of elegant studies that established the general design principles for these sequences as well as classified them as CNnv and RNnv schemes depending on the rotation properties of the spin angular momenta during the rotor-synchronized train of rf pulses.68–71 RNnv -type sequences, employed in this work, are designed to produce a net rotation of the spin angular momenta by π around the x axis of the rotating frame.68 Following the Levitt nomenclature, n and v are small integers and are referred to as the symmetry numbers of the pulse sequence. In the R2nv symmetry-based spin diffusion experiments introduced here, all pairwise combinations of n = 1, 2 and v = 1, 2 are employed to produce four sequences, R211, R212, R221, and R222. The pulse element of the R2 symmetry sequences can be either a basic π pulse or composite pulses such as 90°x90°·y90°x, 90°x270°-x or 90°x180°y90°x. In these R2 sequences, the rf field amplitude and phase alternation are defined according to the R-type symmetry. It should be noted that the basic R211 symmetry scheme has the same pulse type as the rotor synchronized PARIS66 scheme, and that the basic R212 symmetry scheme has the same pulse type as the conventional DARR/RAD scheme. We demonstrate that these different R2-driven spin diffusion schemes (RDSD) exhibit similar transfer efficiencies at moderate MAS rates (<20 kHz) except that R211 and POST-R221 are advantageous for the recoupling of coupled spins with small chemical shift differences. At very fast MAS frequencies, the individual R2 irradiation schemes display distinct dependencies of the polarization transfer efficiencies on the chemical shift differences between coupled carbon spins. Experiments and numerical simulations in [U-13C,15N]-alanine and [U-13C,15N] N-formyl-Met-Leu-Phe (MLF) tripeptide indicate that under very fast MAS the polarization transfer in these R2nv spin diffusion experiments is driven by the broadened second-order rotational resonance condition (ω1 ± nωr - KscωDD ≤ Δωiso ≤ ω1 ± nωr + KscωDD), and we present the theoretical treatment of these sequences that correctly accounts for the polarization transfer dynamics and for the observed chemical shift dependence of the individual R2nv schemes. Our results further indicate that due to these distinct chemical shift dependencies it is advantageous to employ a specific R2 sequence for a given correlation experiment, e.g., CACB vs. CACO.</p><p>We further demonstrate that these R2nv spin diffusion sequences perform very efficiently in proteins and protein assemblies, using three examples: spherical assemblies of U-13C,15N-enriched HIV-1 CA protein, U-13C,15N-enriched dynein light chain DLC8, and sparsely-13C/U-15N-labeled dynactin CAP-Gly domain. We anticipate that the approach presented here for R2nv spin diffusion under fast-MAS conditions will be broadly applicable to studies of proteins and protein assemblies by MAS NMR spectroscopy, which is a rapidly growing area of research.</p><!><p>[U-13C,15N]-alanine and [U-13C,15N] N-formyl-Met-Leu-Phe (MLF) tripeptide were purchased from Cambridge Isotope Laboratories. Both powder samples were used in the subsequent NMR experiments without any further purification or recrystallization. The sample of spherical assemblies of U-13C,15N-enriched HIV-1 capsid protein (CA) was prepared according to established procedures.6 CAP-Gly sparsely enriched in 13C and uniformly labeled with 15N was prepared from E. coli, grown in minimal medium containing 1,3-13C glycerol (Cambridge Isotopes) as the sole carbon source and 15NH4Cl as the sole nitrogen source, as described previously.72–74 The solid-state NMR sample of CAP-Gly was prepared by controlled precipitation from polyethylene glycol,36,44,62,63 slowly adding a solution of 30% PEG-3350 to the solution of 24.3 mg of CAP-Gly (38.5 mg/mL), both dissolved in 10 mM MES buffer (10 mM MgCl2, pH 6.0), as described previously.7 U-13C, 15N-enriched dynein light chain protein (DLC8) was expressed in E.coli and purified as described previously.75 The solid-state NMR sample of DLC8 was prepared by controlled precipitation, slowly adding a solution of 30% PEG-3350 to the solution of 11 mg of DLC8 (30 mg/ml). Prior to the precipitation step, both PEG-3350 and DLC8 were dissolved in 10 mM MES buffer (10 mM MgCl2, pH 6.0) and doped with 50 mM EDTA-Cu(II). The protein samples were packed in 1.8 mm MAS rotors for solid-state NMR experiments.</p><!><p>All NMR experiments were carried out on a Varian InfinityPlus solid-state NMR spectrometer operating at a Larmor frequency of 599.8 MHz for 1H, and 150.8 MHz for 13C. Solid-state NMR experiments were performed using either a 3.2 mm Varian triple-resonance T3 probe (spinning rates of up to 25 kHz) or a 1.8 mm MAS triple-resonance probe developed in the Samoson laboratory (spinning rates up to 50 kHz).</p><p>The pulse schemes for RDSD 2D experiments are shown in Figure 1. During the mixing period, a series of R2nv symmetry pulses is applied on the 1H channel, as illustrated in Figure 1(b)–(e) for each specific R2nv experiment. For R21v symmetry irradiation, the rf field strength equals the MAS frequency, and one rotor period contains two π pulses. For R22v symmetry irradiation, the rf field strength equals half the MAS frequency, and one rotor period contains one π pulse. For POST-R2nv symmetry irradiation, the basic π pulse is replaced by a composite pulse, (π/2)0(3π/2)180, and the rf field strength is twice that of the R2nv symmetry irradiation, as shown in Figure 1(f) for POST R221.</p><p>NMR experiments on the MLF sample packed into a 3.2 mm rotor were recorded at room temperature and MAS frequency of 16 kHz. Typical 90°· pulse lengths were 2.8 µs for 1H and 4.0 µs for 13C. The cross-polarization contact time was 1.4 ms, and the recycle delay was 3.0 s. Two-pulse phase-modulation (TPPM)76 1H decoupling with an rf field strength of 96 kHz was performed during the acquisition, and continuous wave (CW) 1H decoupling with the same rf field strength was applied during the t1 evolution time. 1H irradiation with an rf field strength of 16 and 8 kHz during the mixing time was applied for 2D R21v and R22v (v = 1, 2) correlation experiments, respectively. The 2D spectra were collected as (1000 × 256) (complex x real) matrices with 8 scans; TPPI (time proportional phase incrementation) scheme was used for phase-sensitive detection in the indirect dimension.77 A series of experiments with different mixing times ranging between 1 and 50 ms were performed.</p><p>NMR experiments on an alanine sample packed into a 1.8 mm rotor were performed at the MAS frequency of 40 kHz. To reduce sample heating during fast MAS spinning, nitrogen gas at 0°C was used for cooling, resulting in a final sample temperature of 20°C. The rf field strength on the 1H channel during the mixing time was 40 and 20 kHz for R21v and R22v experiments, respectively. CW 1H decoupling with rf field strength of 9 kHz was employed in the t1 and t2 dimensions. The 2D spectra were collected as (2000 × 512) (complex x real) matrices with 8 scans; TPPI scheme was used for phase-sensitive detection in the indirect dimension. A series of experiments with different mixing times ranging between 5 and 500 ms were performed.</p><p>NMR experiments on protein samples packed into a 1.8 mm rotor were performed at the MAS frequencies of 10 and 40 kHz. To reduce the sample heating due to fast MAS spinning at 40 kHz, a stream of cooling nitrogen gas at −20°C, −25 °C and −35°C was used for the HIV-1 CA, CAP-Gly and DLC8 samples, resulting in final sample temperatures of 0°C, −5°C and −15°C, respectively. For all 2D R2nv 13C-13C correlation experiments, TPPI scheme was used for phase-sensitive detection in the indirect t1 dimension. The other parameters were the same as those used for the experiments on alanine. For all RDSD NMR experiments conducted at the MAS frequency of 10 kHz, a 96 kHz 1H TPPM decoupling was applied during t1 and t2. Detailed acquisition and procession parameters are shown in the Supporting Information.</p><!><p>All numerical simulations were performed in SIMPSON.78 168 REPULSION (α, β) angles and 16 γ angles were used to generate a powder average. The atomic coordinates for the model spin systems employed in the simulations were taken from the SSNMR structure of the leucine residue in the N-f-MLF-OH tripeptide (PDB ID 1Q7O).50 These atomic coordinates are generally regarded as a valid representation for spin systems of other amino acids. For Cα-C′ spin diffusion simulations, a spin cluster containing two carbons and one proton was used. For Cα-Cβ spin diffusion simulations, a spin cluster containing two carbons and three protons was employed. The one-bond dipolar coupling constants for 1H-13C and 13C-13C were 22,690 and 2,251 Hz, respectively. In all simulations, all possible pairwise dipolar couplings were taken into account. J couplings were ignored as their effects are negligible given their small size. Other parameters used in simulations were the same as in the corresponding experiments.</p><!><p>R2nv symmetry-based pulse irradiation on protons can be used to reintroduce hetero- and homonuclear dipolar couplings, which in turn allow for spin diffusion among carbon spins. The space-spin selection rules for the symmetry-allowed first-order average Hamiltonian terms have been reported elsewhere.68 Figure 2 demonstrates space-spin selection diagrams for the interaction Hamiltonians in the R211 sequence. It can be seen that both the 1H-13C heteronuclear dipolar couplings and the 1H-1H homonuclear dipolar couplings are reintroduced by the R211 symmetry irradiation, with six heteronuclear dipolar coupling terms HIS(1) and ten homonuclear dipolar coupling terms HII(1) of the first-order average Hamiltonian being symmetry-allowed. Even though spin diffusion between coupled 13C spins is primarily determined by the 13C-13C coupling strength and the first-order rotational resonance conditions (nωr = Δωiso), the reintroduced 1H-13C and 1H-1H dipolar couplings represent an essential contribution, resulting in greatly broadened RR matching conditions. Furthermore, these dipolar couplings also contain the second-order average Hamiltonian terms. Even though the recoupled second-order average Hamiltonian terms are much smaller than the first-order contributions, they can also drive the 1H-13C and 1H-1H cross relaxation and assist the 13C-13C spin diffusion. Table 1 summarizes the number of the first- and second-order average Hamiltonian terms in the R2nv symmetry sequences. These recoupled hetero- and homo-nuclear dipolar Hamiltonian terms would drive the polarization transfer (see the Supporting Information for details on the recoupled terms for each R2nv sequence). The first-order rotational resonance conditions would be broadened greatly by R2nv re-introduced dipolar interactions, and these can be expressed as, nωr - KscωDD ≤ Δωiso ≤ ± nωr + KscωDD, where KscωDD denotes the size of the recoupled dipolar interactions by R2nv and Ksc is the corresponding scaling factor.</p><p>It is obvious from Table 1 that the number of the symmetry-allowed Hamiltonian terms is different for each recoupling sequence, except for R212 and R211. Even though for R212 and R211 the same number of first- and second-order Hamiltonian terms are symmetry-allowed, the nature of these recoupled Hamiltonian terms {l, m, λ, μ} as well as the corresponding scaling factors are totally different (see the Supporting Information), suggesting that different polarization transfer dynamics might be observed in the corresponding spin diffusion experiments. To examine the 13C-13C transfer rates in each specific sequence at moderate MAS frequencies, we have recorded a series of two-dimensional R2nv (n, v = 1 and 2) correlation spectra with varying mixing times in the MLF tripeptide. The 13C-13C polarization transfer build-up curves acquired at the MAS frequency of 16 kHz are illustrated in Figure 3a for the Cα-Cβ correlation of Leu residue, and in Figure 3b for the Cα-C′ correlation of Met residue.</p><p>The results demonstrate that, despite the different number of symmetry-allowed Hamiltonian terms in each of the R2nv symmetry-based schemes, all of these sequences exhibit similar polarization transfer efficiencies and polarization transfer rates for Cα-Cβ correlations at moderate spinning frequencies. On the other hand, for the Cα-C′ correlation, the R211 scheme shows a slightly higher polarization transfer rate than the other sequences. We also note that, as expected, the POST versions of sequences in which the basic π-pulse of the R-element is replaced with composite (900270180) pulses, display improved transfer efficiency in the 13C-13C correlation experiments, especially for coupled 13C spins with large chemical shift differences.</p><!><p>Either proton-driven or dipolar-assisted spin diffusion can be used to perform broadband homonuclear correlation experiments, but both of these recoupling methods display low tolerance to large resonance frequency offsets, which are most pronounced at fast and very fast MAS rates (>30 kHz). Since we found that at moderate MAS frequencies the performance of the R2nv symmetry-driven sequences is largely independent of the chemical shift offset between the coupled spins, we have proceeded with examining the spin diffusion behavior of these sequences at very fast MAS frequencies. Two idealized models of dipolar-coupled spin networks were constructed that represent the smallest coupled C-C spin systems in proteins, using the atomic coordinates of the leucine residue in N-f-MLF-OH, as shown in Figure 4. We used these models in the numerical simulations, to evaluate the 13Cα-13Cβ and 13Cα-13C′ polarization transfer dynamics.</p><p>Figure 5 demonstrates the 13C-13C polarization transfer dynamics by basic (solid curves) and POST (dotted or dashed curves) R2nv symmetry sequences simulated for the above model spin systems at the MAS frequency of 40 kHz. The build-up curves for the Cα-Cβ and Cα-C′ correlations are shown in Figure 5(a) and 5(b), respectively. As can be appreciated, under the very fast MAS conditions, the various R2nv symmetry driven schemes exhibit very different polarization transfer dynamics, depending greatly on the chemical shift difference between the coupled 13C spins. For the Cα-C′ polarization transfer by the basic R2nv, the R222 sequence with an rf field irradiation of 20 kHz exhibits the fastest polarization transfer rate. On the other hand, there is almost no Cα-C′ polarization transfer observed for the R21v (v = 1, 2) sequences with rf field irradiation of 40 kHz. At the same time, for the Cα-Cβ recoupling, the R211 symmetry scheme yields the most efficient polarization transfer. Even though the Cα-Cβ polarization transfer can also be accomplished with the R222 sequence, the transfer rate is significantly slower. Confirming the experimental results, the current numerical simulations indicate that DARR (referred to as R212 symmetry sequence here) is inefficient for polarization transfer at high MAS frequencies; furthermore, it is very sensitive to the chemical shift differences, rendering DARR experiments impractical under these conditions.</p><p>As discussed above, the replacement of the basic π pulse by composite pulses, gives rise to the various POST R2nv symmetry sequences, and some of these are expected to yield higher transfer efficiency with the appropriate selection of composite pulses. For example, for Cα-Cβ polarization transfer, POST R221 symmetry sequence consisting of (90x90y90x) composite pulses, exhibits transfer efficiency much higher than the basic R211 scheme. In the case of Cα-C′ recoupling, the POST R222 symmetry sequence, consisting of (90x270−x) composite pulses, exhibits a much higher transfer efficiency than the basic R211 scheme. It should be noted, however, that not all POST R2nv sequences perform better than the basic R2nv schemes. For instance, then POST R221 symmetry sequence, consisting of (90x270−x) composite pulses, has a lower transfer efficiency than the basic R221 scheme for the Cα-Cβ polarization transfer.</p><p>In order to understand the behavior of the R2nv symmetry driven sequences as a function of the MAS frequency and the chemical shift difference, one needs to further consider the various factors important in the polarization transfer dynamics. For example, at low or moderate MAS frequencies, DARR (or R212 symmetry sequence) can be regarded as a broadband rotary resonance (RR) based recoupling technique, since the first-order RR matching conditions are broadened greatly by the reintroduced 1H-13C dipolar couplings. Following this logic, one might think that at very fast MAS frequencies, the transfer efficiency in the DARR experiments may be poor since the partly recoupled heteronuclear dipolar interactions are not strong enough to satisfy the first-order RR matching condition. To investigate this possibility, we have examined the extent of dipolar broadening by DARR and by other R2nv symmetry sequences at the MAS frequency of 40 kHz. Figure 6 shows the experimental 13C CP/MAS spectra of alanine spun at 40 kHz, recorded with the 1H rf field irradiation during the acquisition period by (a) R212 (DARR), (b) R211, (c) R221, and (d) R222 symmetry pulses. The results show that 13C NMR lines are strongly broadened in the 1D R21v experiments (Figure 6 (a) and (b)), and that the line shapes for these two cases are similar because both sequences have the same number of symmetry-allowed Hamiltonian terms (see Table 1). Interestingly, even though the R221 symmetry scheme retains the largest number of symmetry-allowed 1H-13C and 1H-1H Hamiltonian terms, the broadening of the 13C NMR resonances by the R221 irradiation is much smaller than that by the R211 and R212 sequences. This is the result of the smaller scaling factor for recoupling of the heteronuclear dipolar couplings by R221. Since no first-order average 1H-13C dipolar Hamiltonian terms are allowed in the R222 symmetry scheme (see Table 1), the 13C MAS spectrum exhibiting the least broadening in the presence of the 1H rf field is obtained.</p><p>It is clear from the above discussion that 13C MAS spectra acquired with the various R2nv irradiation sequences are broadened to different degrees, and in the case of R222 irradiation, no broadening from the recoupled first-order average Hamiltonian interactions ensues. However, these re-introduced dipolar couplings cannot broaden efficiently the first-order rotational resonance conditions (nωr = Δωiso) under very fast MAS, and the polarization transfer profile depends to a greater extent on the second-order rotational resonance conditions (ω1 = nωr ± Δωiso).36 The strength of the rf field irradiation and the size of the recoupled dipolar couplings would jointly dominate the polarization transfer. The degree of broadening in the different R2nv symmetry sequences, therefore, does not directly correspond to the polarization transfer efficiencies, indicating that the dipolar broadening is not the dominant factor in spin diffusion experiments.</p><p>Figure 7 provides the experimental 13C-13C polarization transfer dynamics in the various R2nv symmetry-driven spin diffusion experiments for U-13C, 15N-alanine, spun at 40 kHz. The Cα-Cβ and Cα-C′ build-up curves are plotted in Figure 7 (a) and (b), respectively. The results demonstrate that the R211sequence exhibits the most efficient polarization transfer for the Cα-Cβ spin diffusion experiments. For the Cα-C′ polarization transfer, the R22v sequences yields much higher transfer rates than the R21v symmetry sequences. This finding is consistent with the simulation results shown in Figure 5 except for the differences in the transfer rates, which can be attributed to relaxation effects. Various cross-relaxation times, such as T1HH, T1HC and T1CC contribute to the magnetization transfer process, and were not taken into account in SIMPSON simulations. However, disregarding these relaxation times in the numerical simulations does not affect the comparison of the performances of the different R symmetry sequences, as demonstrated experimentally on protein samples (see below). Despite the difference in the absolute transfer rates between the experimental and simulated data the relative polarization transfer dynamics between the different R2 sequences is captured correctly in the simulations.</p><p>It should be noted, that although the R212 scheme (DARR or RAD) resulted in the largest broadening of the 13C lines (Figure 6 and discussed above), the DARR polarization transfer efficiency at very fast MAS frequencies is very low, especially for the Cα-C′ correlation experiments, since neither the first-order (nωr = Δωiso) nor the second-order (ω1 = nωr ± Δωiso) rotary resonance conditions are fulfilled at the MAS frequency of 40 kHz. In other words, the 1H-13C heteronuclear dipolar couplings are no longer the primary factor in creating uniform 13C-13C polarization transfer at very fast MAS frequencies, and the polarization transfer profile depends on the rf field strength, the chemical shift difference and the size of the recoupled dipolar interactions as well. On the other hand, even though the other R2nv sequences also depend significantly on the chemical shift differences of coupled carbons, each of these experiments exhibits high recoupling efficiency for either Cα-Cβ or Cα-C′ correlations, suggesting that with a pair of experiments, R211 and R222, both types of correlations can be recorded. Alternatively, if a single experiment is desirable to achieve both Cα-Cβ and Cα-C′ correlations, the R221 symmetry sequence can be used. Although this experiment does not exhibit the highest transfer efficiencies for either Cα-Cβ or Cα-C′ correlations, the transfer efficiency is still high enough to attain high sensitivity and uniform excitation for both spectral regions.</p><p>To further evaluate the experimental results, we examined the dependence of chemical shift difference on the polarization transfer dynamics at fast and moderate MAS frequencies by numerical simulations. Figure 8 illustrates the simulated dependence of the 13C-13C polarization transfer efficiency on the chemical shift difference for R212, R212, R222, and R221 sequences at the MAS frequency of 40 kHz. It can be noted that the R211 symmetry scheme exhibits relatively high polarization transfer efficiency for the coupled 13C spins with small chemical shift differences (of less than 47 ppm). This indicates that the R211 symmetry sequence is suitable for recording Cα-Cβ, Cβ-Cγ, and other sidechain correlations in spin diffusion experiments. On the other hand, essentially no polarization transfer is expected for the coupled 13C spins with chemical shift differences larger than 50 ppm in the R211 experiment, while DARR (the R212 sequence) results in very weak polarization transfer. At the same time, the R222 and R221 symmetry sequences give rise to much higher transfer efficiencies for correlations between coupled carbons with large chemical shift differences, such as Cα-C′ spin pairs whose chemical shift differences are of the order of 120 ppm, even multi-bond Cβ/γ-C′ spin pairs with chemical shift differences of 120–170 ppm. This simulated behavior agrees well with the experimental results presented in Figures 5 and 7.</p><p>It is obvious from the above that the polarization transfer under very fast MAS can be driven by the R2nv recoupled dipolar interactions when the second-order rotational resonance conditions are fulfilled, ω1 ± nωr -.KscωDD ≤ Δωiso ≤ ω1 ± nωr + KscωDD, where KscωDD denotes the scaled dipolar couplings re-introduced by R2nv symmetry sequences. However, even when these conditions are fulfilled, it is still difficult to obtain a true broadband recoupling. The transfer efficiency is dependent on the chemical shift difference, and it is therefore anticipated that polarization transfer profile will be dependent on the magnetic field strength. Even though we have not yet performed R-type spin diffusion experiments at magnetic field strengths other than 14.1 T, the magnetic field dependence of the performance of these sequences can be understood by the analysis of Figure 8. The simulated dependence of the chemical shift difference reveals the conditions where strong vs. no correlations would be observed for each R2nv sequence. For instance, at the MAS frequency of 40 kHz, for the R221 sequence there will be no correlations for coupled 13C nuclei with chemical shift differences of 9–14 kHz. Translating the CS difference into ppm, we realize that no correlations are expected between the coupled 13C spins with the chemical shift differences of 90–140 ppm at 9.4 T, of 60–90 ppm at 14.1 T (as confirmed experimentally in this work), and of 40–60 ppm at 21.1 T. In other words, and considering that the R2-driven spin diffusion is accomplished by the second-order rotational resonance conditions, the above considerations imply that the correlation information for the desired chemical shift differences, i.e., aliphatic-to-carbonyl or aliphatic-to-aliphatic regions in proteins, can be obtained at each magnetic field by an appropriate setting of the MAS frequency for each R2nv sequence, i.e. 30 kHz at 9.4 T, 40 kHz at 14.1 T, and 60 kHz at 21.1 T.</p><p>Indeed, numerical simulations of the dependence of the polarization transfer efficiencies on the chemical shift differences for each R2nv sequence, conducted for the magnetic field strength of 21.1 T and the MAS frequency of 60 kHz demonstrate that the transfer profiles (see Figure S4 of the Supporting Information) are in agreement with the above analysis, which was made on the basis of the simulations at 14.1 T and 40 kHz.</p><p>At moderate MAS frequencies (<20 kHz), the numerical simulations indicate that the polarization transfer dynamics is only mildly dependent on the chemical shift differences, except for the rotational resonance conditions (see Supporting Information). Therefore, broadband homonuclear recoupling can be easily achieved by each of the four R2nv schemes, confirming our experimental observations. This behavior is expected to hold at moderate MAS frequencies for any magnetic field strength practically accessible in a modern NMR spectrometer.</p><!><p>As discussed in the previous sections, at very fast MAS frequencies where DARR (R212) experiment fails, the other R2nv symmetry sequences are expected to result in efficient polarization transfer and therefore should be useful for 13C-13C homonuclear correlation spectroscopy of proteins. In Figure 9, we provide the 14.1 T 13C-13C correlation spectra using the various R2nv symmetry schemes for spherical assemblies of U-13C,15N enriched HIV-1 CA capsid protein, spun at the MAS frequency of 40 kHz. We note that it is of great interest to further characterize the structure and dynamic properties of this morphology since it relates to the assembly in the immature HIV-1 virions. At the same time, the inherently limited spectral resolution of the spherical assemblies at moderate MAS frequencies (see Supporting Information showing a 10 kHz DARR spectrum) so far made their solid-state NMR studies challenging.</p><p>As illustrated in Figure 9 (b), at 40 kHz the DARR spectrum exhibits only few cross peaks, even at the mixing time as long as 250 ms. Notably, even cross peaks for directly bonded carbons are not observed in the spectrum, confirming that DARR fails under very fast MAS conditions in proteins. On the other hand, the R211 correlation spectrum displayed in Figure 9 (a) contains a large number of cross peaks in the aliphatic region. However, almost no aliphatic-to-carbonyl correlations are seen, illustrating the inefficient recoupling of the carbon spin pairs with large chemical shift differences by this sequence. Conversely, Figure 9 (d) illustrates that the R222 symmetry irradiation results in very efficient recoupling between the carbonyl and aliphatic carbons with large chemical shift differences, while the polarization transfer within the aliphatic carbons is very weak. The most uniform polarization transfer is achieved by the R221 irradiation, and the corresponding spectrum is provided in Figure 9 (c), exhibiting cross peaks over the entire spectral range. At the same time, the polarization transfer efficiency for spin diffusion within the aliphatic carbons is about 70% of that in the R211 symmetry driven experiment. This is consistent with the numerical simulations presented in Figure 8 and with the model experiments on alanine presented in Figure 7, that show the second highest polarization transfer efficiency for the R221 symmetry scheme among all R2nv sequences, for both aliphatic-to-aliphatic and carbonyl-to-aliphatic carbons. Therefore, the combined results provided in Figure 9 demonstrate that the R222, R221, and R211 symmetry-driven spin diffusion experiments are efficient for 13C-13C correlation spectroscopy in proteins and can be used in place of DARR at very fast MAS frequencies.</p><p>Another observation made on the basis of the experimental 13C-13C spin diffusion spectra of spherical HIV-1 CA assemblies acquired at 40 kHz and at 10 kHz is that the 40 kHz R2-based spectra exhibit higher resolution (as illustrated in Figure S6 of the Supporting Information). To further test this experimental observation, we carried out R2-based experiments for U-13C,15N-enriched DLC8 protein. The resulting 2D 13C-13C correlation spectra acquired with MAS frequencies of 10 kHz (DARR) and 40 kHz (R211) are shown in Figure 10. As can be appreciated, the R211 correlation spectrum recorded at ωr=40 kHz (Figure 10 (b)) exhibits significantly higher resolution without any loss of the correlation information, compared to the DARR spectrum at 10 kHz. The 1D slices extracted along the ω1 and ω2 frequency dimensions of the 2D spectra (Figure 10 (c) and (d)), clearly demonstrate that the lines are narrower along both frequency dimensions at the MAS frequency of 40 kHz, even when low-power CW 1H decoupling with the rf field strength of 9 kHz was performed. This resolution gain is also obvious in the comparison of the frequency domains of the indirect (ω1) dimensions of the 40 and 10 kHz R2 and DARR spectra (see Supporting Information).</p><p>R2nv driven spin diffusion experiments were also conducted on the sparsely-13C, U-15N-enriched CAP-Gly domain of dynactin in which only few neighboring 13C atoms are enriched (for enrichment pattern, see72–74). Figure 11 displays the 2D 13C-13C correlation spectra with the various R2nv symmetry schemes at the MAS frequency of 40 kHz. As can be appreciated, excellent resolution is seen, and the correlation information as well as the transfer efficiencies using the various R2nv sequences for the sparsely labeled CAP-Gly sample are consistent with those obtained for HIV-1 CA protein assemblies and DLC8. Interestingly, in the case of the sparsely enriched CAP-Gly protein, no appreciable resolution enhancements were noted for the MAS frequency of 40 kHz compared to the 10 kHz spectra (see Supporting Information), suggesting that considerable contributions to the linewidths in the 10 kHz spectra of the uniformly enriched proteins are due to 13C-13C dipolar and 13C-15N scalar couplings.</p><p>Overall, the above results on model compounds and on three different protein samples demonstrate that R222, R221, and R211 experiments perform well at very fast MAS rates, while DARR and PDSD sequences fail. These R222, R221, and R211 experiments not only have high transfer efficiencies, but also display greatly enhanced spectral resolution for uniformly enriched samples under very fast MAS conditions. Both of these features are critically important in multidimensional experiments for resonance assignments and measurements of distance restraints in large proteins and protein assemblies, in both uniformly labeled and sparsely or selectively labeled samples.</p><!><p>In summary, we describe a family of rotor-synchronized R2nv symmetry sequences for homonuclear correlation spectroscopy in rotating solids, in which R211, R212, R221, or R222 irradiation is applied to the proton spins during the mixing time to assist 13C-13C polarization transfer. Three of these R2-driven spin diffusion sequences (RDSD), R211, R221, and R222, work efficiently at both moderate and fast MAS frequencies. Our results indicate that RDSD sequences exhibit similar transfer efficiencies at moderate MAS rates (up to 20 kHz), and that R211 and POST R221 sequences are superior for the recoupling of coupled spins that possess large chemical shift differences, such as carbonyl-to-aliphatic carbons. At very fast MAS frequencies (40 kHz), where the PDSD and DARR (R212) experiments fail, the R222, R221, and R211 sequences exhibit high polarization transfer efficiencies. Since the broadened second-order rotational resonance conditions (ω1 ± nωr - KscωDD ≤ Δωiso ≤ ω1 ± nωr+ KscωDD) are dominated by the rf field strength and the size of the re-introduced dipolar interactions, these symmetry-based experiments exhibit different dependencies on the chemical shift differences between coupled spins. The R211 sequence is most efficient for recoupling of the carbons with small chemical shift differences, while the R222 sequence shows highest polarization transfer efficiencies for coupled spins with large chemical shift differences. The R221 symmetry sequence displays high transfer efficiency and uniform excitation for both aliphatic-to-aliphatic and carbonyl-to-aliphatic correlations (see the summary figure in Supporting Information). With the replacement of the basic π pulse by various composite pulses, the appropriate POST R2nv symmetry sequences with appropriate are expected to give faster transfer rate and higher transfer efficiency. Given the advantageous properties of the R2nv symmetry-based spin diffusion schemes introduced here, we believe that they will become indispensible for resonance assignments and structure determination of proteins and protein assemblies.</p>

PubMed Author Manuscript

Molecular recognition of <i>N</i>-acetyltryptophan enantiomers by β-cyclodextrin

The enantioselectivity of β-cyclodextrin (β-CD) towards L-and D-N-acetyltryptophan (NAcTrp) has been studied in aqueous solution and the crystalline state. NMR studies in solution show that β-CD forms complexes of very similar but not identical geometry with both L-and D-NAcTrp and exhibits stronger binding with L-NAcTrp. In the crystalline state, only β-CD-L-NAcTrp crystallizes readily from aqueous solutions as a dimeric complex (two hosts enclosing two guest molecules). In contrast, crystals of the complex β-CD-D-NAcTrp were never obtained, although numerous conditions were tried. In aqueous solution, the orientation of the guest in both complexes is different than in the β-CD-L-NAcTrp complex in the crystal. Overall, the study shows that subtle differences observed between the β-CD-L,D-NAcTrp complexes in aqueous solution are magnified at the onset of crystallization, as a consequence of accumulation of many soft host-guest interactions and of the imposed crystallographic order, thus resulting in very dissimilar propensity of each enantiomer to produce crystals with β-CD.

molecular_recognition_of_<i>n</i>-acetyltryptophan_enantiomers_by_β-cyclodextrin

Introduction<!>NMR studies<!>X-ray crystallography studies<!>Conclusion<!>Experimental Materials and methods<!>NMR spectroscopy<!>X-ray crystallography<!>Molecular modeling

<p>Cyclodextrins (CDs) are cyclic, water-soluble carbohydrates with a rather non-polar cavity that can host a variety of organic molecules (guests) and form inclusion complexes [1]. The guest molecules may be completely or partly enclosed inside the cavity depending on their size and the CD macrocycle's dimensions. The host-guest interactions established in the cavity are of van der Waals type, whereas between parts of the guest extending out of the cavity and the host's hydroxy groups are H-bonding interactions and/or of electrostatic nature. CDs have been studied and used for the enhancement of solubility, bioavailability and stability of drugs [2][3][4][5]. Moreover, being oligomers of α-D-glucopyranose, CDs possess an intrinsic chirality, thus they form diastereomeric inclusion complexes with enantiomeric pairs and frequently they exhibit enantioselectivity in aqueous solution or they can co-precipitate with only one enantiomer (enantioseparation). The separation of enantiomers via cyclodextrin inclusion is particularly important in the case of guests of pharmaceutical interest, since enantiomerically pure drugs are crucial for the pharmaceutical industry [1,6,7].</p><p>It has been proven difficult so far to explain and to predict the recognition abilities of specific CDs towards enantiomers, especially in solution. An interesting attempt is a thermodynamic study in aqueous solution with microcalorimetry of a large number (43) and variety of chiral organic compounds [8] with β-CD at room temperature. It was shown that properties and interactions important for chiral recognition include (i) weak non-bonding interactions rather than polar, (ii) nonsymmetrical non-polar penetrating guests and (iii) large distance of the chiral center from charged/hydrophilic groups. Moreover, trends in enantioselectivity do not follow trends in association constants, i.e., the association constants for the β-CD complexes of both enantiomers of N-acetyltyrosine, N-acetylphenylalanine and N-acetyltryptophan are in decreasing order, whereas their enantioselectivity (ratio of the binding constants, K, of the L-to the D-enantiomer) shows an increasing order (1.04, 1.1 and 1.34, respectively). X-ray crystallography, on the other hand, can improve our understanding of chiral recognition by CDs at the atomic level by providing insight into the interactions and the fit of the guest in the cavity, taking into account that crystal lattice forces may introduce additional and more stringent parameters for the enantiodiscrimination [9,10]. However, the crystallographic structures of diastereomeric complexes of CDs with chiral guest molecules in the literature are scarce. For β-CD with fenoprofen [7], a partial chiral resolution of the racemic mixture occurs, since the obtained crystals contain discrete β-CD dimers enclosing (R)-or (S)-enantiomers in a S/R ratio = 3:1. The enantiomers adopt different orientations in the β-CD dimers and preference of the (S) complex is dictated both by stronger H-bonding of the carboxyl group, as well as more favorable methyl-phenyl interactions inside the cavity. In contrast, no discrimination is shown by β-CD for (R)-and (S)flurbiprofen [11], since the crystals grown from the racemic mixture have both enantiomers enclosed (as a head-to-head dimer) in a β-CD dimer. In the case of substituted CDs, 2,3,6tri-O-methyl-α-CD discriminates between (R)-and (S)-mandelic acid [12] as it forms very different crystals from a racemic mixture. The same host crystallizes exclusively with (R)-(−)-1,7dioxaspiro [5.5]undecane, the Dacus Oleae pheromone, from an aqueous solution of the racemic mixture (enantioseparation) [13] also exhibiting high enantioselectivity in solution. Likewise, heptakis-(2,3,6-tri-O-methyl)-β-CD displays high enantio-selectivity in solution towards (S)-(+)-1,7-dioxaspiro [5.5]undecane and under certain conditions it co-crystallizes only with the (S)-enantiomer [14]. Induced host-guest fit, made possible by the macrocyclic flexibility of the permethylated CDs plays a crucial role in their capacity for chiral discrimination.</p><p>Chiral recognition of amino acids and their derivatives by CDs has been also tested using phase-solubility diagrams [15], NMR spectroscopy [16] and electrochemical methods [17], as well as by X-ray crystallography [18]. Detailed structures of β-CD with L-and D-N-acetylphenylalanine (NAcPhe) grown separately [18] has shown that although the two complexes are isomorphous (same space group, very similar unit cell dimensions and same packing of β-CD dimers) there are differences regarding the positioning of the guest molecules, the D-enantiomer being ordered, whereas the L-enantiomer extensively disordered. This disparity seems to be determined by subtle hydrophobic differences and H-bonding interactions among guests themselves and with the host and co-crystallized water molecules in the lattice. Additional structures of β-CD with different L-phenylalanine derivatives [19,20] confirm the above general result. In the present study, we report on the inclusion of the Land D-enantiomers of N-acetyltryptophan (NAcTrp) in β-CD (Scheme 1) in an effort to contribute to the study of chiral recognition of amino acid derivatives by CDs in the crystalline state and in solution. The guest NAcTrp has been selected because of its large aromatic side chain with appropriate dimensions to fit tightly in the β-CD cavity thus expected to have restricted mobility and limited disorder. Indicative of the interest and possible applications of the CD use in chiral selectivity/discrimination of tryptophan are studies in aqueous solution [21], in electrochemistry for sensor development [17,22,23], as components of solid phases in chromatography [24], or in capillary electrophoresis [25].</p><!><p>In deuterium oxide (D 2 O), each of the NAcTrp enantiomers induced significant chemical shift displacements (shielding) in the 1 H NMR signals of the β-CD cavity protons, namely H3 (near the wider, secondary side) and H5, H6,6' (at the narrower, primary side), signifying cavity inclusion of each enantiomer (Scheme 1). When a racemic mixture of NAcTrp was added to a β-CD solution no differentiation in the signals was observed due to in situ formation of diastereomers, except for a very small splitting of the methyl signal of the N-acetyl group. No differentiation was detected in the 13 C NMR spectrum either. In order to determine the stoichiometry of the complexes continuous variation (Job) plots [26] were drafted. For β-CD protons only the cavity signals due to H5, H3 and H6,6' showed significant shifts upon complexation (Supporting Information File 1, Figure S1). The inflection point of the graphs at 0.5 indicates a 1:1 stoichiometry for both enantiomers. The tryptophan protons were affected differently upon complexation (Supporting Information File 1, Figure S2), i.e., the graphs due to shifts of the indole's benzene ring protons (H3, H4, H5 and H6) indicate a 1:1 host/guest stoichiometry, whereas those of the indole moiety (H8) and of the aliphatic protons (H9,9', H10, H12), with an inflection point at ≈0.3, suggest a host/guest ratio close to 2:1. This behavior reveals the existence of two different complexation modes, one involving the indole phenyl ring with one host only and the aliphatic chain with two host molecules. The fact that the second mode takes place mainly when there is an excess of host concentration indicates that the inclusion of the indole moiety is the predominant mode of interaction. Moreover, it is observed that the magnitude of the shifts of the L-enantiomer are always larger and the slopes of the Job plots steeper than those of the D-enantiomer, suggesting stronger binding of β-CD with L-than with D-NAcTrp. 2D ROESY spectra of each enantiomer with β-CD at a 1:1 mole ratio in D 2 O were obtained under identical conditions (temperature, concentration, acquisition parameters). Strong intermolecular dipolar interactions were observed between indole protons (H3, H4, H5, H6, H8) and the β-CD cavity protons (H5, H6,6', H3) in both enantiomeric guests, confirming full inclusion of the Trp side chain. To facilitate the comparison of the two in situ formed diastereomers and to visualize the small differences (Supporting Information File 1, Figure S3) in dipolar through space intermolecular interactions in each case, 3D correlation maps were employed. They were displayed carefully so as to ensure the same intensity for the reference intramolecular correlations between NAcTrp H9,9' with H6 (average distance ≈3.5 Å) and with H8 (average distance ≈4.0 Å) in each of the enantiomers (Figure 1a), enlarging the points of difference in the magnified maps (Figure 1b). Thus (i) guest-H6,H3,H5-host-H5,H66',H3 interactions are very similar in both enantiomers with guest-H5-host-H3 clearly weaker than the others, and guest-H6/host-H6,6' stronger in L-than in D-, suggesting that guest-H6,H3 are embedded inside the cavity, guest-H5 is closer to the narrow β-CD rim and L-H6 is closer to it than D-H6. (ii) Guest-H8-host-H3 interactions are equally strong in both enantiomers, stronger than the guest-H8/host-H5,H6,6' ones, which in turn are stronger in L-than in D-. Moreover, interactions between guest-Me12-host-H3 are strong for both enantiomers (Supporting Information File 1, Figure S3b), suggesting that the N-acetyl group is in both cases at the wide secondary opening of β-CD, and L-H8, is closer to the primary opening than D-H8 suggesting a difference in tilting. (iii) Guest-H4-host-H5 interactions are similar in both enantiomers but this of guest-H4-host-H6,6' is considerably stronger in D-than in L-, while guest-H4-host-H3 interactions are practically absent for both enantiomers, implying that L-H4 is extended further out of the primary side than D-H4. (iv) Guest-H9,9' and H10 show weak interactions with host-H3 thus they reside mostly closer to the wide opening of the host.</p><p>The above interactions detected by NMR suggest that in the aqueous environment the inclusion modes in each diastereomeric complex are very similar but non-identical. D-H4 is located near the primary side of the host, while L-H4 is completely outside (scarcely communicates with the cavity). On the other hand H8 (at a ≈7 Å distance from H4) is at the secondary side in both enantiomers, slightly closer to H5 of the host only in the L-enantiomer. These interactions suggest a common binding model, with the indole part included in the direction H4 to H8 from primary to secondary opening and with the L-enantiomer having its H4 end exposed and its NAc group at the secondary side in contact with CD-H3. A different degree of tilting with respect to the β-CD axis to accommodate the hydrophobic NAc group in the cavity is inferred by the NMR data in each case, thus explaining the small differences observed in solution. However, as the Job plots suggested, the aliphatic part is influenced by a second host molecule presumably via its secondary side. This implies that in solution, host-guest association is possible through additional orientations and stoichiometry, thus the presence of alternative arrangements in low percentage cannot be excluded.</p><!><p>In the crystalline state, the structure of the inclusion complex of L-NAcTrp in β-CD comprises dimers. The asymmetric unit of the complex contains two crystallographically independent β-CD hosts (A and B) forming a dimer (Figure 2), in which two guest molecules of L-NAcTrp are enclosed in a head-to-head fashion (host:guest ratio, 1:1). The pair of L-NAcTrp molecules inside the dimer are found in orientational disorder, i.e., the guest exhibits a major orientation, molecules C and D (occupancy 65%), and a co-existing minor orientation (molecules E and F, occupancy 35%) in a statistical fashion. The dimers pack along the axis a at an angle of 19° thus forming a broken channel (Intermediate packing) [10,27]. The mean distance of the centers of mass of two consecutive β-CD dimers is 5.78 Å. Co-crystallized with each dimer, 21.45 water molecules are found distributed over 36 sites. The water molecules form the usual water networks of H-bonds, one linking the primary and the other the secondary hydroxy groups [28], many of them stabilizing the crystal lattice (structural water molecules).</p><p>The glucopyranose residues (in 4 C 1 chair) of both A and B β-CD have a rather undistorted conformation (Supporting Information File 1, Table S1) (angles between the glycosidic oxygen atoms O-4n similar to these of the regular heptagon, 128.57°, deviations of the O-4n atoms from their mean plane, close to zero). The tilt of the mean glucopyranose planes towards their 7-fold axis are small and close to their average values (7.1 and 7.7°, respectively). As in all β-CD dimeric complexes [28], the macrocycles' conformation is stabilized by hydrogen bonds connecting (i) intramolecularly, the O-3n and O-2(n+1) atoms of neighboring glucopyranose units (mean 2.73 Å and 2.75 Å for A and B, respectively, 2.78 Å in hydrated β-CD) and (ii) intermolecularly, the O-3nA and O-(8−n)B atoms of monomers A and B, respectively (range of distances 2.7-2.8 Å, Supporting Information File 1, Table S2). At the primary side, only β-CD molecule B exhibits disorder of the C-Ο63Β bond in two conformations, the major (−)-gauche C-Ο63Βa (occupancy 78%) pointing outward and the minor (+)-gauche C-Ο63Βb (22%) pointing towards the interior of the cavity, the latter interacting with guests C and D of neighboring dimers (Figure 3a).</p><p>The aromatic moieties of both guest orientations maintain the same relative position with the host, their planes interacting in a π•••π fashion (Figure 2 and Figure 3) (dihedral angle between D, F) in cavity B are close to the secondary hydroxy level, apparently in order to optimize the π•••π interactions between the indole planes (Figure 2 and Figure 3, Table 1). The above suggest a tight fit of the guest inside the cavity. On the other hand, the aliphatic part of NAcTrp, positioned in the space between dimers, exhibits more freedom: the carboxylic and acetylamino groups of guests D and F inside β-CD B are close and parallel, whereas in β-CD monomer A the acetylamino moiety of the major guest C is close to the carboxyl group of minor guest E, their respective carboxyl and acetylamino groups pointing to opposite directions (Figure 2 and Figure 3). These differences maximize the strong interactions between major guests C and D (Figure 3, Table 1).</p><p>Numerous trials to crystallize the inclusion complex of β-CD with D-NAcTrp have failed to give anything but hydrated β-CD crystals [29], as described in detail in the experimental section, however, some crystals were grown after hydrothermal treatment of the solution (65 °C for duration of 6 days) [30,31]. The structure of the latter could not be solved by isomorphous replacement (using the coordinates of β-CD-glutaric acid complex [32], that is isomorphous to hydrated β-CD [29]. This was an indication that the structure should be quite different from hydrated β-CD. However, no guest could be located during the refinement and the present structure (henceforth "β-CD-D-Table 1: H-bond distances of β-CD-L-NAcTrp complex: (1) between guest molecules themselves and with the host (2) with water molecules, (3) between structural water molecules and the host. NAcTrp") was refined as a β-CD-water complex (Table 2). "β-CD-D-NAcTrp" exhibits the "herringbone" packing of the β-CD monomers (Figure 4) as the hydrated β-CD structures reported so far [29,[33][34][35], as well as several monomeric β-CD complexes [32,36,37]. The conformation of the β-CD macrocycle (Supporting Information File 1, Table S3) is similar to the monomeric β-CD structures [29], but more distorted than in the dimeric β-CD-L-NAcTrp complex: The glucopyranose residues adopt the regular 4 C 1 chair conformation, but the angles be-tween them deviate from the angle of the regular heptagon and the tilt of their average planes towards the 7-fold β-CD axis varies between 5.0 and 25.8°. At the primary side, two hydroxy groups (O61 and O65) point towards the interior of the cavity and two exhibit two-way disorder of the C-Ο63 and C-Ο67 bonds.</p><p>Comparison of the "β-CD-D-NAcTrp" structure to this of hydrated β-CD [29] pinpoints the difficulty of solving the struc- ture as an isomorph. It can be seen (after the appropriate transformation of coordinates due to different origin and axes; Supporting Information File 1, Figures S4 and S5) that the hydrated β-CD macrocycle does not superpose exactly in the lattice of "β-CD-D-NAcTrp", which may render the two structures not quite isomorphous. It is worth noting that many of the hydrated β-CD structures [29,[33][34][35], as well as several monomeric β-CD complexes [32,36,37] are determined in lattices with different origin or interchanged crystallographic axes or even inverse coordinates (Supporting Information File 1, Figure S4). Further, by superposition of one glucopyranose unit of "β-CD-D-NAcTrp" to the equivalent unit of hydrated β-CD [29] the difference in coordinates of the two structures is more apparent (Supporting Information File 1, Figure S6). In contrast, the same kind of superposition applied to monomeric structures mentioned above shows that they superpose completely on hydrated β-CD.</p><p>Although the NMR results have shown that β-CD forms complexes with both L-and D-NAcTrp in aqueous solution at room temperature, it was not possible to crystallize the β-CD-D-NAcTrp complex. In contrast, the β-CD complexes of both enantiomers of N-acetylphenylalanine (NAcPhe) have been determined [18] and they are isomorphous with β-CD-L-NAcTrp.</p><p>Although the isomorphous complexes of L-NAcPhe and D-NAcPhe exhibit identical packing of the β-CD dimers, the relative stability of the guest molecules enclosed in them is controlled by subtle changes in the guest positioning. L-NAcPhe is highly disordered even at 20 K probably due to very weak nonpolar and polar interactions, whereas D-NAcPhe is highly ordered, although the non-polar interactions between the phenyl moieties are also weak. Its stability is gained by the N-acetyl group of one D-NAcPhe guest, which rotates and "hides" inside the dimer cavity [18] (probably because of unfavourable exposure to the aqueous environment). Similarly, β-CD-L-NAcTrp is also more stable than β-CD-L-NAcPhe due to the larger side chain of the guest. L-NAcPhe is shorter than in L-NAcTrp, which has two consequences for the stability of the complex (a) no strong π•••π interactions at 3.5 Å can be established in the middle of the β-CD dimer as in L-NAcTrp (Figure 5); (b) the aliphatic moieties of β-CD-L-NAcPhe protruding from the primary sides between dimers do not interact directly or even indirectly via β-CD hydroxy groups along the channels, as in the L-NAcTrp complex. Modeling the possibility of formation of a dimer β-CD-D-NAcTrp complex by energy minimization of the interactions of D-NAcTrp inside the β-CD dimer (as determined in the β-CD-L-NAcTrp structure) revealed a complex similar to β-CD-L-NAcTrp (Supporting Information File 1, Figure S7). The positioning of the D-indole groups is very similar to these of the L-enantiomer (closest distance 3.46 Å between the aromatic planes). The approaching aliphatic moieties between two β-CD dimers along the channel could be stabilized possibly by an inward pointing hydroxy group Ο63Βb of β-CD (assuming that the β-CD host remains unchanged), which H-bonds to the carboxylic oxygen atom of the D guest and the acetyl O1 atom of the C guest, however, the acetyl methyl group of C is exposed to the water environment. "Hiding" of the latter group inside the cavity, as in the case of the β-CD-D-NAcPhe complex, is not possible due to the bulkier indole group of D-NAcTrp that fills the cavity. This unfavorable environment might be a factor that forbids the formation of a β-CD-D-NAcTrp dimer structure. The difficulty in crystallizing the β-CD-D-NAcTrp may arise from a higher free energy barrier of crystal nucleation compared to other competing processes in solution at room temperature, but under the higher temperature and pressure conditions of the hydrothermal cell the presence of D-NAcTrp or of the complex β-CD-D-NAcTrp may influence the initial crystal nuclei which eventually lead to the grown crystals and differentiates them slight from hydrated β-CD. It is worth noting that hydrothermal treatment in crystallization trials has yielded uncommon structures such as, novel packing of β-CD-ethanol crystals [31] during trials to crystallize the β-CD-N-(1adamantyl)salicylaldimine complex in ethanol, novel association of β-CD monomers in structures of β-CD complexes, e.g., with 4-pyridinealdazine [30], polyethylene glycol [38] or adamantane [39].</p><!><p>This work has been focused on the ability of β-CD to discriminate between the enantiomers of N-acetyltryptophan. NMR studies in aqueous solution show that both enantiomers form similar, but not identical complexes with β-CD. L-NAcTrp induces larger shifts of β-CD cavity protons, suggesting stronger binding. For both enantiomers the prevailing complexation mode involves insertion in the cavity with the N-acetyl group in the secondary side and the indole moiety exiting the primary side, more exposed in L-than in D-NAcTrp. The tendency of the N-acetyl group to hide in the cavity is considered as the major cause for the differences between the two complexes that also results in somewhat folded NAcTrp structures, compared to the conformation observed in the crystal. In addition, both complexes are in contact with a second β-CD molecule suggesting presence of higher stoichiometries and possibility of different inclusion modes at low concentration. Overall, the orientation of both enantiomeric guests with respect to the macrocycle in the solution structures is opposite to the orientation of L-NAcTrp in ther crystal.</p><p>On the other hand, only the complex β-CD-L-NAcTrp crystallizes readily forming a dimeric complex (two host and two guest molecules) packed in broken channels, isomorphous to the known β-CD complexes of the NAcPhe enantiomers. Numerous crystallization trials failed to produce crystals of the β-CD-D-NAcTrp complex yielding only hydrated β-CD crystals. The fact that β-CD-D-NAcTrp could not be crystallized in dimers as the β-CD-L-NAcTrp might be due to destabilization of the interface between dimers, because of exposure of the acetyl group to the water environment of the exterior and the inability to "hide" in the cavity, due to the bulky indole group occupying it. Trials to employ more energetic crystallization conditions resulted in crystals of a slightly different structure than hydrated β-CD crystals. The disagreement between solution and crystal structure in terms of complex formation and orientation/conformation of the guest indicates that the lattice forces and organization in the crystal prevail by far over the soft host-guest contacts established in solution and determines the final orientation of the guest inside the host and the formation of the crystals per se.</p><!><p>N-Acetyl-L-tryptophan (L-NAcTrp), N-acetyl-D-tryptophan (D-NAcTrp) and β-CD were obtained from Sigma-Aldrich. Deuterium oxide was a product of Deutero GmbH.</p><!><p>The spectra were carried out on a 500 MHz Bruker Avance instrument at 300 K using a BBI probe, the library pulse sequences and 300 ms mixing time for the 2D ROESY runs. The compounds were dissolved in unbuffered D 2 O. The data was processed with Topspin.</p><!><p>Crystallisation of β-CD-L-NAcTrp. In an aqueous solution of β-CD (6 mM) an equimolar quantity of L-NAcTrp was added and stirred for an hour until the solution became clear, which indicated formation of a complex. Then the solution was placed in an incubator at 23 °C, where by slow evaporation of the solvent, single crystals appropriate for X-ray data collection were obtained. The crystals had a diamond shape and a slightly pink color.</p><p>Crystallisation trials of β-CD-D-NAcTrp. Trials to crystallize the complex of β-CD with D-NAcTrp under various conditions, including the above, did not result to single crystals of the complex. D-NAcTrp in the presence of β-CD (6 mΜ) at 50-60 °C, required a small quantity of ethanol in order to obtain a clear solution, from which crystals of hydrated native β-CD precipitated. This was proved from data collection from several crystals and structure determination based on isomorphous replacement using the coordinates of the β-CD-glutaric acid complex [32], which is isomorphous to hydrated β-CD [29]. Use of racemic mixtures of NAcTrp produced also native β-CD crystals. However, use of a hydrothermal cell [30,31], in which β-CD (0.050 mM) and D-NAcTrp (0.025 mM) were placed in 2 mL of water and left at 65 °C for 5-7 days, produced crystals that could not be refined by isomorphous replacement using the coordinates of hydrated β-CD or other isomorphous crystals, as above.</p><p>Structure determination. Low temperature X-ray data were collected at synchrotron radiation light sources. A single crystal, covered with a drop of paraffin oil, was mounted on a hair fiber loop and was instantly frozen to 100 K. Crystal data and analysis details are given in Table 2.</p><p>β-CD-L-NAcTrp. Data of the β-CD-L-NAcTrp complex were collected at the beamline X13 of EMBL at DESY, Hamburg, by the oscillation method using a CCD of 165 mm radius detector.</p><p>The DENZO and SCALEPACK [40] software were used for data processing and scaling, respectively. The unit cell parameters and their esds were determined by the least square method from the high resolution frames of the collected data. The structure was solved by the isomorphous replacement method using the host coordinates of the β-CD-1,12-dodecanodioic acid complex [28]. The structure solution and the refinement were carried out with the SHELXL97 program [41]. The coordinates of the guest and solvent atoms were determined by successive cycles of difference maps and refinement. The non-hydrogen β-CD atoms and the oxygen atoms of the co-crystallized water molecules were treated anisotropically. Hydrogen atoms were placed at idealized positions and refined by the riding model (UH = 1.25 UC). The refinement of the structure, by full matrix least squares, converged to R1 = 0.0609, wR2 = 0.1663 and Goodness-of-fit = 1.076, for Fo > 4σ(Fo). Refinement details appear in CCDC 1531988. The structures were rendered in PyMOL [42].</p><p>"β-CD-D-NAcTrp". Diffraction data were collected at the X06DA beamline, Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland. The XDS [43] software package was used to reduce data and determine the unit cell parameters and space group, which were the same as hydrated β-CD. Trials to use isomorphous replacement (using the coordinates of β-CD-glutaric acid complex [32], which is isomorphous to hydrated β-CD [29], to refine the structure was unsuccessful (vide supra). The structure was solved finally by molecular replacement methods [44] using the β-CD-glutaric acid complex coordinates. The refinement was carried out with the same strategy as in β-CD-L-NAcTrp complex. Early in the refine-ment numerous peaks appeared mainly at the primary hydroxy side of the cavity. Some were at bonding distances with each other, but by introducing the strongest of them as water molecules into the refinement did not result in a model of the guest (Table 2). Refinement details appear in CCDC 1531987. The structures were rendered in PyMOL [42].</p><!><p>The molecular models of D-NAcTrp complexes were based (a) on the geometry of the major orientation of β-CD-L-NAcTrp by changing the chirality of the Cα atom and (b) on β-CD non-hydrogen atoms of the corresponding lattice. To relieve steric clashes, restrained energy minimization of D-NAcTrp have been performed, while non-hydrogen atoms of β-CD are kept fixed in space. The XLEaP module of the AMBER 16 suite [45] was used and the GAFF parameters were applied to the β-CD molecules with AM1-BCC atomic charges using the Antechamber module [46], while the ff99SB parameters were employed for NAcTrp. Restraint energy minimizations in implicit solvent were performed for 1,000 steps using a pairwise generalized Born model [47], while all β-CD nonhydrogen atoms were kept fixed in space using harmonic restraints of 10 kcal/mol Å 2 . For the "β-CD-D-NAcTrp" complex, the indole moiety was placed inside the β-CD cavity with the aliphatic part protruding from its primary side towards the empty space formed by three neighboring β-CD monomers of the lattice (Figure 4b), whereas for the β-CD-D-NAcTrp dimer model the crystallographic coordinates of the β-CD-L-NAcTrp dimer were employed after changing the chirality of the L-NAcTrp Cα atom only to generate the D-NAcTrp guest molecule.</p>

Beilstein

Synthesis of tunable porosity of fluorine-enriched porous organic polymer materials with excellent CO2, CH4 and iodine adsorption

We herein report the construction of four the novel fluorine-enriched conjugated microporous polymers (FCMP-600@1-4), which have permanent porous structures and plenty of fluorine atoms in the skeletons as effective sorption sites. Among them, FCMP-600@4 shows considerable adsorption capacity of CO 2 of 5.35 mmol g −1 at 273 K, and 4.18 mmol g −1 at 298 K, which is higher than the reported values for most porous polymers. In addition, FCMP-600@1-4 display high selectivity of CO 2 /N 2 and high CH 4 uptakes.Today, world climate change and environmental problems have become increasingly prominent, so that people have to face the impact of excessive carbon dioxide of atmosphere on humanity, such as the global warming and acid rain. People are eager to find a solution to reduce the concentration of carbon dioxide in the atmosphere, while limiting its emissions, but also studying the ability to capture and storage of new materials. For this purpose, porous organic polymers (POPs) are emerged as the times required, which is a new kind of porous materials with large specific surface area and permanent pore structure. Because of its low density, large specific surface area, adjustable size, and high porosity, as well as a great potential in gas storage, separation, heterogeneous catalysis and other aspects 1,2 . POPs has become one of the hotspots in the recent years and rapid development. People have studied a series of POPs, in addition to traditional zeolites 3 and activated carbons 4 , including polymers of intrinsic microporosity (PIMs) 5 , hypercross-linked polymers (HCPs) 6 , conjugated microporous polymers (CMPs) 7 , and covalent organic frameworks (COFs) 8 . Compared with inorganic microporous materials and metal organic frameworks (MOFs), the synthesis of POPs has just started. But the organic synthesis of chemistry and polymer chemistry have been provided a wide range of development space for the synthesis of such materials. Therefore, from scientific research and practical application, design and synthesis of POPs with good adsorption property of carbon dioxide are of great significance.Among them, CMPs have attracted a high degree of concern in the recent years due to the excellent capture performance of CMPs for carbon dioxide 1,7-13 . CMPs are synthesized via metal-catalyzed cross-coupling chemistry to form cross-linked network. It is a subclass of POPs with conjugated structure, precise adjustment of micropore, large specific surface area and high stability, and the introduction of functional groups in the pore skeleton can effectively improve the capture capacity of carbon dioxide.In particular, the existence of nitrogen atoms in the porous skeleton, the aromatic heterocyclic network, the introduction of ions and so on are all beneficial to improving the adsorption of carbon dioxide on the materials in reported research studies [9][10][11][12] . In order to improve the adsorption properties of carbon dioxide on the polymers, in this paper, fluorine-enriched monomer 4,4′-dibromooctafluorobiphenyl (DBFB) and comonomer containing acetylene bond were selected to synthesize a series of structural tunable CMPs by Sonogashira-higihara reaction under Pd(0) catalysis (Fig.

synthesis_of_tunable_porosity_of_fluorine-enriched_porous_organic_polymer_materials_with_excellent_c

<!>Synthesis and characterization.<!>Discussion<!>Iodine capture.<!>Conclusion<!>Methods

<p>BET surface areas of the polymers increase from 755 to 901 m 2 g −1 , and the total pore volumes of the polymers hole also vary from 0.4242 to 0.6654 cm 3 g −1 . Particularly, FCMP-600@1-4 exhibit the outstanding adsorption of carbon dioxide and methane.</p><!><p>All of the polymer networks were synthesized by palladium(0)-catalyzed Sonogashira-higihara reaction of 4,4′-dibromooctafluorobiphenyl (DBFB) and comonomers containing acetylene moities. All the reactions were carried out at a fixed reaction temperature and reaction time (120 °C/48 h). The general synthetic routes toward FCMP@1-4 polymers are shown in Fig. 1. The insoluble polymers were filtered and washed with water, tetrahydrofunan, chloroform, and methanol, respectively, in order to remove the inorganic salts, organic monomers, residual catalyst, and oligomers. Then the pyrolysis reactions of the FCMP@1-4 were carried out on quartz tubes in an electric furnace under a argon atmosphere. The FCMP@1, 2, 3, and 4 samples were heated from the room temperature to 400 °C, 600 °C, and 800 °C with a heating rate of 3 °C/min, then pyrolyzed at 400 °C, 600 °C, and 800 °C for 2 h in argon gas (400 sccm), respectively. Then, we investigated the CO 2 adsorption capacity of these samples at 273 K and 298 K, respectively. We found these precursors at 600 °C displayed the best results compared to precursors at 400 °C and 800 °C. Therefore, we selected samples processed under 600 °C condition to be carefully investigated. The pyrolysis reactions at 600 °C in argon gas were denoted to FCMP-600@1, FCMP-600@2, FCMP-600@3, and FCMP-600@4. Our aim is to explore the effect of structure and connecting position of linker on pore properties of the resulting porous polymers. All of these polymers are insoluble in common organic solvents because of their highly cross linked structures.</p><p>Formation of FCMP@1-4 was confirmed by the FT-IR analysis. The disappearance of C-Br bonds in spectra of FCMP@1-4 compared with monomer 4,4′-dibromooctafluorobiphenyl demonstrated the success of phenyl-acetylene coupling (ESI, Figure S1). The four infrared spectra of the polymers are basically similar and demonstrate two main adsorption regions: a first absorption band in the 650-1250 cm −1 region, which is assigned as to benzene ring skeleton vibration; while the second peak close to 2900 cm −1 , corresponding to -C-H stretching of benzene ring. In addition, a relatively weak peak at approximate 2202 cm −1 , which referred to -C≡C-stretching of alkynyl moiety of FCMP@1-4, which was further proved that the polymers were synthesized successfully. Elemental analysis indicated that the carbon and hydrogen contents of FCMP@1-4 were close to the theoretical values of an ideal network with a high degree of polycondensation. X-ray diffraction (XRD) showed the amorphous nature of the resulting FCMP@1-4 (ESI, Figure S2a) and FCMP-600@1-4 (ESI, Figure S2b). Transmission electron microscopy (TEM) analyses also showed the amorphous texture of FCMP@1-4 (ESI, Figure S3(a-d)) and FCMP-600@1-4 (ESI, Figure S3(e-h)) materials. Field-emission scanning electron microscopy (FE-SEM; ESI, Figure S4(a-d)) was utilized to investigate the morphology of FCMP@1-4 polymers. The results of FE-SEM show that FCMP@1-4 are irregular sphere shape with particle size 100~300 nm, while FCMP-600@1-4 are irregular lumps with nanometre dimensions (ESI, Figure S4(e-h)). Furthermore, X-ray photoelectron spectroscopy (XPS) results display fluorine elements still exist in FCMP-600@1-4 after pyrolysis (ESI, Figure S5). The surface areas and porous properties of FCMP@1-4 and FCMP-600@1-4 were analyzed by nitrogen sorption analysis at 77.3 K. As shown in Figure S6(a-d), except for FCMP@4, the isotherms of FCMP@1, 2 and 3 showed rapid nitrogen adsorption at low pressure. The Brunauer-Emmett-Teller (BET) surface areas of FCMP@1, 2, 3 and 4 were calculated to be 551, 636, 692, and 88 m 2 g −1 , respectively. The total pore volumes were 0.3865, 0.6983, 0.4074 and 0.1180 cm 3 g −1 , respectively (ESI, Table S1). Compared to FCMP@1-3, FCMP@4 has a significantly low surface area and pore volume. This could be caused by the strong π-π stacking effect between the molecules tetrakis(4-ethynylphenyl)ethene, which lead to formation of planar sheet-like rather than three-dimensional structure 14 . Besides that, the pore size distributions of FCMP@1-4 are very broad (Figure S6(e)). The porosity data of the polymers are summarized in Table S1. In order to overcome this, a successive cross-linking pathway was utilized to improve the BET surface area of the porous polymers. The obtained porous materials FCMP-600@1-4 displayed high surface areas via template-free pyrolysis of FCMPs precursors at 600 °C. The BET surface areas were obtained to be 755, 780, 807 and 901 m 2 g −1 and the total pore volumes were 0.4242, 0.6654, 0.4033 and 0.4331 cm 3 g −1 (micropore volumes calculated from the nitrogen isotherms at P/P 0 = 0.0500 are 0.1951, 0.4502, 0.1636 and 0.1998 cm 3 g −1 ) for FCMP-600@1, FCMP-600@2, FCMP-600@3 and FCMP-600@4, respectively. These results indicated that the surface area and pore volume could be indeed increased by using the pyrolysis of POPs without any templates. As shown in Fig. 2a, FCMP-600@1-4 materials show type I isotherms featured by a sharp uptake at the low-pressure region between P/P 0 = 1 × 10 −5 to 1 × 10 −2 , reflecting the presence of micropores. Distinctly, FCMP-600@1 and 2 possess obvious hysteresis extending to low pressure between the adsorption and desorption isotherms, while FCMP-600@3 displays a relatively tiny hysteresis, which is partly attributed to the swelling in a flexible polymer network, as well as mesopore contribution [11][12][13] . Compared with FCMP-600@1, 2, and 3, FCMP-600@4 exhibits a negligible hysteresis loop in the whole pressure range, suggesting that this polymer possesses a very rigid molecular structure. The increase in nitrogen sorption at a high relative pressure for FCMP-600@1-4 may arise from the interparticulate porosity associated with the mesopores of the samples. The pore size distributions were calculated from the nonlocal density functional theory (NLDFT) using the model of carbon as an adsorbent, and the main micropore size peaked at 1.05, 1.74, 0.78, and 0.84 nm for FCMP-600@1, 2, 3, and 4, respectively (Fig. 2b).</p><!><p>Gas uptake capacity and separation. The CO 2 adsorption capacities of FCMP-600@1-4 under 273 K and 298 K were also measured (Fig. 3), which displayed linear trend at both 273 K and 298 K, respectively. At 273 K and 1.05 bar, the CO 2 capture uptakes of FCMP-600@1, 2, 3, and 4 are 88, 68, 73, and 119 cm 3 g −1 (5.35 mmol g −1 ), respectively (Fig. 3a). The adsorbance also can reach 65, 49, 61 and 93 cm 3 g −1 (4.18 mmol g −1 ) for FCMP-600@1, 2, 3, and 4 at 298 K (Fig. 3b). FCMP-600@1-4 can enhance the CO 2 uptake by 3.3-, 2.3-, 2.7-, and 4.2-fold than those of the corresponding precursor FCMP@1, 2, 3, and 4, respectively (ESI, Figure S7 and Table S1). Among them, FCMP-600@4 dispalys the highest CO 2 capture capacity than those of other three polymers at both 273 K and 298 K, which could be attributed to the narrower micropore size and higher micropore surface area of FCMP-600@4. This value is a little lower than that of recently reported P-PCz (S BET = 1647 m 2 g −1 , 5.57 mmol g −1 ) 9 , FCTF −1 -600 (S BET = 1535 m 2 g −1 , 5.53 mmol g −1 ) 15 , and PPF −1 (S BET = 1740 m 2 g −1 , 6.12 mmol g −1 ) 16 , but can (a) Nitrogen adsorption/desorption isotherms measured at 77.3 K for FCMP-600@1-4, the isotherms of FCMP-600@1-3 are shifted vertically by 300, 200 and 100 cm 3 g −1 for better visibility, respectively; (b) pore size distributions calculated using density functional theory (DFT) method, for clarity, the curves of FCMP-600@1-3 are shifted vertically by 3, 2 and 1 cm 3 g −1 , respectively. compete with the best performing POP-based adsorbents like BILP-4 (S BET = 1135 m 2 g −1 , 5.34 mmol g −1 ) 17 , ALP −1 (S BET = 1235 m 2 g −1 , 5.37 mmol g −1 ) 18 . In particularly, FCMP-600@4 (4.18 mmol g −1 ) also exhibits an excellent CO 2 capacity at 298 K, which is higher than the reported values for most porous polymers at 273 K, such as CPOP-9 (S BET = 2440 m 2 g −1 , 4.14 mmol g −1 ) 19 , CPOP-8 (S BET = 1610 m 2 g −1 , 3.75 mmol g −1 ) 18 , and fl-CTF400 (S BET = 2862 m 2 g −1 , 4.13 mmol g −1 ) 20 . Compared to FCMP-600@1 and 3, FCMP-600@2 has a broader micropore size distribution. Therefore, FCMP-600@2 exhibits lower CO 2 capture capacity than those of FCMP-600@1 and 3 at the same conditions, although FCMP-600@1, 2 and 3 show the similar BET surface areas.</p><p>The isosteric heat (Q st ) of adsorption CO 2 was estimated from adsorption data collected under 273 K and 298 K through the Clausius-Clapeyron equation. At zero coverage, the Q st of FCMP-600@1, 2, 3, and 4 are 23.4, 19.9, 17.3, and 21.4 kJ mol −1 , respectively (Fig. 3c). The Q st are lower than the values reported for imine-linked organic polymers, CTFs, diimide polymers, and so on 9,[15][16][17][18][19][20] . The relatively high CO 2 uptake and binding by FCMP-600@1-4 are most likely due to favorable interactions of the polarizable CO 2 molecules through hydrogen bonding and/or dipole quadrupole interactions that utilize the proton-free fluorine sites of phenyl rings [21][22][23][24][25] .</p><p>In light of high CO 2 capture capacities, high surface areas, fluorine-enriched skeletons, and small pore sizes for FCMP-600@1-4, it is reasonable to study the selective uptake of FCMP-600@1-4 for small gases (CO 2 , CH 4 , and N 2 ) to evaluate their potential use in gas separation. The methane isotherms depicted in Fig. 3d are fully reversible and the uptakes of FCMP-600@1, 2, 3, and 4 reach 36, 27, 30, and 53 cm 3 g −1 at 273 K and 1.0 bar, respectively (ESI, Figure S8 and Table S1). The CH 4 uptakes of FCMP@1-4 were 4.8-6.6 cm 3 g −1 at the same conditions (ESI, Figure S9 and Table S1). Apparently, FCMP-600@1-4 are higher 4.5 −1 1 times than those of the FCMP@1-4 precursors. This result implyed that the FCMP-600@1-4 can efficiently capture CH 4 due to high microsurface area and micropore volume 26,27 . The selectivities of FCMP-600@1-4 toward CO 2 over CH 4 and N 2 were investigated by collecting pure component physisorption isotherms at 273 K (ESI, Figure S8), and then which were predicted from the experimental pure component isotherms using the ideal adsorbed solution theory (IAST). At zero coverage, the high CO 2 /N 2 selectivity was recorded for FCMP-600@1-4 (109-77 at 273 K) (ESI, Figure S10a). Moreover, FCMP-600@1-4 show a moderate level CH 4 /N 2 selectivities: 8-11 (273 K) (ESI, Figure S10b).</p><!><p>In the recent years, the capture of iodine using porous materials has attracted considerable interest. Most interestingly, we found the fluorine-enriched polymers were highly efficient for the iodine adsorption. The absorption of solid iodine was conducted by exposing the samples to nonradioactive iodine vapor in a sealed vessel at 350 K and ambient pressure, which was the typical fuel reprocessing condition. Gravimetric measurement was performed at different time intervals during the iodine loading (Fig. 4a). Except for FCMP-600@2, the maximum iodine uptakes of other three porous materials were reached quickly saturated in the first 4 h. As the synthesized polymers, FCMP-600@2 has the maximum value for iodine uptake reached up to 141 wt.%, followed by FCMP-600@4 (111 wt.%), FCMP-600@1 (108 wt.%), and FCMP-600@3 (90 wt.%). The thermogravimetric analysis (TGA) of the I 2 -loaded FCMP-600@2 and 4 polymers reveal a significant weight loss from 90 to 300 °C (ESI, Figure S11), the calculated iodine mass loss were 152 and 105 wt.% for FCMP-600@2 and FCMP-600@4, respectively, which was close to the saturated adsorption value.</p><p>Additionally, the FCMP-600@1-4 are capable of capture iodine in solution. When the FCMP-600@2 (30 mg) in iodine/hexane solution (4 mg mL −1 , 3 mL), the dark purple solution gradually faded to light purple (Fig. 4b and Figure S12). The UV/Vis absorption intensity of the samples was decreased with the prolonged action time (ESI, Figure S13). It can be observed from the adsorption kinetics of iodine at room temperature that the adsorption process was affected by the contact time (Fig. 4c). In the initial stage, the adsorption capacity increased quickly with the prolonged contact time, and then slow down to equilibrium after about 10 h. The removal efficiencies of polymers achieved for the solution are 81.2-92.4%. The adsorption kinetics of iodine for FCMP-600@1-4 were analyzed through the frequently used pseudo-first-order and pseudo-second-order models were adopted 22 . Results show that the adsorption data fits well in pseudo-second-order kinetic model with good linear correlation coefficient (R 2 ) values of 0.9961, 0.9962, 0.9962 and 0.9962 for iodine solution of FCMP-600@1, 2, 3 and 4, respectively (ESI, Table S2, Figures S14 and S15). This confirmed that the iodine adsorption process in this work was governed by the pseudo-second-order kinetics. The XPS spectrum of fluorine-enriched polymers indicated that the coexistence of elemental iodine and triiodide ion, which suggested a hybrid of physisorption and chemisorption (ESI, Figure S16). Furthermore, it is very easy to remove or release the trapped iodine molecules of the samples via immersion of the iodine-loaded sample in ethanol. When the I 2 -FCMP-600@1-4 were immersed in ethanol, the colour of the solvent were changed from colourless to dark brown (ESI, Figure S17), indicating that the iodine guests were released from the solid. The four samples were recycled easily for at least five times without significant loss of iodine uptake (ESI, Figure S18).</p><p>The saturated iodine adsorption capacities of FCMP-600@1-4 can be determined from the adsorption isotherms (ESI, Table S3, Figures S19 and S20). Two different adsorption stages were observed from the plot of the equilibrium concentration versus the quantities of the adsorbed iodine at equilibrium. At the equilibrium uptake increases linearly with the increase of iodine solution concentration at low concentration. Then, the adsorption reached its maximum value and the adsorption process turned to be independent on the concentration. The simulation results revealed that the iodine adsorption of samples could be well described using Langmuir adsorption isotherm (ESI, Figures S19 and S20), suggesting a monolayer adsorption behavior for iodine molecule on the surface of polymers. From the sorption kinetics, the maximum capacities for iodine uptake reached up to around 550, 729, 520, and 539 mg g −1 for FCMP-600@1, 2, 3, and 4, respectively.</p><!><p>In summary, four the novel fluorine-enriched porous materials were successfully designed and synthesized. The properties of FCMP-600@1-4 were well investigated and discussed. The BET surface areas of FCMP-600@1-4 can be tuned by changing the geometry and size of comonomer. FCMP-600@1-4 have the BET specific surface areas of 755-901 m 2 g −1 as well as permanent microporosity, and the abundant fluorine atoms in the skeleton endow the materials with high CO 2 /N 2 (109-77) and CH 4 /N 2 (8 −1 1) selectivities. At 273 K and 1.05 bar, FCMP-600@4 exhibits the highest CO 2 uptake of 119 cm 3 g −1 , and CH 4 uptake of 53 cm 3 g −1 , and the rest materials are in the range of 68-88 cm 3 g −1 for CO 2 . Meanwhile, FCMP-600@1-4 show good adsorption capacities of 90-141 wt.% toward iodine vapor. We hope this type of fluorine-doped absorbent can be effective for gas storage and will bring new application possibilities.</p><!><p>Synthesis of FCMP@1. 4,4′-Dibromooctafluorobiphenyl (114 mg, 0.25 mmol) and 1,3-diethynylbenzene (47 mg, 0.375 mmol) were put into a 50 mL two-necked round-bottom flask, then the flask exchanged 3 cycles under vacuum/N 2 . Then added to 2 mL DMF and 2 mL triethylamine (Et 3 N), the flask was further degassed by the freeze-pump-thaw for 3 times. When the solution had reached reaction temperature, a slurry of tetrakis(triphenylphosphine)palladium(0) (19.9 mg, 0.017 mmol) in the 1 mL DMF and copper(I) iodine (3.1 mg, 0.017 mmol) in the 1 mL Et 3 N was added, and the reaction was stirred at 120 °C for 48 h under nitrogen atmosphere. The solid product was collected by filtration and washed well with THF, methanol, acetone, and water for 4 times, respectively. Further purification of the polymer was carried out by Soxhlet extraction with methanol, and THF for 24 h, respectively, to give FCMP@1 as yellow powder (88.7% yield). Elemental Analysis (%) C 69.24, H 1.55. Found: C 66.88, H 1.16.</p><p>Synthesis of FCMP@2. 4,4′-Dibromooctafluorobiphenyl (114 mg, 0.25 mmol) and 1,3,5-triethynylbenzene (45 mg, 0.25 mmol) were put into a 50 mL two-necked round-bottom flask, then the flask exchanged 3 cycles under vacuum/N 2 . Then added to 2 mL DMF and 2 mL triethylamine (Et 3 N), the flask was further degassed by the freeze-pump-thaw for 3 times, purged with N 2 . When the solution had reached reaction temperature, a slurry of tetrakis(triphenylphosphine)palladium(0) (27.7 mg, 0.024 mmol) in the 1 mL DMF and copper(I) iodine (5.7 mg, 0.032 mmol) in the 1 mL Et 3 N was added, and the reaction was stirred at 120 °C for 48 h under nitrogen atmosphere. The solid product was collected by filtration and washed well with THF, methanol, acetone, and water for 4 times, respectively. Further purification of the polymer was carried out by Soxhlet extraction with methanol, and THF for 24 h, respectively, to give FCMP@2 as brown solid (93.6% yield). Elemental Analysis (%) Calcd. (Actual value for an infinite 2D polymer) C 70.86, H 1.11. Found: C 68.17, H 0.95. Synthesis of FCMP@3. 4,4′ -Dibromo o c taf luorobipheny l (114 mg, 0.25 mmol) and tetrakis(4-ethynylphenyl)methane (78 mg, 0.188 mmol) were put into a 50 mL two-necked round-bottom flask, then the flask exchanged 3 cycles under vacuum/N 2 . Then added to 2 mL DMF and 2 mL triethylamine (Et 3 N), the flask was further degassed by the freeze-pump-thaw for 3 times. When the solution had reached reaction temperature, a slurry of tetrakis(triphenylphosphine)palladium(0) (17.9 mg, 0.015 mmol) in the 1 mL DMF and copper(I) iodine (3.7 mg, 0.02 mmol) in the 1 mL Et 3 N was added, and the reaction was stirred at 120 °C for 48 h under nitrogen atmosphere. The solid product was collected by filtration and washed well with THF, methanol, acetone, and water for 4 times, respectively. Further purification of the polymer was carried out by Soxhlet extraction with methanol, and THF for 24 h, respectively, to give FCMP@3 as yellowish-brown powder (94.3% yield). Elemental Analysis (%) C 82.44, H 3.07. Found: C 78.28, H 3.43.</p><p>Synthesis of FCMP@4. 4,4′-Dibromooctafluorobiphenyl (114 mg, 0.25 mmol) and 1,1,2,2-tetrakis(4-ethynylphenyl)ethene (71.5 mg, 0.188 mmol) were put into a 50 mL two-necked round-bottom flask, then the flask exchanged 3 cycles under vacuum/N 2 . Then added to 2 mL DMF and 2 mL triethylamine (Et 3 N), the flask was further degassed by the freeze-pump-thaw for 3 times. When the solution had reached reaction temperature, a slurry of tetrakis(triphenylphosphine)palladium(0) (17 mg, 0.015 mmol) in the 1 mL DMF and copper(I) iodine (2.7 mg, 0.015 mmol) in the 1 mL Et 3 N was added, and the reaction was stirred at 120 °C for 48 h under nitrogen atmosphere. The solid product was collected by filtration and washed well with THF, methanol, acetone, and water for 4 times, respectively. Further purification of the polymer was carried out by Soxhlet extraction with methanol, and THF for 24 h, respectively, to give FCMP@4 as pale yellow powder (88.7% yield). Elemental Analysis (%) C 82.83, H 3.01. Found: C 75.79, H 2.12.</p><p>Synthesis of FCMP-600@1-4. The pyrolysis reactions of the FCMP@1-4 were carried out on quartz tubes in an electric furnace under argon atmosphere. The FCMP@1, 2, 3, and 4 samples were heated from the room temperature to 600 °C with a heating rate of 3 °C/min, then pyrolyzed at 600 °C for 2 h in argon gas (400 sccm), respectively. The pyrolysis reactions at 600 °C in argon gas were denoted to FCMP-600@1, FCMP-600@-2, FCMP-600@-3, and FCMP-600@4, respectively.</p>

Scientific Reports - Nature

Polarizable Simulations with Second order Interaction Model (POSSIM) force field: Developing parameters for alanine peptides and protein backbone

A previously introduced POSSIM (POlarizable Simulations with Second order Interaction Model) force field has been extended to include parameters for alanine peptides and protein backbones. New features were introduced into the fitting protocol, as compared to the previous generation of the polarizable force field for proteins. A reduced amount of quantum mechanical data was employed in fitting the electrostatic parameters. Transferability of the electrostatics between our recently developed NMA model and the protein backbone was confirmed. Binding energy and geometry for complexes of alanine dipeptide with a water molecule were estimated and found in a good agreement with high-level quantum mechanical results (for example, the intermolecular distances agreeing within ca. 0.06\xc3\x85). Following the previously devised procedure, we calculated average errors in alanine di- and tetra-peptide conformational energies and backbone angles and found the agreement to be adequate (for example, the alanine tetrapeptide extended-globular conformational energy gap was calculated to be 3.09 kcal/mol quantim mechanically and 3.14 kcal/mol with the POSSIM force field). However, we have now also included simulation of a simple alpha-helix in both gas-phase and water as the ultimate test of the backbone conformational behavior. The resulting alanine and protein backbone force field is currently being employed in further development of the POSSIM fast polarizable force field for proteins.

polarizable_simulations_with_second_order_interaction_model_(possim)_force_field:_developing_paramet

I. Introduction<!>A. Force Field<!>Electrostatic Energy<!>The Rest of the Force Field<!>B. Parameterization of the Force Field<!>C. Calculating Dimerization Energies for the Alanine Dipeptide Complexes with Water<!>D. Gas-Phase and Liquid-State Simulations of the Tridecaalanine peptide<!>A. Alanine Dipeptide and Tetrapeptide Conformational Energies and Angles<!>B. Alanine Dipeptide \xe2\x80\x93 Water Dimerization Energies and Distances<!>C. Gas-Phase and Hydrated Simulations of the Tridecaalanine Peptide (Ala-13)<!>IV. Conclusions

<p>While quantum mechanical calculations offer valuable data in a variety of biological and biomedical calculations, applications of empirical force fields remain the only way of approaching the majority of problems of interest. On one hand, they require less computer resources. On the other hand, the issue of choosing the best level of quantum theory is still a non-trivial one, and the level of quantum mechanical accuracy in a specific application is far from being guaranteed.</p><p>When empirical force fields are employed, accurate assessment of energy often requires explicit treatment of the electrostatic polarization.1 The properties which depend on it include dimerization energies and acidity constants of small molecules, energies of protein-ligand interactions, protein pKa values, or even the very thermodynamic stability of complexes in solutions. For example, we have demonstrated that that pKa values for acidic and basic residues of the OMTKY3 can be reproduced within 0.6 and 0.7 pH units of the experimental data with a polarizable force field. The corresponding errors with the non-polarizable OPLS were 3.3 and 2.2 pH units.2 Formation of sugar-protein complexes represents yet another example when polarization is critical for predicting a thermodynamically stable structure.3 It is generally acknowledged that polarization is an important component in many computational studies of proteins and protein-ligand complexes, although it is sometimes included in surrogate forms, such as, for example, conformation-specific protein charges.4</p><p>There are two main issues related to the empirical polarizable force field development. The first one is in the functional form of the electrostatic polarization. Using fluctuating charges saves time and is computationally efficient in simulating uniform systems such as pure liquid water.5 However, it causes problems when out-of-plane polarization response is required or when a bifurcated hydrogen bond is formed. Therefore, the inducible dipoles approach is more adequate when arbitrary systems have to be simulated with a high degree of accuracy. On the other hand, it is known that the inducible dipole technique slows down polariable calculations very significantly. In order to reduce the severity of this problem, we are applying the second-order approximation in treatment of the electrostatic polarization. It has been demonstrated to increase the speed by ca. an order of magnitude without sacrificing the accuracy.6 Moreover, this approximation makes the so called polarization catastrophe (the resonance-like infinite growth of the induced dipole moment values) impossible. Our previous paper described development of the POSSIM (POlarizable Simulations with Second order Interaction Model) software and force field parameters for a series of small molecules, including water and NMA. In this work, we describe creation of alanine and protein backbone parameter sets in the POSSIM framework.</p><p>The second issue is choosing the source of fitting data for a polarizable force field. High-level quantum mechanical results are very attractive in this respect,7,8 but experimental data can be more robust. We follow the middle-of-the-road path by relying on experimental data whenever possible and making heavy use of quantum mechanical calculations when needed. One important issue is the standard procedure of producing torsional parameters for peptides by fitting to conformational energies of di- and tetrapeptides.8 We include it in our work and are describing an improved procedure for creation of the torsional parameters in the Methods section below. At the same time, the quantum mechanical conformers employed in such calculations are created by gas-phase quantum mechanical optimizations and they often belong to parts of the conformational space which are not found in experimental protein structures. Therefore, we have included an additional conformational test in the alanine and backbone parameter fitting. It is known that the tridecaalanine peptide (ala-13) forms a stable–α helix.9 Therefore, we studied the stability of our POSSIM ala-13 α–helix and compared it to that of the OPLS-AA8 for benchmarking. We have also discovered that the quality of the force field in reproducing the quantum mechanical di- and tetrapeptide conformational energies has a relatively weak effect on the stability of the tridecaalanine peptide in water.</p><p>Overall, the following has been derived, developed or otherwise calculated in this work: (i) the torsional parameters for the alanine residues and protein backbones have been produced; (ii) binding energies of a water molecule with the alanine dipeptide as calculated with the POSSIM and OPLS-AA force fields have been compared with the quantum mechanical data to confirm transferability of the non-bonded parameters and to justify using the latter from the POSSIM NMA model in protein studies; (iii) the resulting parameters were employed in gas-phase and aqueous solution simulations of an α-helix to validate the resulting POSSIM parameters as acceptable in protein and peptide simulations. Moreover, the additional optimizations in the step (i) above and the steps (ii) and (iii) altogether represent a novel development in our methodology of protein force field production, as compared to that used to create the previous version of the force field.</p><p>The rest of the paper is organized as follows: Given in Section II is description of the methodology involved. Section III contains results and discussion. Finally, conclusions are presented.</p><!><p>The total energy Etot is a sum of the electrostatic interactions Eelectrostatic, the van-der-Waals energy EvdW, harmonic bond stretching and angle bending Estretch and Ebend and the torsional term Etorsion: (1)Etot=Eelectrostatic+EvdW+Estretch+Ebend+Etorsion</p><!><p>The electrostatic polarization energy as calculated with inducible point dipoles μ is: (2)Epol=−12∑iμiEi0, where E0 is the electrostatic field in the absence of the dipoles.</p><p> (3)μi=αiEi0+αi∑j≠iTijμj where α are scalar polarizabilities and Tij is the dipole-dipole interaction tensor. The self-consistent Equation 3 is usually solved iteratively. Let us explicitly write down the first two iterations: (4a)μiI=αiEi0 (4b)μiII=αiEi0+αi∑j≠iTijμjI=αiEi0+αi∑j≠iTijαjEj0</p><p>We are using the second-order expression in Equation (4b). It has been previously shown to yield a ca. order of magnitude increase of the computational speed with no loss of accuracy.6 The electrostatic energy also includes the pairwise-additive contribution from interactions of permanent charges: (5)Eadditive=∑i≠jqiqjRijfij,</p><p>The factor fij is set to zero for 1,2- and 1,3-pairs (atoms which belong to the same valence bond or angle), to 0.5 for 1,4-interactions (atoms in the same dihedral angle) and to 1.0 otherwise.</p><p>To avoid unphysical increase of the electrostatic interactions at short distances, each atom type has a cutoff parameter Rcut. When the overall distance Rij is smaller than the sum of these parameters Rminij=Rcuti+Rcutj for the atoms i and j, Rij is replaced by an effective smooth function</p><p>The following important points about the second-order approximation in equation 4b should be made. First of all, we do not fit parameters using the full-scale polarization solution to Equation 3 to later employ Equation 4b as an approximate technique during the simulations. For our practical purposes, Equation 4b is, in fact, the representation of the many-body interactions. It does differ from the true physical point-dipole approximation, and thus we always carefully monitor whether any errors are introduced by not computing inducible dipoles with the complete iterative procedure. So far, simulations of gas-phase dimers, quantum mechanical electrostatic three-body energies, pure liquids, solutions and peptides have given us no indication that the second-order approximation leads to any deficient physical results, and we have always been able to produce fitting to quantum mechanical and experimental data which was as good as for the full-scale polarization.6,10 Moreover, application of the second-order approximation given by Equation 4b turns the expression for the inducible dipoles into an analytical one, thus eliminating the possibility of the polarization catastrophe. This can also become a very useful feature in future developments, e. g. in creating a continuum dielectric model, as convergence issues are known to be of importance for continuum solvation techniques.</p><!><p>We are using the standard Lennard-Jones formalism for the van-der-Waals energy: (7)EvdW=∑i≠j4εij[(σijRij)12−(σijRij)6]fij</p><p>Geometric combining rules are applied (εij = (εi · εj)1/2, σij= (σi · σj)1/2). Bond stretching and angle bending are computed with the usual harmonic formalism, and the torsional term is calculated as: (8)Etorsion=∑iV1i2[1+cos(φi)]+V2i2[1−cos(2φi)]+V3i2[1+cos(3φi)],</p><p>The fixed-charges OPLS-AA force field used for benchmarking is functionally the same, except that it lacks the polarization part of the electrostatic energy.</p><!><p>Whenever possible, the force field parameters for the alanine peptides were adopted directly from the previously created NMA parameter values.10 The only completely new parameters were those for the backbone torsions. This is different from the previous version of the PFF for proteins in which electrostatic parameters for the alanine (and thus for the backbone) were also refitted.7 Therefore, we believe that the present work demonstrates a greater degree of utilizing parameter transferability.</p><p>Fitting of torsional parameters for the protein backbone φ and ψ angles (Figure 1) cannot be done separately from each other as the torsions are coupled.</p><p>The initial part of our torsional fitting was the same as used before.7,8 (i) The fitting was done to an ab initio data obtained previously8 at the LMP2/cc-pVTZ(-f)//HF-6-31G** level with Jaguar software suite.11 (ii) The choice of the fitting subspace is illustrated on Figure 2. Out of the six alanine dipeptide local minima previously used,7,8 only two are shown for the sake of clarity.</p><p>(iii) We used the following non-Boltzmann weighting scheme for the error at the fitting points: (9)Wi=A·exp(−b·Gi)</p><p>Here Gi is the magnitude of the torsional surface gradient at the point i, and Wi is the weight. This way more importance was given to the points with low gradients (near the minima).</p><p>In the presented work, we used the procedure described above only to produce the initial guess for the torsional parameters to be employed in Equation 8. After that, the following approach was taken. The errors in the conformational energies were combined with the errors in the conformational angles φ and ψ to produce the error function as shown in Equation 10: (10)erf=∑i(Ei0−Ei)2+∑j(φj0−φj)2+(ψj0−ψj)2</p><p>Here Ei0 and Ei are the quantum mechanical and empirical conformational energies for all the conformers i, and the second sum contains the values of the backbone angles φ and ψ. The error function was minimized as a function of the torsional parameters in Equation 8.</p><!><p>In order to test the transferability of the NMA nonbonded parameters employed for our alanine and protein backbone model, we calculated energies of interaction of the alanine-dipeptide with a water molecule. Structures and energies obtained for these systems with the POSSIM program were compared to the quantum mechanical results obtained with Jaguar.11 For hydrogen bonds, a good level of accuracy can be achieved via MP2 calculations extrapolated to the basis set limit, where the contribution of higher level excitations (e.g. CCSD (T)) has been shown to be negligible (except for some cases, such as pi stacking of aromatic rings, where the MP2 level has been shown to not be sufficient).</p><p>Briefly, dimer geometries were obtained by LMP2 optimizations with a cc-pVTZ(-f) basis set. The final quantum mechanical dimer binding energy Ebind, as used in this work, is a linear combination of the LMP2 binding energy for a smaller cc-pVTZ(-f) basis set (Eccpvtz) and the LMP2 binding energy with a larger cc-pVQZ(-g) basis set (Eccpvq).15 This method has been previously demonstrated to produce a high-quality fitting and benchmarking data for force field development.7,8</p><!><p>In order to give our alanine and backbone model a final test, we carried out simulations of a tridecaalanine (ala-13) peptide both in gas-phase and in aqueous solution at 25°C and 1 atm. The initial structure was set at the α-helix conformation, with φ = 296° and ψ = 319°, and the simulations proceeded with all the degrees of freedom completely unconstrained. It is known experimentally that an α-helix represents a stable conformation of alanine peptides, including ala-13, both in gas-phase and in aqueous solution.9 We intended to show that our POSSIM force field for the alanine and backbone protein systems performs reasonably well under these conditions and is thus sufficiently robust to be successfully employed in protein and protein-ligand studies.</p><p>Gas-phase and hydrated simulations consisted of at least 18 × 106 and 25 × 106 Monte Carlo configurations, respectfully, to ensure convergence. A 7 Å dipole-dipole cutoff was used. An 8 Å cutoff was employed for the intermolecular interactions in solution (including both the solute-solvent and solvent-solvent interactions). The standard correction procedure to account for the Lennard-Jones interactions beyond the cutoff was used. The electrostatic interactions were quadratically feathered over the last 0.5 Å before the cutoff distance. A rectangular box with periodic boundary conditions was used. The box contained 948 water molecules. The initial box setup was done to have 10 Å of water on each side of the hydrated ala-13 molecule. After that, the isobaric-isothermal (NPT) ensemble was used, with Metropolis Monte Carlo technique. In the case of the OPLS simulations, a three-site model was used with TIP3P13 non-bonded parameters and flexible bond lengths and bond angles. A flexible three-site POSSIM water model10 was employed in the polarizable runs.</p><p>All the calculations which did not involve quantum mechanics (i. e., geometry optimizations and Monte Carlo runs) were performed with our previously introduced POSSIM software suite.10 Whenever possible, comparison with the fixed-charges OPLS-AA force field was done, and the OPLS-AA results were also calculated with the POSSIM program.</p><!><p>We have followed the previously established procedure of calculating the alanine di- and tetrapeptide conformational energies and φ and ψ values as the initial assessment of the quality of the parameters for the alanine and protein backbones. The same set of the conformers that was employed in the previous studies was used.7,8 The production of the torsional parameters proceeded as described in the Methods section. First, weighted fitting to rotamer energies was carried out. The resulting parameters are shown in Table 1 (torsional parameters which are not listed were the same as in the NMA model10). We denote this set of parameters as tors.1, as opposed to the final set tors.final. Given in Table 2 are conformational energies and φ and ψ values, as computed with the quantum mechanics, POSSIM and OPLS. In addition to the six conformers used in previous studies, we have also added PII and αR which are more relevant in aqueous solution.14 Quantum mechanical optimizations were done at the LMP2/cc-pVTZ(-f) level. In both OPLS and POSSIM calculations, conformers β2, αL, PII and αR had the backbone dihedral angles fixed at the quantum mechanical values. It is known that molecular mechanics usually does not reproduce these conformers well. Overall, the performance of both POSSIM and OPLS is satisfactory. The POSSIM results have a slightly lower error in the conformational energies, while the OPLS results are closer to the quantum mechanics in terms of the geometries.</p><p>We have also calculated relative energies of the extended and globular conformations of the alanine tetrapeptide (shown on Figures 3 and 4, respectively). We determined the quantum mechanical energy difference for these conformers to be 3.09 kcal/mol, the globular form being the global energy minimum. At the same time, this quantity is known to have a relatively large range of calculated quantum mechanical energies. For example, Reference 15 lists the globular – extended energy gap for the alanine tetrapeptide to be between 2.88 and 4.99 kcal/mol. The POSSIM result with the tors.1 torsional parameters set was 2.53 kcal/mol, and the OPLS result was 3.51 kcal/mol.7,8</p><p>We then further refined the backbone torsional parameters as described in the Methods section. The resulting values of the torsional Fourier coefficients and the conformational energies and angles are given in Tables 3 and 4, respectively. This set of the torsional parameters is termed tors.final, and this is the final set for the POSSIM protein backbone φ and ψ. The average dipeptide conformational energy error is now slightly higher at 0.97 kcal/mol, but the average errors in the backbone angles φ and ψ are reduced to 1.6° and 12.9°, respectively. Moreover, the globular – extended energy gap in the tetrapeptide is 3.14 kcal/mol, in a better agreement with the quantum mechanical results (3.09 kcal/mol with our calculations and 2.88–4.99 kcal/mol from the data Reference 15). The value of the ψ for the C7eq conformer is lower now, but this part of the conformational space is not relevant in practical protein applications. The overall average error in both backbone angles was reduced.</p><!><p>There are four possible water hydrogen bonding sites in the alanine dipeptide – two NH hydrogens and two carbonyl oxygen atoms. However, our quantum mechanical energy minimizations have demonstrated that water molecules prefer to make two hydrogen bonds at the same time, one with the H and one with the O atom. Therefore, there are only two water-alanine dipeptide heterodimer structures, as shown on Figures 4 and 5.</p><p>The quantum mechanical structures were used as the initial guesses for the POSSIM optimizations. Both POSSIM and OPLS-AA were utilized. We compared the binding energies, as well as the geometries of the complexes. Both hydrogen bonding distances (O…H-N) and H…O=C) and the φ and ψ angles of the alanine dipeptide backbone were used for the comparison. The results of these calculations are presented in Table 5. The quantum mechanical energy of the dimerization is reproduced slightly better with the OPLS, the average error being 0.89 kcal/mol vs. 1.12 kcal/mol with POSSIM. The latter tends to underestimate the magnitude of the binding energy. This is not unexpected. The non-bonded parameters for the alanine dipeptide have been adopted from NMA fitting.</p><p>And the same tendency was also present in the NMA case, with the POSSIM underestimating the NMA-water binding energy by an average of 0.89 kcal/mol.10 The overall performance of the NMA parameters was very good. This included reproducing liquid NMA heat of vaporization and density. Which lead us to the conclusion that our quantum mechanical NMA-water binding energies are probably somewhat overestimated. Therefore, a similar trend in the alanine dipeptide complex formation with water could have been expected and is not at all an indication of problems with the protein POSSIM force field. Moreover, it can be easily seen from the data in Table 5 that the POSSIM performed noticeably better than the OPLS in reproducing the hydrogen bond lengths, which are probably given much more accurate than the energies by the quantum mechanics. The average errors in these lengths are 0.15 and 0.06Å with the OPLS and POSSIM calculations, respectively.</p><p>It is also worth noting that the values of the φ and ψ backbone angles in this complex, as computed with the POSSIM, are much closer to the resulting quantum mechanical values of these angles than their OPLS counterparts, with the average error of only 5.3° vs. 12.8°. This is so even though the POSSIM gives the lowest-energy monomer conformer (C7eq) ψ angle of only 34.4° vs. the quantum mechanical 88.1° and the OPLS 61.8°. We believe that this fact confirms that (i) the conformational energy surface is rather flat at that region, and so the precise location of the minimum is not entirely crucial; (ii) the POSSIM force field is robust and adequate in reproducing important binding geometries.</p><p>We have further investigated the alanine dipeptide – water binding properties by running calculations, in which the values of φ and ψ were kept the same as in the fully optimized quantum mechanical dimers in all the cases (quantum mechanical, OPLS and POSSIM monomers and also the OPLS and POSSIM dimers). The results are presented in Table 6. The structure B dimerization energy as computed with the POSSIM is slightly greater than the quantum mechanical one in this case (−12.2 vs. −11.7 kca/mol), otherwise the trends are the same as in the fully relaxed geometry optimizations. The average errors in the dimerization energies with the POSSIM and OPLS are 0.76 and 0.39 kcal/mol, respectively. The POSSIM and OPLS errors in the hydrogen bonding distances are 0.09 and 0.05Å. Interestingly, the improvement in geometry achieved by fixing the backbone angles is greater with the OPLS than it is with the POSSIM. Once again, we believe this indicates that, even though the C7eq conformational geometry is better reproduced with the OPLS, the more important binding properties are better assessed with the POSSIM force field.</p><!><p>We have carried out Monte Carlo simulations of the ala-13 in order to test the robustness of the POSSIM force field by assessing stability of this experimentally known α-helical peptide. While quantum mechanical gas-phase alanine dipeptide conformational energies and geometries are important in fitting, these simulations provided a direct comparison with the available experimental observations. In particular, we were assessing the general stability of the helix and the average values of the backbone φ and ψ angles. Figures 6 and 7 show graphs of the average values of these angles as a function of the simulation length (in millions of Monte Carlo configurations) for the OPLS force field, as well as with POSSIM, using both the tors.1 and tors.final torsional parameters. Each angle value represents averaging over the last 200,000 configurations before the indicated simulation length.</p><p>The experimental values of the backbone φ and ψ in an α-helix are 296° and 319°, respectively, with a 7° uncertainty.16 In finding the average values of the backbone angles, we disregarded one residue on each end of the helix.</p><p>Two conclusions can be made from the presented results. First, the final version of the POSSIM, as well as the OPLS force field, yield better agreement with the experimental data than the POSSIM version with the tors.1 parameters. Second, the φ values are more stable than those of the angle ψ with all the force fields tested.</p><p>But one should keep in mind that the experimental data represent crystallographic results, and thus the thermal motion allowed in the Monte Carlo calculations can cause oscillations beyond the ±7° experimental lines. Overall, we can conclude that the gas-phase simulations confirm that the newly developed POSSIM force field is stable and robust. They reproduce the experimentally observed α-helix gas-phase stability. The stability of the simulated helixes can also be evaluated by studying the final structure of the system shown on Figures 8–10. One can see that, while the OPLS and POSSIM with tors.final produce a stable α-helix, the POSSIM/tors.1 helix denaturates. At the same time, the average φ and ψ angles in the tors.1 version of POSSIM are not extremely far from the experimental data, therefore the helicity of the structure is at least partially conserved.</p><p>Average values of the φ and ψ angles as a function of the simulation length for the tridecaalanine (ala-13) peptide in water are shown on Figures 11 and 12. In this case, as can be expected, the stability of the both angles is greater, and the deviations are smaller. It should be noted that the angle φ tends to be too low compared to the experimental crystallographic values, while the angle ψ is somewhat too high, thus their sum stays roughly at the same spot as the experimental one (255° or −105°), and the α-helicity of the structure for all the force fields employed is good.</p><p>Structures of the ala-13 peptide after 25 × 106 Monte Carlo configurations in water are given on Figures 13–15. Water molecules are not removed for clarity. It can be seen from the figures, in combination with the graphs and the table for the liquid-state simulations, that in this case (hydrated ala-13) all three force fields (OPLS and the two versions of POSSIM) perform adequately, and no denaturation of the tridecaalanine α-helix is observed.</p><!><p>We have presented results of developing a fast polarizable POSSIM force field for alanine and protein backbones. The quantum mechanical data set used for fitting was streamlined and simplified as compared to the previous version of the complete polarizable force field for proteins, and a high degree of transferability of the potential energy parameters has been demonstrated.</p><p>We have included a previously unused step of calculating dipeptide dimerization energies with a water molecule as an additional proof of validity of the technique and the resulting force field. The POSSIM force field performs well in this test.</p><p>The torsional fitting procedure has been augmented by a new step, a direct optimization-type fitting of the torsional parameters to the quantum mechanical conformational energies and structures.</p><p>At the same time, we believe that quantum mechanical dipeptide conformers in themselves are not a sufficient tool in validation of a force field. One of the reasons for this assumption is that most of these conformers belong to parts of the total conformational space which are rarely found in experimentally known proteins. Therefore, we have included an additional step to further test the robustness of the POSSIM force field. We have simulated the tridecaalanine peptide (ala-13) in both gas-phase and aqueous solution with the Monte Carlo technique. This peptide is experimentally known to form an α-helix under these conditions. The POSSIM ala-13 (and the OPLS used for benchmarking) was found to maintain a stable α-helical conformation as well.</p><p>We conclude that the resulting polarizable POSSIM force field is adequately accurate and we will use this model for the alanine and protein backbones as the basis for further development of a complete polarizable POSSIM force field for proteins.</p>

PubMed Author Manuscript

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified.Graphical abstract.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03797-4.

combination_of_strong_anion_exchange_liquid_chromatography_with_microchip_capillary_electrophoresis_

<!>Introduction<!>General<!>HPLC system and fraction collector<!>MCE-SDS<!>Protein concentration<!>Sf9 cytosol lysate<!>Samples from CHO cell culture containing an IgG antibody<!>Fresh-frozen human plasma<!>Fraction collection and preparation for MCE-SDS<!>Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis<!><!>Validation of peak identity<!><!>Fresh-frozen human plasma<!>Repeatability<!>Expected sensitivity<!>Peak capacity<!>Speed<!>Size and pI range<!>Conclusion<!>

<p>Open Access funding enabled and organized by Projekt DEAL.</p><!><p>The use of multi-dimensional separations for complex samples is a proven and tested strategy to facilitate an improved separation and easier analysis of complex samples, which are often insufficiently resolved using only one separation technique [1]. In the last century, two-dimensional gel electrophoresis (2-DE) emerged as one of the most successful technique for two-dimensional separations of complex protein mixtures [2].</p><p>With the advancement of analytical instruments, 2-DE has been replaced in many cases by automated instrumental approaches. Commonly employed strategies include high-performance liquid chromatography (HPLC) coupled to ( ×) HPLC, capillary electrophoresis (CE) × CE, and HPLC × CE approaches [3–8]. As pointed out by Ranjbar et al. [3], the popularity of HPLC × CE is much lower than HPLC × HPLC, despite the high orthogonality that can be achieved [3, 4, 6]. Such combinations provide a unique selectivity and may offer an alternative to other approaches. Even though, both capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and ion exchange chromatography (IEX) are frequently employed for the characterization of proteins [9–12], the coupling of both techniques has not received much attention [13].</p><p>Within our group, an HPLC × CE approach for the separation of protein mixtures has been recently developed [14, 15]. It is based on the general principle of 2-DE, that is, a charge-based separation in the first dimension followed by a size-based separation.</p><p>The charge-based separation in the first dimension is realized by strong anion exchange (SAX) HPLC. The employed stationary phase is based on a poly(styrene–divinylbenzene) polymer. It is stable over a wide pH and pressure range and compatible with many commonly found additives and buffer excipients. Furthermore, it has a high loading capacity [16, 17]. An RP-1 guard column protects the SAX column from lipophilic contaminants. Sample preparation is minimal and is usually limited to dilution and filtration. For a comprehensive offline coupling, the eluate is automatically collected in individual fractions by a fraction collector. The resulting fractions are subsequently analyzed by microchip capillary electrophoresis sodium dodecyl sulfate (MCE-SDS). The MCE-SDS system is compatible with common buffer additives used in IEX [18]. Another advantage of the chip-based assay is time expenditure. For each run in the first dimension, a manifold of runs is required in the second dimension. Therefore, the second dimension is often the speed-limiting step during analysis. Regular CE-SDS runs take about 15 to 35 min, which is in the order of magnitude of a usual HPLC run. Therefore, several runs would represent a significant prolongation of an analysis. One possibility to circumvent this problem is the use of multiplexed capillary arrays [13]. In the presented approach, the chip-based separation is completed within 1 min, effectively eliminating this issue.</p><p>Our approach is intended as a platform approach, applicable to a wide range of samples with minor method adaptions. In this study, three different samples were investigated.</p><p>A commonly used production system for recombinant proteins are Spodoptera frugiperda (Sf9) cells [19]. In our case, the Sf9 cells were used for the production of soluble guanylyl cyclase (sGC), a heterodimeric heme protein which is the main receptor for nitric oxide [20]. Cell lysates of transfected and untransfected cells were analyzed. The applicability of our approach for biotechnological process control or analysis was evaluated. The identification of the sGC and the general practicability were the main concern.</p><p>The supernatant obtained from a Chinese hamster ovary (CHO) cell culture sample and its intermediate after a protein A purification step were subjected to analysis by our approach. The cell culture produced an immunoglobulin G1 (IgG) monoclonal antibody (mAb), which was secreted into the surrounding medium. Besides the product, several other proteins, commonly referred to as host cell proteins (HCP), are secreted by the cells. The HCP amount is usually regarded as critical quality attribute and monitored closely. Its minimization is an important objective during downstream processing [21–24]. Several analytical methods are used for HCP characterization, each with individual strengths and weaknesses [21, 25, 26]. Our approach might serve as an alternative analytical technique for the comparison or optimization of purification steps or as an orthogonal technique for the HCP determination.</p><p>Human plasma is a well-controlled product with a consistent composition [27–30]. Its proteome is well characterized and many proteins serve as biomarkers in diagnostic applications [30–33]. The proteins, whose individual concentration can differ over several orders of magnitude [31], are embedded in a complex matrix consisting of lipids, ions, carbohydrates, and more [29, 30]. Therefore, human plasma represents an ideal model to compare various approaches.</p><p>The goal of this study is to demonstrate the feasibility and applicability of the approach.</p><!><p>Bovine serum albumin (BSA; Fraction V (pH 7.0) for Western blotting), dithiothreitol (DTT; BioChemica grade), ethylenediaminetetraacetic acid (EDTA; molecular biology grade), glycerol (molecular biology grade), and sodium chloride (NaCl; for analysis) were obtained from PanReac AppliChem GmbH (Darmstadt, Germany). Sodium hydroxide (NaOH; EMSURE grade) and Amicon® Ultra-0.5 Ultracel-10 K centrifugal filter devices were purchased from Merck (Darmstadt, Germany). Sodium chloride (HPLC grade) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS; electrophoresis grade) were obtained from VWR International GmbH (Darmstadt, Germany) and exclusively used for the HPLC experiments. Hydrochloric acid (HCl, p.a. grade), albumin from human serum (≥ 97%, HSA), albumin from chicken egg white (≥ 98%, ovalbumin), bovine serum albumin (≥ 96%, BSA), and β-lactoglobulin from bovine milk (≥ 85%) were acquired from Sigma-Aldrich (Steinheim, Germany). Matuzumab was received as a gift from Merck KGaA (Darmstadt, Germany). Syringe filters (polyvinylidene fluoride, 0.22 µm) and β-mercaptoethanol (β-ME, p.a. grade) were obtained from Carl Roth (Karlsruhe, Germany). Ultrapure water (conductivity 0.055 µS/cm) was supplied by an arium® pro VF system from Sartorius (Goettingen, Germany). Nylon membrane filters (0.2 µm pore size, 47 mm diameter) were supplied by GE Healthcare (Buckinghamshire, UK). The centrifuges 5417C and 5430 were purchased from Eppendorf (Hamburg, Germany). The pH measurements were done with a FiveEasy™ FE20 pH-meter and either an LE438 pH electrode (buffers) or an InLab Micro electrode (samples), all from Mettler Toledo (Gießen, Germany). All other chemicals were obtained from Sigma-Aldrich (Steinheim, Germany) in the highest grade of purity. Figures were plotted in OriginPro 2021 (9.8.0.200) from OriginLab Cooperation (Northampton, MA, USA). Calculations were performed using Microsoft® Excel® 2019 (v. 1809) from Microsoft Corporation (Redmond, WA, USA).</p><!><p>The HPLC system (dwell volume 7.6 mL) consisted of a VWR Hitachi L-2130 quarternary pump, an L-2200 autosampler fitted with a 5.0 mL syringe kit, an L-2450 diode-array detector (all VWR International GmbH, Darmstadt, Germany), and a Techlab T1 column oven (Techlab, Braunschweig, Germany). The HPLC was controlled by EZChrom Elite (3.3.2 SP2, VWR International GmbH) and it was further used for data handling and integration. Directly coupled to the system was a Foxy R1 fraction collector (Knauer, Berlin, Germany), whose control was integrated into the HPLC software. The stationary phase was a semi-preparative PL-SAX 1000 Å, 8 µm, 50 × 7.5 mm column (Agilent Technologies, Waldbronn, Germany) protected by a 4 × 3.0 mm RP-1 guard column (Phenomenex, Aschaffenburg, Germany).</p><p>Mobile phase A (MPA) consisted of 20 mM TRIS pH 8.5, mobile phase B (MPB) consisted of 20 mM TRIS and 0.75 M NaCl pH 8.5, and mobile phase C (MPC) was composed of 20 mM TRIS and 1.5 M NaCl pH 8.5. The pH was adjusted to ± 0.05 units with 6 M HCl. All mobile phases were filtered through nylon membrane filters prior to use. If not otherwise indicated, the following method parameters were used. The column oven was thermostated at 30 °C, the flow rate was set to 1.3 mL/min, and UV absorption at 200 nm, 214 nm, and 280 nm (4 nm bandwidth, 2.5 Hz) was recorded. The injection volume is indicated at the respective samples. The following gradient was used for elution: from 0.0 to 5.0 min 100% MPA, followed by a linear gradient from 0% at 5.0 min to 100% MPB at 45.0 min. From 45.1 to 55.0 min 100% MPC and from 55.1 to 70.0 min 100% MPA was pumped through the system.</p><!><p>A LabChip® GX II Touch™ HT instrument, controlled with LabChip® GX Touch™ software (v. 1.7.819.0), was used with Protein Express LabChips and associated Protein Express kits (all PerkinElmer, Waltham, MA, USA). The HT Protein Express 200 high sensitivity assay was used. The proprietary chips are made of fused silica with a footprint of 37.25 mm × 37.25 mm. A plastic case (49.6 mm × 49.6 mm footprint, height up to 14 mm) is glued to the top of the chip and provides eight cylindrical wells for reagents and waste. A fused silica capillary (20–50 µm diameter, 24 mm length) is attached to the bottom of the chip as a "sipper" [34]. The "sipper" is used to hydrodynamically aspirate approximately 100–150 nL of the sample. The sample is subsequently moved electrokinetically into the separation channel (length 14 mm, width 31 µm) [35, 36]. A TRIS-Tricine buffer containing a non-cross-linked, linear, high molecular mass poly(dimethylmethacrylate) polymer, sodium dodecyl sulfate (SDS), and a proprietary dye is used as a sieving matrix for the size-based separation in an entangled polymer network at 30 °C [36, 37]. Laser-induced fluorescence (excitation 635 nm, emission 700 nm) is utilized for detection [38]. Further information about the detailed mode of operation is given by Chow [36] and in the associated patent [37]. In the preceding publication by Bousse et al. [39], a field strength of 200–300 V/cm is employed for the separations. The data was evaluated with the LabChip® GX Reviewer software (v. 5.5.2312.0, PerkinElmer).</p><!><p>Total protein concentrations were determined by the method of Bradford [40] using BSA as standard and Roti-Quant® (Carl Roth, Karlsruhe, Germany) as dye. Absorption at 470 and 595 nm was recorded by a Sunrise™ Absorbance Reader controlled and evaluated by Magellan™ software (both Tecan Deutschland GmbH, Crailsheim, Germany).</p><!><p>Sf9 cells were obtained from the DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany). Soluble guanylyl cyclase subunits were recombinantly expressed using the baculovirus/Sf9 cell system. Recombinant viruses were generated as described in the Bac-to-Bac® Baculovirus Expression System manual [41] (Invitrogen™, Thermo Fisher Scientific, Waltham, USA). Sf9 cells were cultivated in Sf-900™ II Serum Free Medium (Gibco® by life technologies™, Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. Spinner cultures were grown at 27 °C and 140 rpm on a shaking incubator. For experiments, Sf9 cell density was adjusted to 2 × 106 cells/mL and a volume of 100 mL was co-infected with baculoviruses encoding the sGC subunits: N-terminally twin strep tagged (TST) α1 [42, 43] and β1 [44] subunits. Fusion protein β1YFPα1 was cloned as previously described in [45]. Conjoined sGC β1α1 fusion construct was cloned as described in [46].</p><p>After 72 h of incubation, Sf9 cells were harvested by centrifugation (3020 × g at 4 °C for 2 min). The cell pellet was resuspended in lysis buffer (50 mM triethanolamine/HCl, 10 mM DTT, 1 mM EDTA, pH 7.4) containing complete™ protease inhibitor cocktail (Roche, Mannheim, Germany) and homogenized by ultrasound sonication. The samples were cleared by centrifugation (21,000 × g at 4 °C for 30 min). The cell lysates were diluted with MPA (1 + 1) and filtered through syringe filters. Injection volume was adjusted to the determined protein content so the injected protein amount remained comparable.</p><!><p>Cell culture supernatant and protein A purified IgG antibody, derived from a fed batch of a ready-to-use biosimilar CHO cell line using First CHOice® medium and feeds, were kindly provided by UGA Biopharma GmbH (Hennigsdorf, Germany). The frozen (− 20 °C) samples were thawed, vortexed (15 s), diluted with MPA (1 + 1), and filtered through syringe filters.</p><!><p>Anonymized leukocyte-depleted fresh-frozen human plasma was obtained from Städtisches Klinikum Braunschweig gGmbH and stored at − 32 °C. It was thawed in a tempered water-bath at 37 °C. The plasma was aliquoted and the aliquots were stored at − 32 °C. For use, aliquots were thawed as previously described and diluted with MPA (1 + 1), vortexed for 15 s, and filtered through syringe filters. Adjustments of the pH were done with 1% NaOH before filtration.</p><!><p>If not otherwise indicated, in each HPLC run, 30 fractions evenly spread over 45 min were collected (equals to 1.5 min or 1.95 mL per fraction). If the fractions were not processed immediately after collection, they were stored at − 20 °C and thawed for measurement. All fractions were vortexed 3 × 5 s and centrifuged at 10,000 × g for 3 min. Then, samples were prepared according to the manufacturer's protocol [18]. Briefly, to 7 µL of sample buffer containing 3.4% (V/V) β-ME, 5 µL of each fraction were added in a 96-well plate. For non-reduced conditions, the sample buffer was used without the addition of β-ME. The plate was sealed and heated to 100 °C for 5 min. After cooling to room temperature, the plate was centrifuged at 1250 × g for 5 min; then, 32 µL water were added and mixed thoroughly. The plate was centrifuged again at 1250 × g for 5 min; then, the samples were measured with the LabChip®.</p><!><p>For desalting, 500 µL from each sample's 21st fraction were transferred to an Amicon® filter device. Each capped device was centrifuged at 14,000 × g for 5 min. Then, 250 µL of MPA were added. Centrifugation at 14,000 × g for 5 min followed. This was repeated three more times. Finally, MPA was added to an approximate volume of 250 µL, the filter device inverted, and the desalted concentrate recovered by centrifugation for 2 min at 2060 × g.</p><p>SDS sample buffer (1% SDS, 50 mM TRIS/HCl, 100 mM DTT, 30% glycerol, pH 7.5) was added to each sample (raw lysates and desalted fractions) in a 1:1 ratio. Samples were vortexed for homogenization and heated to 99 °C for 3 min. After cooling, 80 µg of total protein was separated on a 10% SDS polyacrylamide electrophoresis gel. SDS-PAGEs were performed on the Mini-PROTEAN® Tetra System by Bio-Rad in a TRIS/glycine/SDS running buffer (25 mM TRIS, 192 mM glycine, 0.1% SDS, pH 8.3) and an applied voltage of 80 V for 4 h. The apparent molecular masses were determined by using PageRuler™ Unstained Protein Ladder and PageRuler™ Prestained Protein Ladder (both from ThermoFisher Scientific, Waltham, USA). After completion of the electrophoresis, the gels were washed with deionized water and proteins were transferred to nitrocellulose membranes using the Semi-Dry electro blotter Sedec™ M (Peqlab Biotechnology, Erlangen) with transfer buffer (25 mM TRIS, 192 mM glycine, 0.02% SDS, pH 8.3). Ponceau S staining, blocking of non-specific binding sites, and antibody incubation were performed as described in [45]. The following primary antibodies were used for detection: α1 (1:5000; Sigma-Aldrich G4280, Steinheim, Germany) and β1 (1:2000; Sigma-Aldrich G4405, Steinheim, Germany). Anti-rabbit IgG horseradish peroxidase–linked antibody (1:2000; Cell-Signaling Technologies 7074, Danvers, MA, USA) was used as secondary antibody for detection.</p><!><p>Two-dimensional separations (reduced conditions) of Sf9 cytosol lysate from a untransfected cells, 750 µl injected; b transfected cells expressing the sGC subunits TSTα1 and β1, 1500 µL injected; c transfected cells, expressing sGC fusion protein β1α1, 1423 µL injected; d shows the individual electropherograms of the 21st fraction of each separation, bottom (black) untransfected sample, middle (red) transfected sample TSTα1 / β1 (offset 400 FU), and top (blue) transfected fusion protein sample (offset 1250 FU); peaks 1–6 are attributed to the sGC</p><p>Cutouts of the immunoblots of the 21st fraction of each separation depicted in Fig. 1 ("after separation") and of the corresponding raw Sf9 cytosol lysates ("raw material"), with primary antibody for α1 subunit (a) and with primary antibody for β1 subunit (b); position of the ladder proteins and their nominal molecular mass are indicated on the left side of the images</p><!><p>Commonly, the peak identity is confirmed by mass spectrometry (MS) [4, 48–50]. MCE-SDS and CE-SDS in general are considered as incompatible with MS due to the intrinsic properties of the employed gel matrix. Nevertheless, approaches to address this issue have been published [51–53], with the most recent one [51] showing promising results. These approaches are currently limited to regular CE-SDS and do not include chip-based formats. However, recent progresses in microchip-based CIEF-MS [49] raise hope that similar successes may be achieved with MCE-SDS.</p><p>During the SAX-HPLC run, the employed salt gradient for the elution introduces a large amount of nonvolatile ions, effectively eliminating the possibility of using MS. However, the individual fractions contain only a fractional amount of the initial total protein amount, reducing the complexity of the sample. By means of buffer exchange to a volatile, MS compatible buffer, the individual fractions could be analyzed with MS. Direct coupling of cation [50] and anion exchange chromatography [12] with MS has been reported. The employed strategies could also be applied for further developments of the first dimension if MS compatibility is desired.</p><!><p>Two-dimensional separations (non-reduced conditions) of CHO cell culture supernatant before (a) and after protein A purification (purified antibody, b); injection volume 3000 µL for both; in c, two exemplary electropherograms of the 14th fraction of each separation are depicted, the upper red trace from the supernatant and the lower, black trace from the purified mAb. The intensity of the impurity peaks (10–165 kDa) is greatly reduced in the black trace</p><p>Two-dimensional separations (reduced conditions) of 500 µL human plasma under standard conditions (a) and with modified elution: flow rate 1 mL/min, linear gradient from 0 to 100% MPB over 40 min (b), the peaks are presumed to be albumin (1), Ig light chains (2), Ig heavy chains (3), serotransferrin (4), ceruloplasmin (5), and apolipoprotein A1 (6); detailed view of two electropherograms from fraction 17 of the separation in 4a (c) and from fraction 15 of the separation in 4b (d), peaks 1 and 5 are indicated in both traces</p><!><p>The human plasma proteome is well known [30, 31, 55] and classical 2-DE employs a comparable separation mechanism. A detailed depiction of a 2-DE separation of human plasma is provided in [56]. Further 2-DE separations may be found in several other publications [57, 58]. Generally, the elution order in our approach is the opposite as in 2-DE, from basic to acidic proteins. The proposed assignment of the peaks is based on their relative abundance, their apparent molecular mass, and their relative positions to each other. The most abundant protein is albumin [31] (calculated molecular mass based on amino acid sequence (cMM) 66 kDa [30]), which corresponds to peaks "1" (blue box) around 64 kDa in fractions 15 to 19. In fraction 2 and in 10 to 12, the peaks "2" (green box) around 24 and 27 kDa presumably are immunoglobulin light chains (cMM 23 kDa [30]) and peaks "3" (orange box) around 65 kDa (cMM 53–75 kDa [30]) the corresponding heavy chains. The peak "4" (red box) at 90 kDa in fraction 12 could be serotransferrin (cMM 75 kDa [30]). Both, immunoglobulins and serotransferrin, can be found at more basic pIs than albumin in 2-DE [56]. Furthermore, the peak "5" (petrol box) at 134 kDa in fraction 17 could be ceruloplasmin (cMM 120 kDa [30]) and the peak "6" (purple box) in fraction 15 around 24 kDa might be apolipoprotein A1 (cMM 28 kDa [30]). Overall, the pattern seems similar to those of the aforementioned 2-DE separations, if the assignment is correct. For a definitive statement, confirmation of peak identity would be necessary.</p><p>Several changes of the method were conducted and tested. The short isocratic hold time (5 min) at the beginning of the method was removed. The dwell volume of the used HPLC system is large (7.6 mL). Therefore, the gradient needs about 5.85 min at 1.3 mL/min (or 7.6 min at 1 mL/min) until it reaches the column inlet. This should be a sufficient isocratic hold time for a gradient method. As a result, the same separation in a shorter timeframe (40 min) was obtained. The number of fractions remained constant, effectively reducing the individual fraction's volume and increasing the sensitivity. Second, the flow rate was reduced to 1 mL/min. This yielded a higher sensitivity without impairing the separation. The combined effect of both changes can be seen in Fig. 4b. In comparison to Fig. 4a, the separation remains unaffected but is shifted leftwards, two fractions earlier. Additionally, the intensity of the peaks increases. This is very well visible, but, due to the chosen gray-scale, only for the low abundant peaks. For example, the intensity of the peak "5" around 133 kDa in fraction 17 (standard conditions, Fig. 4c), respective 15 (modified conditions, Fig. 4d), increased from 215 to 382 FU. Nevertheless, it applies also to the high abundant peaks, e.g., the peak "1" around 67 kDa in fraction 17 (standard conditions) has an intensity of ≈ 3000 FU, while in fraction 15 (modified conditions) the intensity is at about 5700 FU. The effect is also visible in the chromatograms. The peak intensity is increased and the peaks are shifted to earlier retention times while preserving the original peak pattern (SI Fig. 4).</p><p>The pH value of the sample might influence the retention and therefore the separation. The sample's pH was increased from 8.24 to 9.59 and 10.55. Higher pH led to an unchanged separation, with some decrease in peak intensity (data not shown). This might be due to precipitation. Overall, the separation is robust to a basic shift in the sample's pH value.</p><p>Due to the limited protein capacity of the column, the high abundance of albumin creates a challenge for the detection of proteins with much lower concentrations. The proven and tested strategies used in 2-DE for the depletion of the most abundant proteins [59, 60] could also be applied here and should allow for more individual proteins to be detected.</p><!><p>Three consecutive injections of 750 µL Sf9 cytosol lysate (53.4 mg/mL before dilution) were analyzed. The following method parameters differed from the described parameters: 1 + 1 dilution with lysis buffer, 25 fractions over 37.5 min were collected. A peak with a comparatively low intensity (signal to noise ratio (S/N) ≈ 5) and a peak with a normal intensity (S/N > 100) were investigated. The low intensity peak can be seen as a "worst-case scenario," while the normal intensity peak represents a normal use case. The S/N was calculated according to the European Pharmacopoeia [61]. The electropherograms were integrated and all peaks within the assay's specified range (10–200 kDa) included. Time corrected areas (corr. area) were obtained from the LabChip's software and used for further calculation. The relative standard deviation (RSD) of the corr. area and of the percentage of the corr. area of the individual peak to the total corr. area of all peaks in the fraction (%area) was calculated. The RSDs for the low intensity peak were 15.5% (corr. area) and 21.9% (%area), while for the normal intensity peak they were 20.6% (corr. area) and 10.1% (%area). These values are in the order of magnitude of previously reported values for this particular LabChip® assay [62] and within the specifications given by the manufacturer (up to 30% RSD for quantitation) [18]. Improved values might be obtained by averaging the values from multiple MCE-SDS injections of the same sample at the expense of speed [62]. For this sample, injecting every fraction twice improved the RSD for the normal intensity peak to 17.7% (corr. area) and 9.73% (%area). The RSDs for the low intensity peak did not benefit from this, with values of 23.2% (corr. area) and 21.7% (%area). In this case, the low S/N has a major influence on precision, since the S/N needs to be > 100 for optimal precision [63]. Further improvements may be achieved with the ProteinEXact assay, which has tighter specification limits for quantification [64, 65].</p><!><p>The MCE-SDS assay's linear concentration range starts at 5 ng/µL [18], and Kahle et al. [62] reported an S/N of 44 at 10 ng/µL for carbonic anhydrase. One fraction contains about 1950 µL eluate, division through the aforementioned 5 ng/µL leads to at least 9.75 µg of protein required per fraction. Assuming a recovery of 85%, at least 11.5 µg need to be injected \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$(rac{9.75 \mu g}{85\%} =11.5 \mu g)$$\end{document}(9.75μg85%=11.5μg). The typical injection volume ranges from 100 to 3000 µL. Assuming 1000 µL are injected, the protein's concentration should be 11.5 ng/µL or higher to be detectable.</p><p>Porebski et al. [66] report an influence of the salt concentration on peak areas during CE-SDS. Since the LabChip® moves the sample electrokinetically into the separation channel [35], an influence on the areas can also be reasonably expected here. This was confirmed by adding equal amounts of BSA to each fraction of a blind run. The peak area is negatively correlated with the salt concentration (SI Fig. 5). This should be continued to be investigated in conjunction with further method changes. The previously described method changes represent a good starting point. Another possibility is the use of the Protein Pico Assay. Here, a sensitivity of approximately 10–50 pg/µL, which corresponds to a 100-fold improvement over the Protein Express Assay, can be expected [67]. The drawback is that a buffer exchange would be necessary. Other two-dimensional HPLC × CE approaches use evaporation and reconstitution [68]. In our case, this would unfavorably increase the salt concentration.</p><!><p>An easy and convenient way for the assessment of the peak capacity is simply counting peaks. Each MCE-SDS run's electropherogram was integrated and all peaks with an apparent molecular mass ≥ 10 kDa were counted. The results from all 30 electropherograms were summed. For the most complex sample, namely Sf9 cytosol lysate, between 330 (sample Fig. 1a) and 419 peaks (sample Fig. 1b) were achieved. Human plasma (sample Fig. 4b) counted 218 peaks. One possibility to increase the peak capacity could be an increased amount of fractions. For HPLC × HPLC separations, the requirement was stated that ideally each peak in the first dimension should be sampled at least three to four times [69]. This concept has been refined in subsequent publications [70, 71], and moreover, it has been frequently cited. However, to be precise, this concept and derived concepts are only applicable for HPLC × HPLC separations. Since the theories about peak capacity, sampling frequency, etc. are not as well developed for CE based separations [72], it can only be assumed that a similar impact can be achieved here. Further refinements of the first dimension may provide an additional option to increase the peak capacity.</p><!><p>The presented approach uses commercially available instruments, which can be set up and operated in any lab. This facilitates an easy implementation. The approach is fast and might be accelerated even more. The expected run time for one sample is approximately 3 h. The time is based on the following assumptions: 45 min HPLC preparation, 15 min sample preparation, 45 min HPLC separation, 10 min fraction handling, 30 min fraction preparation for MCE-SDS, 5 min chip preparation and cleaning, 30 min MCE-SDS runs. An online approach is not easily feasible due to the required heating during sample preparation for the 2nd dimension. Several other possibilities arise to streamline the process. Running consecutive analyses and the second dimension in parallel with the first dimension of the next samples reduces analysis time to approximately 1.5 h per sample with the present setup. The initial publication describing the stationary phase points out the possibility of using high linear velocities [16]. In a preliminary experiment using human plasma, the flow rate was doubled from 1 to 2 mL/min. Consequently, the HPLC run was completed within 35 min. The resulting chromatogram and two-dimensional separation are shown in SI Fig. 6. This is another promising possibility to increase the separation speed and will be the scope of further investigations.</p><p>Currently, the fractions are collected in 2-mL microcentrifuge tubes. With the appropriate, commercially available equipment, fraction collection directly in 96-well plates is possible. The required reduced flow rate could be either obtained using a flow splitter or by miniaturization of the separation process. The collection into a 96-well plate would speed up the preparation process for the MCE-SDS analysis considerably. Another possibility is the automation of the remaining manual steps. Particularly pipetting could be sped up by automation and reduce hands-on time. If one is routinely analyzing the same sample and is only interested in particular proteins, an adaption of the gradient and reduction of the fraction amount could further increase the speed. This implies a switch from a comprehensive to a heart-cutting approach.</p><!><p>The size-based assay is suited for apparent molecular masses between 10 and 200 kDa. Below 10 kDa, system peaks interfere with potential sample peaks. In the different samples, proteins over the whole range were found. The Protein Low Molecular Weight Assay could offer an alternative for proteins within the 5–80 kDa range, but a buffer exchange is required for fractions containing more than 0.5 M NaCl [73].</p><p>Generally speaking, proteins need to possess a negative charge to be retained on the column. As a rule of thumb, this necessitates a pI below 7.5–6.5 [9], the lower the better the retention. Several individual proteins were analyzed by SAX chromatography at a concentration of 1 mg/mL in MPA. The resulting chromatograms are presented in SI Fig. 7. Matuzumab is a mAb with a pI of 8.3 (main species)–7.5 (SI Fig. 8). The peak's maximum is at 14.7 min, which is shortly after the beginning of the salt gradient. The other proteins possess a lower pI, Ovalbumin 4.7–4.9 [74, 75], BSA 4.6–5 [76–78], HSA 4.7–5.7 [79–81], and β-Lactoglobulin 5.2 [82, 83]. Their retention times were 21.3 min (Ovalbumin), 24.3 min (BSA), 24.8 min (HSA), and 26.1 min (β-Lactoglobulin). The proteins identified in the human plasma sample are found between pI 4 and 8 in 2-DE [58]. Based on these results, the estimated covered pI range is from about 4 to 8. However, non-retained proteins are not lost, but simply found in the first fractions.</p><p>These considerations should also take into account that the net charge alone is not always sufficient for the explanation of the retention. The (unequal) surface charge distribution, the displacing salt, stationary phase, and protein–protein interactions may influence the retention and sometimes complicate general assumptions [84, 85].</p><p>The aforementioned use of a cation exchange column instead of an anion exchange column could expand the covered range through the altered selectivity. This might be relevant if the attention is on basic proteins and will be subject of further investigations.</p><!><p>The presented approach was very well applicable to the three different examples, clearly demonstrating its feasibility. The IgG containing cell culture supernatant as well as the Sf9 cytosol lysate demonstrated the use of the approach for biotechnological process control or analysis. The latter one focused on a specific protein in a complex matrix and the former focused on the changes during a process. The protein sGC could be confidently identified. The results from the human plasma separation and their comparison to conventional 2-DE results revealed that the underlying comparable separation mechanism might lead to similar but not directly transferable results.</p><p>In all cases, quick two-dimensional separations were achieved. The potential for further acceleration of the separation process was highlighted. Method improvements, especially in the first dimension, should further improve this approach. Potential steps were outlined and briefly discussed. Using best-suited separation systems in the first dimension can also customize this approach for individual requirements and sample properties. The second dimension offers fewer opportunities for refinement.</p><p>With refined parameters, the determination of additional relevant method parameters, e.g., reproducibility, linearity, robustness, etc., is of interest. Further research with the objective of expanding the applications to e.g. impurity analysis, diagnostics, or process analytics will help to gain a more thorough understanding of the technique's properties.</p><!><p>Supplementary file1 (PDF 624 kb)</p><p>Publisher's note</p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p>

PubMed Open Access

POST-HOC ANALYSIS OF VITAMIN D STATUS AND REDUCED RISK OF PRETERM BIRTH IN TWO VITAMIN D PREGNANCY COHORTS COMPARED WITH SOUTH CAROLINA MARCH OF DIMES 2009-2011 RATES

Background Two vitamin D pregnancy supplementation trials were recently undertaken in South Carolina: The NICHD (n=346) and Thrasher Research Fund (TRF, n=163) studies. The findings suggest increased dosages of supplemental vitamin D were associated with improved health outcomes of both mother and newborn, including risk of preterm birth (<37 weeks gestation). How that risk was associated with 25(OH)D serum concentration, a better indicator of vitamin D status than dosage, by race/ethnic group and the potential impact in the community was not previously explored. While a recent IOM report suggested a concentration of 20 ng/mL should be targeted, more recent work suggests optimal conversion of 25(OH)D to 1,25(OH)2D takes place at 40 ng/mL in pregnant women. Objective Post-hoc analysis of the relationship between 25(OH)D concentration and preterm birth rates in the NICHD and TRF studies with comparison to Charleston County, South Carolina March of Dimes (CC-MOD) published rates of preterm birth to assess potential risk reduction in the community. Methods Using the combined cohort datasets (n=509), preterm birth rates both for the overall population and for the subpopulations achieving 25(OH)D concentrations of \xe2\x89\xa420 ng/mL, >20 to <40 ng/mL, and \xe2\x89\xa540 ng/mL were calculated; subpopulations broken down by race/ethnicity were also examined. Log-binomial regression was used to test if an association between 25(OH)D serum concentration and preterm birth was present when adjusted for covariates; locally weighted regression (LOESS) was used to explore the relationship between 25(OH)D concentration and gestational age (weeks) at delivery in more detail. These rates were compared with 2009-2011 CC-MOD data to assess potential risk reductions in preterm birth. Results Women with serum 25(OH)D concentrations \xe2\x89\xa540 ng/mL (n=233) had a 57% lower risk of preterm birth compared to those with concentrations \xe2\x89\xa420 ng/mL [n=82; RR=0.43, 95% confidence interval (CI)=0.22,0.83]; this lower risk was essentially unchanged after adjusting for covariates (RR=0.41, 95% CI=0.20,0.86). The fitted LOESS curve shows gestation week at birth initially rising steadily with increasing 25(OH)D and then plateauing at ~40 ng/mL. Broken down by race/ethnicity, there was a 79% lower risk of preterm birth among Hispanic women with 25(OH)D concentrations \xe2\x89\xa540 ng/mL (n=92) compared to those with 25(OH)D concentrations \xe2\x89\xa420 ng/mL (n=29; RR=0.21, 95% CI=0.06,0.69) and a 45% lower risk among Black women (n=52 and n=50; RR=0.55, 95% CI=0.17,1.76). There were too few white women with low 25(OH)D concentrations for assessment (n=3). Differences by race/ethnicity were not statistically significant with 25(OH)D included as a covariate. Compared to the CC-MOD reference group, women with serum concentrations \xe2\x89\xa540 ng/mL in the combined cohort had a 46% lower rate of preterm birth overall (n=233, p=0.004) with a 66% lower rate among Hispanic women (n=92, p=0.01) and a 58% lower rate among black women (n=52, p=0.04). Conclusions In this post-hoc analysis, achieving a 25(OH)D serum concentration \xe2\x89\xa540 ng/mL significantly decreased the risk of preterm birth compared to \xe2\x89\xa420 ng/mL. These findings suggest the importance of raising 25(OH)D levels substantially above 20 ng/mL; reaching 40 ng/mL during pregnancy would reduce the risk of preterm birth and achieve the maximal production of the active hormone.

post-hoc_analysis_of_vitamin_d_status_and_reduced_risk_of_preterm_birth_in_two_vitamin_d_pregnancy_c

Introduction<!>Study Design<!>Definition of Preterm Birth<!>Laboratory measurements<!>Statistical Analyses<!>Results<!>Discussion

<p>Since its discovery a hundred years ago, vitamin D has emerged as one of the most controversial nutrients and prohormones of the 21st century. Its role in calcium metabolism and bone health is undisputed but its role in immune function and long-term health is still debated. There are clear indicators from in vitro and animal in vivo studies that point to the role of vitamin D in both innate and adaptive immunity (1-3), and an emerging number of observational and cohort studies that support vitamin D's role in pregnancy outcomes (4, 5); however, translation of these findings to clinical practice, including the care of pregnant women, has not yet fully materialized. Until recently, there has been a paucity of data from randomized controlled trials to establish clear-cut beneficial effects of vitamin D supplementation or concentration of circulating 25(OH)D during pregnancy.</p><p>The current vitamin D requirements during pregnancy as established by the Institute of Medicine's (IOM) 2010 guidelines are 400 IU/day as the Estimated Average Requirement (EAR) and 600 IU/day as the Recommended Dietary Allowance (RDA) (6). Sufficiency is defined by the IOM as a total circulating 25(OH)D concentration of at least 20 ng/mL (6). While the IOM guidelines focus specifically on bone health, these recommendations are widely interpreted as covering all health conditions. Recent observational studies have shown that there exists a large proportion of women who, despite achieving the EAR intake for vitamin D, have 25(OH)D concentrations below 20 ng/mL, including a disproportionately high number of African American and Hispanic women (7-9).</p><p>Two vitamin D pregnancy supplementation trials were recently conducted in South Carolina to determine the optimal vitamin D supplementation regimen necessary to achieve sufficiency in pregnant women [defined a priori in the NICHD and Thrasher Research Fund (TRF) studies (10-12) as a total circulating 25(OH)D level 32 ng/mL or greater; deficiency as less than 20 ng/mL; and insufficiency as 20 to <32 ng/mL]. In those trials, 4000 IU vitamin D/day was found to safely achieve a level of at least 32 ng/mL by early in the second trimester in a diverse group of pregnant women. While 1,25(OH)2D can be normal when 25(OH)D is quite low in non-pregnant individuals, only during pregnancy is 25(OH)D directly related to 1,25(OH)2D. Specifically, in the NICHD trial, it was found that the conversion of 25(OH)D to the active form of the hormone, 1,25(OH)2D, plateaued around 40 ng/mL (10). Further, the impact on pregnancy health, including risk of preterm birth (<37 weeks gestation), was explored with findings suggestive that improved vitamin D status was associated with improved health outcomes of both mother and newborn (12, 13).</p><p>While higher vitamin D dose regimens were suggestive of a reduced risk of preterm birth (10, 12, 13), the relationship between preterm birth and 25(OH)D serum concentration was not previously explored in detail. Due to multiple input sources and inter-individual variability in dose response (14), 25(OH)D serum concentration is a better indicator of vitamin D status than dose. The objective of this post-hoc analysis was to examine if an association between 25(OH)D concentration and preterm birth incidence exists within the combined NICHD and TRF cohort both overall and among specific race/ethnic groups, and if so, to compare the rates to the Charleston County, South Carolina March of Dimes (CCMOD) published rates of preterm birth (most recently updated in 2011 at the time of this analysis) to assess potential risk reduction in the community (15).</p><!><p>As previously reported, datasets from the NICHD and TRF vitamin D supplementation trials were combined for this analysis using a common data dictionary (13). Details about both clinical trials and results based on dosage have been published previously (10, 12). Briefly, the studies were conducted concurrently and administered identical questionnaires to produce comparable sociodemographic and clinical characteristics using the same criteria. In the NICHD trial, women with baseline 25(OH)D concentrations ≤40 ng/mL were randomized to one of three treatment groups: control (400 IU vitamin D3/day), 2000 IU/day, or 4000 IU/day. Women with concentrations >40-60 ng/mL (n=22) were randomized to 400 or 2000 IU/day and women with concentrations >60 ng/mL (n=1) were given 400 IU/day. In the TRF trial, women were randomized to either 2000 IU/day or 4000 IU/day without exclusion. Outcome measures for both clinical trials included the following: [1] maternal baseline and delivery 25(OH)D; [2] neonatal 25(OH)D concentration; and [3] gestational age at delivery in weeks. This post-hoc analysis included women who participated in either the NICHD trial or the TRF trial and were followed through delivery and had blood samples available within 6 weeks of delivery (n=509).</p><!><p>Information on gestational age was based on the mother's report of the first day of her last menstrual period generating the obstetrical Expected Date of Confinement or Delivery (EDC or EDD) with ultrasound confirmation at the time of mother's first obstetrical visit. All women were enrolled in either the NICHD trial or the TRF trial <16 weeks of gestation and thus ultrasound confirmation of dating was within the first trimester/early second trimester. Preterm birth was defined as delivery of a liveborn infant at <37 completed weeks of gestation.</p><!><p>Maternal and cord blood/neonatal total circulating 25(OH)D assays—A rapid, direct RIA developed in the Hollis laboratory and manufactured by Diasorin Corporation (Stillwater, MN) was used to measure total circulating 25(OH)D concentration in serum samples as previously described (10). The laboratory participated in an independent quality assessment/assurance program (DEQAS) using National Institute of Standards and Technology (NIST) standards in place throughout both clinical trials (16). The inter- and intra-assay coefficient of variation was ≤10%.</p><!><p>Baseline demographic characteristics for the NICHD and TRF cohorts were summarized and compared using chi-square tests for categorical variables (race/ethnicity, insurance status, marital status, education level, and preterm birth), Mann-Whitney tests for maternal age, Poisson regression for count data (parity and gravidity), and t-tests for baseline maternal 25(OH)D. Serum 25(OH)D concentrations were plotted against gestation week at birth and locally weighted regression (LOESS) was used to explore the relationship in more detail.</p><p>Two 25(OH)D concentrations were of special interest: 20 ng/mL, from the IOM guidelines, and 40 ng/mL, from the 25(OH)D concentration found to achieve maximal production of the active hormone 1,25(OH)2D (10). Preterm birth rates were calculated both for the overall population and among women achieving 25(OH)D concentrations of ≤20 ng/mL, >20 to <40 ng/mL, and ≥40 ng/mL within 6 weeks of delivery. The incidence of preterm birth across 25(OH)D group was tested for a trend and risk ratios and 95% confidence intervals were calculated.</p><p>Univariate log-binomial regression was used to identify covariates for multivariate analysis. Covariates tested included race/ethnicity, maternal age, parity, gravidity, insurance status, marital status, education level, and study. Multivariable log-binomial regression was conducted within the combined cohort to estimate risk ratios and 95% confidence intervals for the association between 25(OH)D serum concentration and the risk of preterm birth, adjusting for the effect of covariates with a univariate association of p<0.20. Participants with a race of "other" were excluded due to small cell sizes. To identify differences by race/ethnic group, preterm birth rates were calculated for each race/ethnic group for those with serum concentrations ≤20 ng/mL vs. ≥40 ng/mL. These rates were then compared to the CC-MOD 2009-2011 data to assess potential risk reduction in the community (15).</p><!><p>The characteristics of the combined NICHD/TRF cohort are found in Table 1. In these diverse groups of women, there were significant differences between the NICHD and TRF cohorts by race/ethnicity, maternal age, parity, gravidity, insurance status, marital status, and education level but not baseline 25(OH)D or proportion of preterm births.</p><p>Within the combined cohort, there were 50 preterm births (<37 weeks) (10%), of which 7 were very preterm (<32 weeks), 5 were moderately preterm (32 to <34 weeks), and 38 were late preterm (34 to <37 weeks). A plot of gestation week at birth as a function of 25(OH)D concentration with preterm status and cut points of interest highlighted is shown in Figure 1, with the fitted LOESS curve superimposed.</p><p>Preterm birth rates were 17% in women with 25(OH)D ≤20 ng/mL (N=82); 10% in women with 25(OH)D >20 to <40 ng/mL (N=194), and 7% in women with 25(OH)D ≥40 ng/mL (N=233). Those with serum 25(OH)D concentrations ≥40 ng/mL within 6 weeks of delivery had a 57% lower risk of preterm birth compared to those with concentrations ≤20 ng/mL (RR=0.43, 95% CI=0.22,0.83), without adjustment for covariates (Table 2). Among women with low baseline 25(OH)D concentrations (≤20 ng/mL) at the beginning of their pregnancy (n=208), those who achieved ≥40 ng/mL within 6 weeks of delivery (n=60) had a 78% lower risk of preterm birth compared to those who did not (RR=0.22, 95% CI=0.05,0.92).</p><p>A zoom on the fitted LOESS curve with confidence bounds superimposed to better display the average behavior across its dynamic range shows gestational age at birth initially rising steadily with increasing 25(OH)D concentration and then plateauing at approximately 40 ng/mL (Figure 2). Box plots showing 25(OH)D concentration by birth category are shown in Supplementary Figure 1. The median 25(OH)D within 6 weeks of delivery for those with a preterm birth (<37 weeks) was 31 ng/mL compared to 39 ng/mL for those with a term birth (p=0.01).</p><p>Within the combined cohort, serum 25(OH)D, race/ethnicity, maternal age, and insurance status each had associations of p<0.20 with preterm birth in univariate log-binomial regression analyses and were included in the multivariable log-binomial regression model. Parity, gravidity, marital status, education level, and study had univariate associations of p≥0.20. In multivariable log-binomial regression, the only variable significantly associated with preterm birth was 25(OH)D serum concentration. After adjusting for race/ethnicity, maternal age, and insurance status, participants with 25(OH)D concentrations ≥40 ng/mL had a 59% lower risk of preterm birth than participants with concentrations ≤20 ng/mL (RR=0.41, 95% CI=0.20,0.86) (Table 2). There was a non-significant decrease in risk (41%) between those with concentrations >20 to <40 ng/mL compared to those with concentrations ≤20 ng/mL (RR=0.59, 95% CI=0.31,1.14).</p><p>Among race/ethnic groups in the combined cohort, the percentage of preterm birth was 8% among Hispanic women, 13% among black women, and 9% among white women. Supplementary Figure 2 shows the plots of gestational age at birth as a function of 25(OH)D concentration by race/ethnic group, with fitted LOESS curves superimposed. There was a 79% reduction in preterm birth among Hispanic women who attained ≥40 ng/mL compared to those ≤20 ng/mL (RR=0.21, 95% CI=0.06,0.69), and a 45% reduction among black women (RR=0.55, 95% CI=0.17,1.76). In white women, there were not enough women with low 25(OH)D concentrations within 6 weeks of delivery for assessment (n=3). Differences by race/ethnicity were not statistically significant when 25(OH)D concentration was included as a covariate in multivariable regression analysis.</p><p>Compared to the CC-MOD reference group, women in the combined cohort with serum 25(OH)D concentrations ≥40 ng/mL had a 46% lower rate of preterm birth (p=0.004) (Figure 3). Among race/ethnic groups, rates were reduced by 66% for Hispanic women (p=0.01), 58% for black women (p=0.04), and 11% for white women (p=0.77). While the 25(OH)D status of the CC-MOD population is not known, as a surrogate measurement for that population, we found that women in the combined cohort control group who received 400 IU/day (the current standard of vitamin D supplementation for pregnant women) and who had a median achieved 25(OH)D concentration of 30 ng/mL had rates of preterm birth that were not significantly different from the CC-MOD population: 9%, 14%, 11%, and 11% for Hispanic, black, white, and all women combined respectively. The data and code used to generate the analyses are available to qualified researchers upon request.</p><!><p>In this post-hoc analysis of the combined cohort dataset from two pregnancy vitamin D supplementation trials, there was a clear association between 25(OH)D serum concentration within six weeks of delivery and preterm birth. Achieving a 25(OH)D serum concentration of ≥40 ng/mL significantly decreased the risk of preterm birth compared to ≤20 ng/mL. While those women with 25(OH)D concentrations between 20 and 40 ng/mL also had a decreased risk of preterm birth, it was less of a decrease (41% vs. 59% in those women with 25(OH)D >40 ng/mL) and it was not statistically significant. These findings support the premise that 25(OH)D concentrations of 40 ng/mL and above are needed to significantly reduce the risk of preterm birth. In subgroup analyses, the reduction in risk was most notable among those with baseline 25(OH)D ≤20 ng/mL and among Hispanic women. When compared to published 2009-2011 CC-MOD rates of preterm birth (15), there were significant reductions in risk for Hispanic and black women ≥40 ng/mL but there was not a statistically significant reduction for white women. These results suggest the importance of vitamin D status during pregnancy.</p><p>A concern that women who deliver prematurely also have lower 25(OH)D concentrations due to less time during gestation for vitamin D supplementation to improve vitamin D status has been raised (17). Women taking 400 IU/day of vitamin D, as well as those on higher doses, reach steady-state by 2 months of supplementation (10). The association in this post-hoc and earlier analyses utilized the 25(OH)D within six weeks of delivery. In a recent publication (4), it was shown that the 25(OH)D concentration closest to the time of delivery, especially in Hispanic women, had a stronger association with gestational age and prematurity than baseline or second trimester 25(OH)D, arguing that the effect is mutable and that even in those women who were deficient at baseline, vitamin D supplementation can affect outcome.</p><p>Other studies have found a similar reduction in risk of preterm birth. A recent study by Bodnar et al. (5) found a 1.8-fold (95% CI=1.3,2.6) increase in the risk of preterm birth among those with 25(OH)D <20 ng/mL compared to ≥30 ng/mL and that the risk of preterm birth significantly decreased as 25(OH)D concentration increased to about 36 ng/mL. In NICHD pregnancy vitamin D supplementation trial by Hollis et al., the conversion of 25(OH)D to 1,25(OH)2D, the active hormonal form of vitamin D, was optimized at ≥40 ng/mL (10). There were no reported safety issues at such higher 25(OH)D concentrations. The Institute of Medicine recommends that pregnant women attain a total circulating 25(OH)D concentration of at least 20 ng/mL (6); however, these findings suggest that higher levels can be beneficial for birth outcomes. This post hoc analysis supports this premise.</p><p>The lack of a visible risk reduction trend among white participants was likely due to the lack of women with low 25(OH)D levels within 6 weeks of delivery in this race group. Also, white participants in this combined cohort started out with relatively high baseline 25(OH)D concentrations (median = 30 ng/mL), which likely left little opportunity for improvement. Black women had a median baseline 25(OH)D of 16 ng/mL, but only increased to a median of 30 ng/mL, which was likely too low to see a statistically significant change in risk. Comparatively, Hispanic women started with a median baseline 25(OH)D of 24 ng/mL and achieved a median 25(OH)D of 38 ng/mL, a change in nutrient status that better spans the response region seen in Bodnar et al. (5, 18, 19).</p><p>Strengths of this analysis include using serum 25(OH)D concentration as the predictor variable, which is statistically more powerful than using treatment group because it accounts for all input sources including cutaneous, food, and supplementation whereas treatment group only accounts for supplemental dose. Also, using 25(OH)D concentration overcomes the inherent bias of compliance and inter-individual variability in response to dose associated with treatment group analyses. Additionally, this randomized trial used supplementation doses ten times higher than the IOM recommendation of 400 IU/day, and therefore a larger spectrum of 25(OH)D concentrations could be assessed in relation to preterm birth risk.</p><p>The limitations of this post-hoc analysis are that this was a post-study analysis that was not able to control for some covariates related to preterm birth such as body mass index (BMI) and other sociodemographic factors such as smoking and alcohol use (which was extremely low in this cohort). Observed differences by race for those ≤20 ng/mL vs. ≥40 ng/mL within the combined cohort indicate that socioeconomic factors may play a role and while this analysis adjusted for insurance status as a proxy for socioeconomic status, additional indicators were not available. The second issue is one of generalizability and whether or not the diversity of socioeconomic backgrounds and racial/ethnic groups of the women in the study are representative of the larger population. There is also the limitation of sample size and power, with a larger sample size necessary to investigate 25(OH)D and race/ethnic trends further, particularly for white women. Additionally, the serum concentrations of the CC-MOD comparison population were unknown. Since the rate of preterm birth in the CC-MOD population was not significantly different from the combined cohort control group, it is reasonable to expect that women in the CC-MOD population had a similar average intake of 400 IU vitamin D per day (the standard amount found in prenatal vitamins) and serum 25(OH)D concentration averaging 30 ng/mL, the average 25(OH)D concentration in the control group taking 400 IU vitamin D per day. Furthermore, knowing that some women opt not to take prenatal vitamins, it is likely that the mean baseline 25(OH)D concentration of 23 ng/mL of the combined cohort is even more representative of the CC-MOD population. The similar rates of preterm birth strengthen the finding that improved vitamin D status was associated with lower rates of preterm births.</p><p>This post-hoc analysis supports the importance of attaining a 25(OH)D blood concentration substantially above 20 ng/mL during pregnancy for the prevention of preterm birth. Reaching 40 ng/mL would achieve the optimal conversion of 25(OH)D to 1,25(OH)2D (10) and lower preterm birth risk. An intake amount substantially higher than the IOM recommendation of 400 IU/day is needed for most people to attain this level. Less than one third (29%) of participants in the control group receiving 400 IU/day achieved a 25(OH)D concentration of 40 ng/mL compared to 57% in the 4000 IU treatment group, a dose amount with no observed adverse effects. Individuals should aim for a desired 25(OH)D level instead of taking a generic dose amount since 25(OH)D serum concentration is a better indicator of vitamin D status. Serum 25(OH)D testing is recommended to determine the specific intake amount an individual needs to attain a specific 25(OH)D concentration.</p><p>The March of Dimes estimates that the annual cost of preterm births in the United States is $12 billion (for 455,918 children) (20). If approximately 50% of preterm births could be prevented in the general population, as this analysis suggests is possible, there could be $6 billion available for other services and, more than 225,000 children and families spared this trauma. In light of this, practice guidelines at the Medical University of South Carolina (MUSC) and other institutions are currently being changed to prospectively target 40 ng/mL for pregnant women with the goal of dramatically lowering preterm birth rates.</p>

PubMed Author Manuscript

Copper-catalyzed CuAAC/intramolecular C–H arylation sequence: Synthesis of annulated 1,2,3-triazoles

Step-economical syntheses of annulated 1,2,3-triazoles were accomplished through copper-catalyzed intramolecular direct arylations in sustainable one-pot reactions. Thus, catalyzed cascade reactions involving [3 + 2]-azide-alkyne cycloadditions (CuAAC) and C-H bond functionalizations provided direct access to fully substituted 1,2,3-triazoles with excellent chemo-and regioselectivities. Likewise, the optimized catalytic system proved applicable to the direct preparation of 1,2-diarylated azoles through a one-pot C-H/N-H arylation reaction.

copper-catalyzed_cuaac/intramolecular_c–h_arylation_sequence:_synthesis_of_annulated_1,2,3-triazoles

Introduction<!>Results and Discussion<!>Conclusion<!>Experimental General information

<p>Transition-metal-catalyzed C-H bond functionalizations are increasingly viable tools for step-economical syntheses of various valuable bioactive compounds [1][2][3], which avoid the preparation and use of preactivated substrates [4][5][6][7][8][9][10][11][12][13][14][15][16]. This streamlining of organic synthesis has predominantly been accomplished with palladium [4][5][6][7][8][9][10][11][12][13][14][15][16], rhodium [17][18][19] or ruthenium [20][21][22] complexes [4][5][6][7][8][9][10][11][12][13][14][15][16]. However, less expensive nickel, cobalt, iron or copper catalysts bear great potential for the development of economically attractive transformations . In this context, we previously reported on the use of costeffective copper(I) catalysts for direct arylations of 1,2,3-triazoles. Thus, we showed that intermolecular copper-catalyzed C-H bond functionalizations could be combined with the Huisgen [51] copper(I)-catalyzed [52,53] [3 + 2]-azide-alkyne cycloaddition (CuAAC) [54], while C-H bond arylations of 1,2,3-triazoles were previously only accomplished with more expensive palladium [55][56][57][58][59][60][61][62] or ruthenium [63][64][65][66] catalysts. Notably, this strategy allowed for the atom-economical synthesis of fully substituted 1,2,3-triazoles in a highly regioselective fashion [54,67]. While the research groups of Rutjes [68] as well as Sharpless [69] elegantly devised alternative approaches exploiting 1-haloalkynes [70], we became interested in exploring a single [71][72][73] inexpensive copper catalyst for onepot reaction sequences comprising a 1,3-dipolar cycloaddition along with an intramolecular C-H bond arylation; in particular, because of the notable biological activities exerted by fully substituted 1,2,3-triazoles [74][75][76][77][78][79][80][81][82][83][84][85][86][87][88]. As a consequence, we wish to present herein novel cascade reactions, in which cost-effective copper(I) compounds serve as the catalyst for two mechanistically distinct transformations for the synthesis of fully substituted annulated 1,2,3-triazoles as well as for twofold N-H/C-H bond arylations. Notable features of our strategy include (i) the development of a chemoselective C-H arylationbased three-component reaction, as well as (ii) the use of inex-pensive CuI for the formation of up to one C-C and three C-N bonds in a site-selective fashion (Scheme 1).</p><!><p>We initiated our studies by exploring reaction conditions for the key copper-catalyzed intramolecular direct C-H bond arylation, employing substrate 3a (Table 1). Notably, the envisioned C-H bond functionalization occurred readily with the aryl iodide 3a when catalytic amounts of CuI were used, even at a reaction temperature as low as 60 °C, with optimal yields being obtained With optimized reaction conditions for the intramolecular direct arylation in hand, we tested the possibility of its implementation in a sequential synthesis of 1,4-dihydrochromeno [3,4d][1,2,3]triazole (4b, Scheme 2). We were delighted to observe that the desired reaction sequence consisting of a coppercatalyzed 1,3-dipolar cycloaddition and an intramolecular C-H bond arylation converted alkyne 1a to the desired product 4b with high catalytic efficacy.</p><p>Subsequently, we explored the extension of this approach to the development of a chemoselective three-component one-pot reaction. Thus, we found that alkyl bromides 2 could be directly employed as user-friendly substrates for the in situ formation of the corresponding organic azides (Scheme 3). Notably, the catalytic system proved broadly applicable, and a variety of organic electrophiles 2, thereby, delivered differently decorated</p><p>Importantly, performing the one-pot reaction in a sequential fashion was not found to be mandatory. Indeed, our strategy turned out to be viable in a nonsequential manner by directly employing equimolar amounts of the three substrates. Hence, inexpensive CuI allowed the direct assembly of aryl iodides 1, alkyl bromides 2 and NaN 3 with excellent chemo-and regioselectivities (Scheme 4). Thereby, a variety of annulated 1,2,3triazoles 4 were obtained, featuring six-or seven-membered rings as key structural motifs. It is particularly noteworthy that the copper-catalyzed transformation enabled the formation of one C-C and three C-N bonds in a chemoselective manner, and thereby provided atom-and step-economical access to annulated carbo-as well as O-and N-heterocycles.</p><p>Finally, we found that the catalytic system also proved to be applicable to the one-pot copper-catalyzed direct arylation of various azoles 5 through N-H/C-H bond cleavages with aryl iodides 6 as the organic electrophiles (Scheme 5).</p><!><p>In summary, we have reported on the use of inexpensive copper(I) complexes for step-and atom-economical sequential catalytic transformations involving direct C-H bond arylations. Thus, CuI enabled the synthesis of fully substituted 1,2,3-tria-zoles through cascade reactions consisting of copper(I)catalyzed [3 + 2]-azide-alkyne cycloadditions (CuAAC) and intramolecular C-H bond arylations. Notably, the optimized copper catalyst accelerated two mechanistically distinct transformations, which set the stage for the formation of up to one C-C and three C-N bonds in a chemo-and regioselective fashion, and also allowed for twofold C-H/N-H bond arylations on various azoles.</p><!><p>Catalytic reactions were carried out under an inert atmosphere of nitrogen using predried glassware. All chemicals were used as received without further purification unless otherwise specified. DMF was dried over CaH 2 . Alkynes 1 [89][90][91][92] and triazoles 3 [93] were synthesized according to previously described methods. CuI (99.999%) was purchased from ABCR with the following specifications: Ag <3 ppm, Ca = 2 ppm, Fe = 1 ppm, Mg <1 ppm, Zn <1 ppm. Yields refer to isolated compounds, estimated to be >95 % pure, as determined by 1 H NMR. Thin- General procedure for the synthesis of triazoles 4</p><p>NaN 3 (1.05 equiv), CuI (10 mol %), LiOt-Bu (2.00 equiv), alkyne 1 (1.00 equiv) and alkyl bromide 2 (1.00 equiv) were dissolved in DMF (3.0 mL) and stirred at 80 °C for 20 h. Then, H 2 O (50 mL) was added at ambient temperature, and the resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with saturated aq NH 4 Cl (50 mL), H 2 O (50 mL) and brine (50 mL), dried over Na 2 SO 4 , filtered and concentrated in vacuo. The remaining residue was purified by column chromatography on silica gel (n-hexane/ EtOAc).</p>

Beilstein

Analysis of Photosynthetic Characteristics and UV-B Absorbing Compounds in Mung Bean Using UV-B and Red LED Radiation

Mung bean has been reported to have antioxidant, antidiabetic, anti-inflammatory, and antitumor activities. Various factors have important effects on the types and contents of plant chemical components. In order to study quality of mung bean from different light sources, mung bean seedlings were exposed to red light-emitting diodes (LEDs) and ultraviolet-B (UV-B). Changes in the growth parameters, photosynthetic characteristics, the concentrations of chlorophyll a and chlorophyll b and the content of UV-B absorbing compounds were measured. The results showed that photosynthetic characteristics and chlorophyll a and chlorophyll b concentrations were enhanced by red LEDs. The concentrations of UV-B absorbing compounds were enhanced by UV-B on the 20th day, while photosynthetic characteristics, plant length, and the concentrations of chlorophyll a and chlorophyll b were reduced by UV-B on the 40th day; at the same time the values of the stem diameter, plant fresh weight, dry weight, and the concentrations of UV-B absorbing compounds were enhanced. It is suggested that red LEDs promote the elongation of plant root growth and photosynthetic characteristics, while UV-B promotes horizontal growth of stems and the synthesis of UV-B absorbing compounds.

analysis_of_photosynthetic_characteristics_and_uv-b_absorbing_compounds_in_mung_bean_using_uv-b_and_

1. Introduction<!>2.1. Plant Materials<!>2.2. Supplementary Light Treatments<!>2.3.1. Growth Parameter<!>2.3.2. Photosynthetic Characteristics<!>2.3.3. Determination of Chlorophyll a (chl a) and Chlorophyll (chl b) and UV-B Absorbing Compounds<!>2.4. Statistical Analysis<!>3.1. Growth Parameters<!>3.2. Photosynthetic Characteristics<!>3.3. Determination of chl a and chl b<!>3.4. Determination of UV-B Absorbing Compounds<!>4. Discussion

<p>Mung bean (Phaseolus radiatus L.) is a leguminous species grown in different parts of the world, primarily especially in Asia including China, India, Burma, and Thailand. Mung bean commonly is a common source of protein in the Asian diet or nutrient supplements [1]. Mung bean has been reported to possess antioxidant, antidiabetic, anti-inflammatory, antitumor and antimelanocytes, and antiangiotensin I-converting enzyme activities [2–8]. Mung bean contains free phenolic acids, bound phenolic acids, total phenolic, and anthocyanin. Correlation analyses between bioactivities and phytochemicals demonstrated that antioxidant bioactivity may be mainly contributed to phenolic compounds, whereas anthocyanins play an important role in the antidiabetic bioactivities [3]. Other reports showed that flavonoids including vitexin and isovitexin were the dominant components in mung bean [2, 9] and the content of vitexin was much higher than that of isovitexin in ethanol extracts [10]. It has been reported that mung bean has a strong antioxidant activity and isovitexin and vitexin contribute to most of the 1,1-diphenyl-2-picrylhydrazyl, ferric-reducing antioxidant power or 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical scavenging ability [2].</p><p>Various factors including geographical location, climate change, temperature, and illumination time have important effects on the types and contents of plant chemical components, which are related to their bioactivity, functionality, and applications. Light quality is one of the most important factors in the regulation of plant growth, morphogenesis, photosynthesis, metabolism, and gene expression [11, 12]. For the photobiological research, ultraviolet-visible spectrum between 200 nm and 800 nm wavelength plays an important role in changes of chemical compounds of the organisms by irradiating them, especially compounds with ultraviolet (UV) absorption property [13, 14].</p><p>Compared to the ordinary fluorescent light source, the light-emitting diode (LED) light sources can provide a single wavelength of light quality with high photoelectric conversion efficiency, fixed wavelength, and low heat. LED light source is considered to be a new important light source in the field of plant physiology and plant cultivation. Previous studies indicated that the LED light sources were used in the research of photomorphogenesis [15], chlorophyll synthesis [16], and photosynthesis [17]. Recently the studies of this field attract more and more researchers focusing on the work [18, 19].</p><p>Many studies indicated that there were the practical problems of insufficient light intensity and limited spectral wavelength during the process of plant cultivation in the laboratory [20–22]. It is necessary to find effective ways to replace or assist the ordinary fluorescent light source, to improve research method for the plant, and to promote the plant quality. In this study, we used the red LEDs and ultraviolet-B (UV-B) radiation as additional light sources for the process of plant cultivation in the laboratory to determine the role of the different qualities of light source on growth and photosynthetic characteristics of mung bean.</p><!><p>Mung bean (Phaseolus radiatus L. cv. Qindou 20) seeds were selected for uniform size. Mung bean seeds were obtained from Yangling Breeding Center of National Bean Engineering Research Center of China (Shaanxi, China).</p><!><p>The ordinary fluorescent light source (power 40 W) was purchased from Philips Inc. The light directly irradiated the seedling of mung bean from am 7:00 to pm 7:00 each day. The UV-B radiation was provided by filter Qin brand (Baoji Lamp Factroy, China) 30 W fluorescence sunlamps. They were filtered with 0.13 mm thick cellulose diacetate (transmission down to 290 nm) for UV-B radiation. The dose of UV-B irradiation was 0.861 kJ/m2 per day. The supplementary light treatments were shown in Table 1. The lamps were suspended above the plant at the height of 40 cm perpendicular to the ground.</p><p>Firstly, seeds were sterilized for 10 min by 0.1% HgCl2 and were grown in Petri dish (diameter 18 cm) after being washed for 50 min by flowing water. Until seeds were germinated, they were transplanted in basin (diameter 25 cm) which was filled with the ratio of peat : vermiculite : perlite for 3 : 1 : 1. One week after seed germination, the supplementary light treatments carried out seed germination. On the 20th day and 40th day of supplementary light treatments, organisms were sampled, respectively, for various analyses.</p><!><p>The morphology including plant height, fresh weight, dry weight, root length, and stem diameter was measured. Mung bean seedlings were oven dried at 80°C until constant weight and being weighed using electronic scale as biomass (g).</p><!><p>Photosynthetic characteristics were measured with a photosynthesis meter (Photosynthesis Meter I-301, CID. Inc.). The water use efficiency was the ratio of photosynthesis and transpiration. The results of stomatal conductance, photosynthesis and water use efficiency were the mean values of the day.</p><!><p>The method for the measure of the concentration of chl a and chl b was extracted by acetone and determined following the reported methods [23]. Intact leaf samples of seedlings (fresh weight 0.5 g), which were at 5-6 leaves stage of development, were placed in a mortar and followed by the addition of silica of 0.2 g, CaCO3 of 0.2 g, and 15 mL 80% acetone. After thorough grinding, the samples were filtrated with two layers of filter paper by pump air and fixed to 25 mL with 80% acetone, and then the absorbance at 663 and 645 nm was determined, respectively. Chlorophyll concentration was calculated and expressed as mg/g FW.</p><p>Fresh samples of 0.5 g were taken from the epicotyls and extracted in 10 mL acidified methanol (methanol-water-hydrochloric acid, 79 : 20 : 1, v/v) for UV-B absorbing compounds, according to the procedure of Mirecki and Teramura [24]. The hydrochloric acid was 36% HCl. Extract absorbance at 300 nm was measured with a spectrophotometer (UV-2100; Shimadzu, Columbia, MD, USA) and the absorbance was arbitrarily used for analysis.</p><!><p>All experiments were performed in six times repeatedly. Statistical analyses were performed with SPSS 11.5 for windows. The results were expressed as the means ± standard error (SE) of triplicate. The data were subjected to one-way analysis of variance (ANOVA) and the significance of difference between samples means was calculated by Duncans' multiple range test and P values less than 0.05 were considered significant.</p><!><p>It was observed that the values of the growth parameters of mung bean seedlings were irradiated for 20th day by red LEDs and UV-B was not significantly different compared with that of the ordinary fluorescent light (Table 2). However, the red LEDs treatment caused a significant increase (P < 0.05) of the values of plant height, fresh weight, dry weight, and root length compared with that of the ordinary fluorescent light for 40th day. With the UV-B radiation for 40th day, an obvious decrease (P < 0.05) of plant height was observed, while it induced a marked increase (P < 0.05) in fresh weight, dry weight, and stem diameter.</p><!><p>The red LEDs treatment induced a significant increase (P < 0.05) in the values of the photosynthetic characteristics for the two durations (Table 3). However, a significant decrease (P < 0.05) was observed in the values of the photosynthetic characteristics of the UV-B radiation for 40th day.</p><!><p>The concentrations of chl a and chl b may affect the values of the photosynthetic characteristics to a certain extent. It was obvious that the red LEDs treatment induced statistically significant increases (P < 0.05) not only in the values of the photosynthetic characteristics (Table 3) but also in the concentrations of chl a and chl b (Figure 1) for the two durations. Compared with the ordinary fluorescent light, the UV-B radiation did not cause significant differences.</p><!><p>With the treatment of UV-B radiation, the concentrations of UV-absorbing compounds were increased dramatically and the same trend of results was shown in Figure 2. UV-B radiation-treated seedlings resulted in a notably increase in the concentrations of UV-absorbing compounds for the two durations (P < 0.05). However, red LEDs did not cause significant difference in comparison to the ordinary fluorescent light.</p><!><p>LEDs are a promising irradiation source for plant growth in space for long life, minimal mass, volume, and being a solid state device. The red LEDs (wavelength 650 nm) were used as a supplementary light source for the greenhouse tomato in 1982, which was reported earlier by Japan's Mitsubishi Corporation [25]. During the process of laboratory cultivation, it has been reported that the ordinary fluorescent light lacked the ultraviolet part of the solar spectrum background, which was essential growth factor to play an important biological role [20, 26].</p><p>However, it was difficult to use a mixed-use LED light sources during the process of plant cultivation completely [27]. Therefore, we used red LED and UV-B as supplementary light sources for the ordinary fluorescent light to study the role of these light sources in plant cultivation. Our results showed that these light sources were obviously increased for growth and photosynthetic characteristics of mung bean. The red LED light source promoted the growth of the mung bean root (Table 2), which was useful to absorb the nutrients and water of the soil.</p><p>Chlorophyll concentrated in the chloroplast grana is the main pigment to capture the energy for photosynthesis in green plants. The results showed that the red LED light source can significantly increase the concentrations of the chlorophyll (Figure 1), which effectively promoted the photosynthesis and water use efficiency (Table 3). On the contrary, UV-B radiation induced a notably decrease in the photosynthesis and water use efficiency. It was suggested that the UV-B treatment will reduce the stomatal opening degree of mung bean and then affect the gas exchange in photosynthesis.</p><p>UV absorption compounds in leaves are an important class of pigments including flavonoid, flavonol, cinnamon, and anthocyanin, which determine the color changes of many plants and are very sensitive to light. They play an important role in protective effect as a class of secondary metabolites [28, 29], which are related to antioxidant, antidiabetic, anti-inflammatory, antitumor and antimelanocytes and antiangiotensin I-converting enzyme activities [2–8]. The previous studies have reported that vitexin and isovitexin were major flavonoid in the ethanol extract of mung bean and vitexin content was much higher than isovitexin in ethanol extracts from mung bean sprout [3, 9]. However, another report showed that no significant difference in levels of vitexin and isovitexin was observed in mung bean sprout of the same cultivar tested in the experiments [2]. Since UV absorption compounds have an absorption peak in UV-B radiation scope, it could be found that the UV-B treatment caused a significant increase in UV absorption compounds (Figure 2). Compared with the ordinary fluorescent light, red LEDs did not induce significant differences in UV absorption compounds. Red LED and UV-B as supplementary light sources have an important effect on plant growth and chemical components.</p>

PubMed Open Access

Tandem rhodium catalysis:Exploiting sulfoxides for asymmetric transition-metal catalysis

Sulfoxides are uncommon substrates for transition-metal catalysis due to their propensity to inhibit catalyst turnover. In a collaborative effort with Ken Houk, we developed the first dynamic kinetic resolution (DKR) of allylic sulfoxides using asymmetric rhodium-catalyzed hydrogenation. Detailed mechanistic analysis of this transformation using both experimental and theoretical methods revealed rhodium to be a tandem catalyst that promoted both hydrogenation of the alkene and racemization of the allylic sulfoxide. Using a combination of deuterium labelling and DFT studies, a novel mode of allylic sulfoxide racemization via a Rh(III)-\xcf\x80-allyl intermediate was identified.

tandem_rhodium_catalysis:exploiting_sulfoxides_for_asymmetric_transition-metal_catalysis

Introduction<!>Development of a DKR of allylic sulfoxides<!>A new mode of allylic sulfoxide racemization by rhodium catalysis<!>Conclusions and Future Outlook

<p>Chiral sulfoxides are structural components found in the world's top selling pharmaceutical drugs, organocatalysts, chiral auxiliaries and ligands for use in asymmetric catalysis (Figure 1).1–6 From a synthetic standpoint, molecules containing the sulfone or sulfoxide functional handle can be exploited for carbon—carbon bond formation via the Julia olefination, an aldol-type reaction, or a Mislow-Braverman-Evans rearrangement. However, organosulfur compounds, including sulfoxides, are often challenging substrates for catalysis because they tend to poison metal catalysts by forming stable organometallic complexes.7,8 Specifically, sulfoxides coordinate to transition metals via the sulfur and/or oxygen heteroatoms.1,9–11 Totland and Alper showed that vinyl sulfone 1 can be hydroformylated under rhodium catalysis to generate aldehydes 2 and 3 in high yields and good branched-to-linear selectivities (Scheme 1).7 However, when the analogous vinyl sulfoxide 4 was subjected to similar reaction conditions, only 50% conversion of the substrate was achieved after extended reaction times, most likely due to catalyst poisoning. While the sulfoxide functionality shows promise as a directing group for formation of the branched regioisomer 5, its strong coordinating coordinating ability inhibits catalyst turnover.</p><p>During our own studies on heteroatom-directed Rh-catalyzed intramolecular hydroacylations, we found that sulfoxide-containing alkenal 7 can direct a highly diastereoselective cyclization to form medium ring ketone 8 (Scheme 2).12 Thus, we became interested in studying the use of chiral sulfoxides as directing groups for stereoselective transformations.</p><!><p>Inspired by the early reports of Mislow and coworkers,13–15 we envisioned a DKR strategy for the functionalization of allylic sulfoxides. Unlike typical sulfoxides (i.e., dialkyl, diaryl, and alkyl-aryl sulfoxides) that are configurationally stable under normal conditions,13 (chiral) allylic sulfoxides 9 are thermolabile and can racemize rapidly at temperatures between 40–70°C. This racemization occurs by a reversible [2,3]-sigmatropic rearrangements through the intermediacy of an achiral sulfenate ester 10.14 The Mislow-Braverman-Evans sequence16,17 which furnishes substituted allyl alcohols 11, stands as the only practical application of the [2,3]-sigmatropic rearrangement of allylic sulfoxides (Scheme 3).</p><p>In considering this limitation, rather than trapping the sulfenate ester 10 with stoichiometric reductants, we propose to use the [2,3]-sigmatropic rearrangement of allylic sulfoxides as the racemization step in a DKR. Asymmetric transformations on the olefin will halt racemization and thus enable access to configurationally stable products. This protocol would provide an alternative to standard oxidation procedures for synthesizing enantioenriched sulfoxides.1 The use of isomerizations or sigmatropic rearrangements as the racemization element in DKRs are rare. Akai and coworkers have shown that allylic alcohols can be racemized in the presence of a vanadium oxo-reagent via 1,3-allylic transpositions, and they achieve a DKR by using lipase-catalyzed acylation (Scheme 4).18,19</p><p>To achieve the desired DKR, the following three criteria has to be met: 1) the chiral catalyst, [Rh]*, must react preferentially with one enantiomer, 2) the rate of racemization must be fast relative to the rate of hydrogenation,20 and 3) the product sulfoxide must not be prone to epimerization (Scheme 5). During this study, we found that tandem catalysis21 is operative because one rhodium complex catalyzes two distinct steps in a cascade process that involves racemization followed by hydrogenation.</p><p>We found that [Rh((S,S)-Ph-BPE)]BF4 catalyzes hydrogenation of racemic 12 to aryl(propyl)sulfoxide (13) in PhMe/DCM in 66 % yield and 90 % ee (Scheme 6). However, sulfenate ester 8 was formed in 28 % yield, presumably due to hydrogenation of allyloxy(aryl)sulfenate ester 10. To reduce byproduct formation, the use of polar solvents (i.e., methanol) that stabilize the polar sulfoxide relative to the non-polar sulfenate ester is desirable. However, polar solvents are reported to significantly lower the rates of sulfoxide epimerization.15</p><p>To enhance the rates of sulfoxide racemization, we investigated the possibility of catalysing the [2,3]-sigmatropic rearrangement. Palladium(II) salts have been reported to catalyse the [2,3]-sigmatropic rearrangement of allylic amine N-oxides,22 and [3,3]-sigmatropic rearrangements, including the Overman,23 Claisen,24 and aza-phospha-oxa-Cope25 processes. On the contrary, metal catalyzed [2,3]-sigmatropic rearrangements of allylic sulfoxides are unprecedented. As part of an effort to elicit antibodies that catalyze pericyclic reactions, the Hilvert group has reported two antibodies that catalyse sulfoxide-sulfenate rearrangements.26 We performed a series of kinetics experiments and found that [Rh((S,S)-Ph-BPE)]BF4] enhances the rate of racemization in methanol by a factor of 33 (t1/2 = 9.6 h).11</p><p>With the discovery that the rhodium complex behaves as a tandem catalyst for both sulfoxide racemization and olefin hydrogenation, it became evident that the relative rates of racemization and hydrogenation could not be controlled simply by changing the catalyst loading. Therefore, we subjected sulfoxide (±)-12 to Rh-catalyzed DKR hydrogenation in methanol at lower pressures of H2 (Scheme 7). By lowering the hydrogen pressure to 0.1 atm, the rate of racemization relative to the rate of hydrogenation is increased. Under these conditions, DKR of allylic sulfoxide 12 occurs to generate enantioenriched sulfoxide 13 in 92 % yield and 88 % ee. Performing the reaction in methanol suppresses undesired byproduct formation, and sulfenate ester 14 is formed in only 8 % yield.</p><!><p>To understand the Rh-catalyzed racemization of allylic sulfoxides, γ-deuterated allylic sulfoxide (±)-12-D was prepared and subjected to hydrogenation in methanol (Scheme 8). After 36 h, deuterated product 13-D was isolated in 75 % yield and 91 % ee. Importantly, a significant amount of deuterium had been scrambled to the α-position. Complete scrambling of the deuterium label was also observed in the recovered starting material. These results support a mechanism involving a Rh(III)-π-allyl intermediate A. Rate acceleration in methanol is consistent with the relatively polar nature of the Rh(III) intermediate.</p><p>Additional insight into this unique mode of racemization was gathered by computing the free energy profile (Scheme 8). We find that oxidative insertion occurs via a concerted 6-membered transition state (D-TS) with a barrier of 16.4 kcal/mol to generate Rh(III)-π-allyl intermediate E. Sulfoxide epimerization occurs by rotation of the sulfinyl unit, and this has a barrier of 15.3 kcal/mol (F-TS). Consistent with our deuterium scrambling results, allyl group rotation via H-TS is facile and exhibits a barrier of 16.6 kcal/mol. A final C—S reductive elimination regenerates the epimerized allylic sulfoxide. The highest barrier (16.6 kcal/mol) within this pathway is significantly lower in energy than the transition-state energy of the uncatalyzed pathway, which is computed to be 23.5 kcal/mol. Throughout the catalytic cycle for sulfoxide-directed hydrogenation, DFT calculations11 support a mechanism where the oxygen atom of the sulfoxide is bound in all of the lowest energy ground states and transition states (Scheme 9).</p><!><p>This study contributes to our emerging interest in using tandem catalysis to address challenges in organic synthesis, including tandem Ru-catalyzed hydroacylations27,28 and tandem Ru-catalyzed aminations.29 The first demonstration of a catalytic asymmetric transformation of racemic allylic sulfoxides is achieved through Rh-catalyzed hydrogenation. This reaction is made possible by a tandem rhodium catalyst, [Rh((S,S)-Ph-BPE)]BF4, that plays a dual role in accelerating the rate of allylic sulfoxide epimerization and catalyzing olefin hydrogenation (Scheme 10). It is also the first small molecule that catalyzes allylic sulfoxide racemization. The mechanism of the newly discovered Rh-catalyzed racemization of allylic sulfoxides was probed by deuterium labelling and DFT and features a Rh(III)-π-allyl intermediate.</p><p>The sulfoxide has great potential for stereoselective transition-metal-catalyzed transformations due to its strong coordinating ability and inherent chirality. While there is concern for sulfoxides to undergo undesired side-reactions or poison catalysts, both of these undesirable processes can be minimized under the appropriate reaction conditions (i.e., solvent, pressure). In demonstrating a successful DKR of allylic sulfoxides, we reveal mechanistic insights that could enable the development of other sulfoxide-directed metal processes. Future studies from our laboratory will focus on complexity-forming reactions, such as hydroacylation.</p>

PubMed Author Manuscript

Disorder and defects are not intrinsic to boron carbide

A unique combination of useful properties in boron-carbide, such as extreme hardness, excellent fracture toughness, a low density, a high melting point, thermoelectricity, semi-conducting behavior, catalytic activity and a remarkably good chemical stability, makes it an ideal material for a wide range of technological applications. Explaining these properties in terms of chemical bonding has remained a major challenge in boron chemistry. Here we report the synthesis of fully ordered, stoichiometric boron-carbide B 13 C 2 by high-pressure-high-temperature techniques. Our experimental electron-density study using high-resolution single-crystal synchrotron X-ray diffraction data conclusively demonstrates that disorder and defects are not intrinsic to boron carbide, contrary to what was hitherto supposed. A detailed analysis of the electron density distribution reveals charge transfer between structural units in B 13 C 2 and a new type of electron-deficient bond with formally unpaired electrons on the C-B-C group in B 13 C 2 . Unprecedented bonding features contribute to the fundamental chemistry and materials science of boron compounds that is of great interest for understanding structure-property relationships and development of novel functional materials.Boron carbide is one of the hardest substances, surpassed only by diamond and boron nitride 1 . The high mechanical and thermal stability, low density and low costs of fabrication have made boron carbide the prime choice in a series of technological applications [1][2][3][4][5][6][7] . Boron carbide preserves the same structure for a range of compositions, and details of this crystal structure have been discussed in terms of chemical disorder of boron and carbon atoms as well as the presence of vacancies 1,[8][9][10][11] . Electronic-structure calculations suggest that the properties of boron carbide depend on the stoichiometry and the details of the disorder 2,7,12,13 .Experimentally, chemical bonding can be accessed through single-crystal x-ray diffraction. Reliable information on the distribution of the electron density in the unit cell can be obtained only for good-quality single crystals with minimal structural disorder 14 . Synthesis of defect-free material is the most challenging task in boron carbide chemistry. We have succeeded in growing small single crystals of the stoichiometric composition B 13 C 2 by high-pressure-high-temperature techniques (see Methods). The material is transparent with a dark red or maroon color, indicating an insulator or a large-band-gap semiconductor. This is in agreement with some literature data 15 , but it is inconsistent with the relatively high electrical conductivity reported for boron carbide 1 . To the best or our knowledge, dark red transparent boron carbide has not been reported before.A multipole (MP) model has been obtained for the crystal structure of B 13 C 2 by refinement against accurately measured intensities of Bragg reflections (see Methods and Supplementary Information Section S1) 14 . The excellent fit to the diffraction data with R 1 = 0.0197 provides strong evidence for the stoichiometry of B 13 C 2 , in agreement with the composition obtained by Energy-dispersive x-ray (EDX) analysis (see Methods). The excellent fit furthermore indicates the absence of disorder: B 13 C 2 is composed of B 12 icosahedral clusters and CBC linear chains (Fig. 1 and Supplementary Information Section S2). Lattice parameters and values of atomic displacement parameters (ADPs) fall within a range previously assigned to the composition B 12 C 3 1,8-10 . The possibility of different compositions was investigated by additional MP refinements with small amounts of carbon at the B P site, corresponding to B 12 + x C 3 − x stoichiometries with x = 0.44 and x = − 0.11, respectively (see Supplementary Information Section S1 for details). Both models gave a slightly worse fit to the diffraction data than the B 13 C 2 model. More importantly, the number of valence electrons of C at the B P site refined to zero, thus showing that the MP refinement has effectively removed carbon from the B P site, providing further support for the ordered

disorder_and_defects_are_not_intrinsic_to_boron_carbide

<!>Methods summary<!>X-ray diffraction experiment for/and electron density analysis.

<p>stoichiometric character of the investigated crystal. Interestingly, a refinement of the independent atom model (IAM) including the site occupancy factors of C at the B P and B E sites resulted in 19% occupancy of the B P site by carbon (x = − 0.11). Contrary to the MP model (R 1 = 0.0197), the IAM with disorder (R 1 = 0.0287) leads to only a small improvement of the fit to the data (Table S4). These results suggest that the charge transfer towards B P in the MP model is mimicked in the disordered IAM by a fractional occupancy of the B P site by C.</p><p>Discrepancies between the present values of the lattice parameters and ADPs and those reported in the literature 1,[8][9][10] for the same composition may be the result of different degrees of disorder and defects between different samples. The single MP refinement 16 reported previously for B 13 C 2 gave a much worse fit to their XRD data (R 1 = 0.0440), which questions the reliability of that model. The single MP refinement 17 for B 12 C 3 also led to a substantially worse fit to their XRD data (R 1 = 0.0250) than we have obtained for our model against the present XRD data (R 1 = 0.0197). Thus, a highly precise MP refinement refutes recent less accurate diffraction studies 13 and theoretical electronic-structure calculations 2,12 , where a disorderly replacement by carbon of a certain fraction of the boron atoms of the B 12 clusters was considered as absolutely essential for the stability of B 13 C 2 .</p><p>The MP model extends the independent atom model (IAM) of spherical atoms by parameters describing the reorganization of electron density due to chemical bonding. Previous electron-density studies on boron carbide 18,19 have been restricted to a discussion of the qualitative features of the electron densities. Quantitative information about chemical bonding can be extracted from the static electron density of the MP model through its topological properties according to Bader's quantum theory of atoms in molecules (QTAIM) 14,20 . Critical points (CPs) are defined as the positions where the gradient of the electron density is zero [∇ρ (r) = 0] 20 . They are classified according to the number of positive eigenvalues of the Hessian matrix of second derivatives as local maxima (3 positive eigenvalues), bond critical points BCPs (2), ring critical points RCPs (1) and local minima (0 positive eigenvalues) 20 .</p><p>All atomic positions of the present MP model can be identified with local maxima in the static electron density, while additional local maxima do not exist. BCPs and RCPs have been found between the atoms of the B 12 cluster in a similar pattern as for α-boron 21 , and with comparable values for the electron densities and Laplacians (Table 1). Together, these features indicate similar bonding by molecular-type orbitals on the B 12 clusters in B 13 C 2 and α-boron 21 . According to Wade's rule 22 , this bonding involves 26 of the 36 valence electrons of the twelve boron atoms of this closo-cluster, thus leaving for each boron atom one orbital but only 5/6 electrons for exo-cluster bonding 21,23 .</p><p>The crystal structure of B 13 C 2 comprises four crystallographically independent atoms. CBC chains contain the carbon atom and a boron atom denoted as B C ; the B 12 cluster is made of six polar and six equatorial atoms, denoted as B P and B E , respectively (Fig. 1). According to the QTAIM 20 , bonding between a pair of atoms exists, if the electron density possesses a BCP between those atoms. For B 13 C 2 , we have found BCPs between pairs of B P atoms from neighboring clusters. The distance B P -B P is slightly larger and the magnitudes of the electron density, ρ BCP , and Laplacian, ∇ 2 ρ BCP , are slightly smaller than those of the corresponding inter-cluster bonds in α-boron 21 and γ -boron 24 (Table 1). The high value of ρ BCP together with a negative value of ∇ 2 ρ BCP of large magnitude indicate a strong covalent interaction between these atoms 20 . The similarities with bonding in α-boron 21 (Table 1) allow this bond to be classified as a 2-electron-2-center (2e2c) bond. Further evidence for this interpretation comes from the QTAIM theory, which assigns a charge to each atom by integration of the electron density over the atomic basins. A charge of − 0.21 electrons has been obtained by integrating the experimental static electron density over the atomic basin of B P (Table 2). This value is in good agreement with electron counting. With 5/6 electrons per boron atom available for exo-cluster bonding, a formal charge of − 0.17 is obtained for B P involved in a 2e2c B P -B P exo-cluster bond.</p><p>Bond-critical points are also found between a B E atom and the closest C atom. Large magnitudes of ρ BCP and the negative Laplacian ∇ 2 ρ BCP indicate a strong covalent interaction and a 2e2c C-B E bond. An equal split of these electrons between C and B E again gives a formal charge of − 0.17 for B E , and it would result in a (B 12 ) 2− group 2 However, carbon is more electronegative than boron and should attract most of the bonding electrons. Indeed, the integration of the electron density over the atomic basins leads to a positive atom B E and a strongly negative C atom (Table 2). A detailed analysis of the electron density shows that the positive charge of B E is the result of a strong polar-covalent character of the C-B E bond, with the BCP much closer to B E than to C (Fig. 2; Table 1), but with a large value of ρ BCP as opposed to an expected small value for ionic bonding 20 .</p><p>With the interpretation of B P -B P and C-B E bonds as 2e2c bonds, only three electrons are left for the two C-B C bonds of the CBC group (see Supplementary Material Section S3). These bonds can therefore be described as a three-electron-three-center (3e3c) bond or as resonance between two equivalent combinations of one 2e2c and one 1e2c bond (Fig. 3). The large values of the electron density along the bond path (Fig. 2a) correlate with the short bond length, which is explained by the internal pressure on the CBC group 2 . Large magnitudes of ρ BCP and ∇ 2 ρ BCP indicate a covalent interaction. The electron deficient character of this bond is in complete agreement with the ionic charge of + 2.30 of B C . The latter value is the result of the extremely small volume of the atomic basin of this atom, which demonstrates that the internal pressure has squeezed out most of the electrons of B C , reminiscent of the effect of pressure on the electrons in lithium metal 25 .</p><p>A 3e3c C-B C -C bond contains one unpaired electron per formula unit B 13 C 2 . Experimentally, unpaired spins have been observed at much lower concentrations in boron carbides of different compositions 2,4,5,26,27 . One explanation lies in chemical disorder and vacancies, which are necessarily present for other compositions than stoichiometric B 13 C 2 , and which reduce the number of unpaired spins. On the other hand, the itinerant character of the electron states or localization as bipolarons may be in agreement with low concentrations of unpaired spins 2,5,12 . The presence of an unsaturated bond on the CBC chains should result in a high chemical reactivity of this bond. However, we have found that B C is extremely small (Table 2) and well shielded from the outside by C atoms and bulky B 12 clusters. Steric effects hindering access to reactive sites is known to stabilize radicals 28,29 . High temperatures can overcome these barriers, and a high reactivity at elevated temperatures towards oxidizing agents has been described for boron carbide 30 . Recently, amorphisation 6,31 of boron carbide B 12 C 3 has been explained on the basis of the presence of carbon atoms at a small fraction of the B P sites 32 . Stoichiometric B 13 C 2 is a form of boron carbide that lacks this detrimental property of technical boron carbide with compositions on the carbon-rich side of B 13 C 2 .</p><p>In summary, we have synthesized stoichiometric boron carbide B 13 C 2 , which is free of intrinsic disorder, and is built of B 12 icosahedral clusters and C-B C -C chains. Unlike band-structure calculations 2,12 on fully ordered B 13 C 2 , the ordered stoichiometric compound is an insulator or large band-gap semiconductor. An experimental electron-density study by X-ray diffraction conclusively determines that B 13 C 2 is an electron-precise material. The electron-deficient character is explained by B C being stripped of two of its valence electrons and the existence of a unique, electron deficient 3e3c bond on the C-B C -C chains. The low chemical reactivity follows from the extremely small volume of B C . Table 2. Atomic basins (volume V Basin ) and ionic charges for the four crystallographically independent atoms in B 13 C 2 along with their multiplicity in the unit cell.</p><!><p>Crystal growth. Single crystals of boron-carbide were grown at high pressures of 8.5-9 GPa and high temperatures of 1873-2073 K using a 1200-ton (Sumitomo) multi-anvil hydraulic press at the Bayerisches Geoinstitut. Energy-dispersive x-ray (EDX) analysis has been employed to determine the composition as B 6.51 (12) C, in agreement with stoichiometric B 13 C 2 . The presence of other elements could be excluded.</p><!><p>A single crystal of boron-carbide of dimensions 0.09 × 0.08 × 0.05 mm 3 was chosen for an x-ray diffraction experiment with synchrotron radiation at beamline F1 of Hasylab, DESY in Hamburg, Germany. The sample was kept at a temperature of 100 K, while a complete data set of accurate intensities was measured for Bragg reflections up to sin(θ )/λ = 1.116 Å −1 . The diffraction data were integrated using the computer program EVAL 33 . Structure refinements have been performed with the software XD2006 34 . A topological analysis of the static electron density has been performed by the modules TOPXD and XDPROP of the computer program XD2006. Two-dimensional density maps have been generated by the module XDGRAPH. See the Supplementary Information for details on procedures and the MP model.</p>

Scientific Reports - Nature

A novel exploratory chemometric approach to environmental monitorring by combining block clustering with Partial Least Square (PLS) analysis

BackgroundGiven the serious threats posed to terrestrial ecosystems by industrial contamination, environmental monitoring is a standard procedure used for assessing the current status of an environment or trends in environmental parameters. Measurement of metal concentrations at different trophic levels followed by their statistical analysis using exploratory multivariate methods can provide meaningful information on the status of environmental quality. In this context, the present paper proposes a novel chemometric approach to standard statistical methods by combining the Block clustering with Partial least square (PLS) analysis to investigate the accumulation patterns of metals in anthropized terrestrial ecosystems. The present study focused on copper, zinc, manganese, iron, cobalt, cadmium, nickel, and lead transfer along a soil-plant-snai food chain, and the hepatopancreas of the Roman snail (Helix pomatia) was used as a biological end-point of metal accumulation.ResultsBlock clustering deliniates between the areas exposed to industrial and vehicular contamination. The toxic metals have similar distributions in the nettle leaves and snail hepatopancreas. PLS analysis showed that (1) zinc and copper concentrations at the lower trophic levels are the most important latent factors that contribute to metal accumulation in land snails; (2) cadmium and lead are the main determinants of pollution pattern in areas exposed to industrial contamination; (3) at the sites located near roads lead is the most threatfull metal for terrestrial ecosystems.ConclusionThere were three major benefits by applying block clustering with PLS for processing the obtained data: firstly, it helped in grouping sites depending on the type of contamination. Secondly, it was valuable for identifying the latent factors that contribute the most to metal accumulation in land snails. Finally, it optimized the number and type of data that are best for monitoring the status of metallic contamination in terrestrial ecosystems exposed to different kinds of anthropic polution.

a_novel_exploratory_chemometric_approach_to_environmental_monitorring_by_combining_block_clustering_

Background<!>Results and disscusions<!><!>Results and disscusions<!><!>Results and disscusions<!><!>Results and disscusions<!>Conclusions<!>Experimental<!>Statistical analysis<!>Abbreviations<!>Competing interests<!>Authors’ contributions

<p>In recent years it has become increasingly clear that industrial contamination is leading to serious threats to terrestrial ecosystems, thus endangering human and environmental health. Generally speaking, industrial contamination is related to any type of waste released into the environment from human industrial activities [1]. Metal contamination is, however, of particular interest because of metal highly toxic properties and their potential side-effects on ecosystem function and integrity [2]. Although this form of contamination dates back to antiquity, widespread industrial contamination accelerated rapidly with the start of the Industrial Revolution (the 1800s) and is currently regarded to be a serious problem in many countries [1]. Among the major sources of metal release we mentione several key human activities, such as mining operations, siderurgy, burning coal and oil in power plants, chemical industry, vehicular traffic, and intensive agriculture [1].</p><p>In this context, environmental monitoring is a standard procedure used for assessing the current status of an environment or trends in environmental parameters [2]. To determine the risks posed by metals on terrestrial ecosystems one should understand their fate along food chains. Briefly, metals are easily accumulated in soils, wherein they may persist over long periods of time [3]. The transfer process begins with the uptake of metals by the primary producers (green plants and bacteria), and continues to the next trophic level, the primary consumers (i.e., herbivores). Measurement of metal concentrations at these trophic levels can provide meaningful information concerning the status of environmental quality and ecosystem health at a specific moment of time [2,4], but only if the large amount of data resulted from such work are handled using appropriate chemometric approaches. Therefore, the present article deals with the most appropriate statistical methods to understand the factors contributing to contamination and metal accumulation in terrestrial ecosystems. Such questions are important in modern environmental science, especially for preliminary environmental impact assessment when researchers use descriptive applications to identify the underlying relationships between metal concentrations at different/same trophic levels.</p><p>To this end, many studies have relied on exploratory multivariate analysis to extract reliable information for environmental quality assessment [5-8]. In most cases the research question of interest in environmental chemistry and monitoring is expressed in terms of variables and cases (observations). A commonly used method to assess the similarity among different cases/variables is Hierarchical cluster analysis (HCA), also known as Tree clustering. This statistical technique reveals natural grouping (or clusters) within relatively large data sets based on measured characteristics. The graphical output is a dendrogram that shows how variables/cases are merged on one axis, whereas the other axis gives the distance at which any two clusters are joined [9]. However, this statistical technique does not allow environmental researchers to simultaneously merge the grouping of both cases and variables. The clustering of both by applying two-way joining clustering (syn. Block clustering) may yield relevant results not only for detecting clusters of cases with a similar magnitude of the measured variables, but also to explore the underlying relationships between these variables. Therefore, we propose that Block clustering (BC) may provide an interesting and powerful statistical approach in environmental monitoring if the researchers may want to simultaneously identify the similarity between different cases and variables.</p><p>When investigating large sets of data is beneficial to reduce their dimensionality in order to improve the efficiency and accuracy of data analysis [10]. Principal component analysis (PCA) is commonly used for this purpose in environmental research [7,11,12], but does not allow scientists to separate between the predictor and response variables [9]. Another statistical method, Exploratory factor analysis (EFA), uncovers the underlying structure for large sets of variables based on the shared variances among factors [13,14], but is sensitive to sample size, i.e., the sample size must be at least three-fold higher than the number of variables [15]. Partial least square (PLS) may represent a solution where such multivariate methods fail. This technique is routinely used in chemometric analysis when a large number of independent variables (>1000) are obtained with respect to a small number of samples (10 to 100) [16]. Depending on the objective of the study, PLS can serve either as a principal component technique, correlation technique, path modeling technique, or canonical correlation technique [17]. Overall, we suggest that this statistical method is a potential approach in environmental monitoring surveys for exploratory modeling of data sets with a large number of variables, but a moderate sample size (n = 20–50). Such situations are often encountered in baseline field surveys, which document the environmental conditions that exist at a specific moment in time to provide background in case of unknown changes in the future [18].</p><p>The present study enlarges our earlier survey [19] to include four additional metals (i.e., Mn, Fe, Ni, Co in addition to Cu, Zn, Cd, Pb) when investigating metal accumulation along soil-plant-snail food chain. These metals were chosen because they are known to serve as vital and/or toxic elements, depending on concentration, chemical and physical form. Nickel is regarded as having no obvious physiological role in plants and animals [20]. In contrast, manganese, iron, and cobalt act mainly as essential micronutrients, but their occurrence at high levels can represents a potentially serious hazard for environmental health [20]. This is of particular interest for Co and Mn, which, together with Ni, rank among the most dangerous 200 chemical compounds released in the environment from human activities according to the 2011 Substance Priority List of the US Agency for Toxic Substances and Disease Registry [21].</p><p>The Roman snail (Helix pomatia) was considered in the present study because this terrestrial gastropod concentrates high metal levels in its soft tissues without revealing any major metabolical disorders and serves as a major herbivore in terrestrial ecosystems [19]. The main purpose of this paper was to introduce a novel chemometric approach in environmental monitoring for understanding the similarities among sites/cases and determining the principal latent factors that influence metal accumulation in biological end-points by combining Block clustering with PLS and using a soil-plant snail food chain as study system.</p><!><p>The levels to which metals accumulate at different trophic levels, the normal content (NC) and alert threshold level (ATV) in soil for each investigated metal are shown as absolute values in Table 1. The standardized values at each trophic levels were normally distributed for all investigated metals (p > 0.05). It was found that the concentrations of metals in soils were within the normal levels at all sites, excepting Cd which occasionally did exceed the reference value from the Romanian Soil Quality regulations [22], but did not reach the corresponding alert threeshold level (ATV). These results showed that anthropic activities have a relatively low impact on soil metal concentrations in the study areas (Figures 1 and 2).</p><!><p>Metal concentrations in soils, nettle leaves, and snail hepatopancreas in the study area (expressed as mg kg-1 d.w.)</p><p>Abbreviations: THM1 - Timisoara city Landfill (Parta-Sag/Timis county); THM2 - South Industrial Platform (Timisoara/Timis county); THM3 - North Industrial Platform (Timisoara/Timis county); THM4 - East Industrial Platform (Timisoara/Timis county); THM5 - Timisoara, Communal Road 64 (DC64) (Timisoara/Timis county).; THM6 - National Road Resita-Caransebes (DN58) (Caras-Severin county); THM7 - Principal Street (Otelu Rosu/Caras-Severin county); THR - Salbagelu Nou (Caras-Severin county); NC–normal metal content in the soil; ATV–alert threshold level for metal concentrations in the soil; MVL–average metal level for all sites taken together.</p><p>General map showing the locations of soil, nettle leaves, and snail sampling sites. Legend. 1–site THM1 (45.6765° lat. N; 21.1626° long. E); 2–site THM2 (45.7112° lat. N; 21.1968° long. E); 3–site THM3 (45.7420° lat. N; 21.1886° long. E); 4–site THM4 (45.7763° lat. N; 21.2517° long. E); 5–site THM5 (45.7787° lat. N; 21.2762° long. E); 6–site THM6 (45.3496° lat. N; 21.9196° long. E); 7–site THM7 (45.5053 lat. N; 22.3371 long. E); R–site THR (45.5667° lat. N; 22.0812° long. E).</p><p>Detailed map showing the locations of soil, nettle leaves, and snail sampling sites (source: Google Maps). Legend. 1–site THM1 (45.6765° lat. N; 21.1626° long. E); 2–site THM2 (45.7112° lat. N; 21.1968° long. E); 3–site THM3 (45.7420° lat. N; 21.1886° long. E); 4–site THM4 (45.7763° lat. N; 21.2517° long. E); 5–site THM5 (45.7787° lat. N; 21.2762° long. E); 6–site THM6 (45.3496° lat. N; 21.9196° long. E); 7–site THM7 (45.5053 lat. N; 22.3371 long. E); R–site THR (45.5667° lat. N ; 22.0812° long. E).</p><!><p>The dendrogram groups the environmental variables on the x-axis using the squared Euclidean distance as a criterion of similarity (Figure 3). At the abiotic level we can observe similar distributions in the soil among the total concentrations for Cu, Zn, Mn, and Ni, and Cd and Co, respectively. Such relationships may reflect the fact that these metals share common anthropogenic sources, such as combustion of coal and heavy fuel oil, municipal waste incineration, vehicular traffic, chemical plants, ferrous and non-ferrous metal production [23]. As a result of having no functional role in plants and terrestrial gastropods [24,25], the measured values for Ni, Cd, and Pb fall close to each other on the x-axis both in the nettle leaves and snail hepatopancreas. Similar associations are also found between the essential trace metals. Because Cu, Co, and Mn are essential regulators of plant growth and development [24], they are clustered near each other in the nettle leaves. There is a close association between the Mn and Fe accumulation in the hepatopancreas; this is mainly related to the fact that these essential microelements follow similar metabolic pathways in land snails [25]. Copper is exclusively regulated in H. pomatia by a specific metallothionein [26], and therefore, its distribution in the hepatopancreas is independent of any other metals. This element is essential for land snails because it is a a component of the chromoprotein hemocyanin, which is essential to their respiration [25].</p><!><p>Block clustering of the metal concentrations in soil, nettle leaves, and snail hepatopancreas. Legend. Cu(S)–copper concentrations in soil; Cu(U)–copper concentrations in nettle leaves; Cu(HP)–copper concentrations in snail hepatopancreas; Zn(S)–zinc concentrations in soil; Zn(U)–zinc concentrations in nettle leaves; Zn(HP)–zinc concentrations in snail hepatopancreas; Mn(S)–manganese concentrations in soil; Mn (U)–manganese concentrations in nettle leaves; Mn(HP)–manganese concentrations in snail hepatopancreas; Fe(S)–iron concentrations in soil; Fe (U)–iron concentrations in nettle leaves; Fe (HP)–iron concentrations in snail hepatopancreas; Cd(S)–cadmium concentrations in soil; Cd(U)–cadmium concentrations in nettle leaves; Cd(HP)–cadmium concentrations in snail hepatopancreas; Co(S)–cobalt concentrations in soil; Co(U)–cobalt concentrations in nettle leaves; Co(HP)–cobalt concentrations in snail hepatopancreas; Co(S)–nickel concentrations in soil; Co(U)–nickel concentrations in nettle leaves; Co(HP)–nickel concentrations in snail hepatopancreas; Pb(S)–lead concentrations in soil; Pb(U)–lead concentrations in nettle leaves; Pb(HP)–lead concentrations in snail hepatopancreas.</p><!><p>The y-axis clusters the sites with similar distribution of metals at different trophic levels (Figure 3). Combining the x- and y-axes reveals information about the underlying similarities among sites, which cannot be provided by applying the Tree Clustering method. Green colors represent lower than average values and yellow to brown the opposite. The first sampling point (THM1) is located near the Sag-Parta landfill, whereas the second sampling point lies within the South Industrial Platform, about 100 m far away from the Timisoara Sud Power Plant. As a result of being located close one to each other (about 1km), these sites showed a similar pattern of metal accumulation at all trophic levels, which is associated with exposure to the same source of environmental pollution (i.e., South Industrial Platform Timisoara).</p><p>The site THM1 regularly exhibited the highest metal concentrations among different locations, irrespective of trophic level. This site does not have engineered systems for collecting landfill leachate or gases, and as a consequence, it is considered as a class B landfill that is suitable to accept only general domestic and commercial waste [27]. Although this landfill was officially closed in 2009 [28], it remains a serious pollution hotspot in the Timisoara area, as shown by our results (Table 1). Similarly, the site THM2 revealed higher metal concentrations along soil-plant-snail food chain as compared to the other investigated sites. This site lies near multiple sources of anthropic pollution, and as a consequence, such findings are not surprising. Our results are in line with recent studies, which found high levels of metals in vegetables from areas adjacent to the South Industrial Platform Timisoara [29].</p><p>The third sampling site (THM3) lies near the former "Solventul Timisoara" petrochemical works. This site fall close to the fourth sampling point (THM 4), which is located within the East Industrial Platform Timisoara. These locations share a similar pattern of Zn accumulation in the soil and nettle leaves (Figure 3). Because both sites are known for long-term exposure to chemical and petrochemical industries, the routine use of Zn compounds in these industries (e.g., zinc oxide as pigment in paint industry or catalysts in the manufacture of rubber) may therefore explain our findings [30].</p><p>The fourth sampling point (THM5) is placed in a wooden area, near the Communal Road DC64 (Timisoara-Ghiroda). The seventh site (THM7) lies in the city of Otelu Rosu, about 50 m far away from the National Road DN68 (Caransebes–Hateg), and 150 m of the former Otelu Rosu steel works, respectively. Interestingly, these two sites are clustered near each other on the vertical axis although they are placed in different counties (i.e., the site THM5 lies more than 100 km away from the site THM7). Although manganese is one of the most abundant metals in soils, its deposition in soil was also shown to be associated with trafficked roads [31]; therefore, the moderate concentrations of Mn that are found in the soil at both sites may be linked to a similar intensity of vehicular traffic along the DC64 and DN68 roads (Figure 3).</p><p>The reference site (THR) corresponds to an area located away from major sources of pollution [32], about 100 m away from the communal road which connects the National Road DN58 to the village of Salbagelu Nou, whereas the sixth site (THM6) lies along the National Road DN58 (Resita-Caransebes). The latter location generally shows higher metal levels than the site THR, which are related to the cumulative action of long-term exposure to vehicular traffic and metallic contamination (Resita steel works). Overall, we can observe that the dendrogram obtained by using the Block clustering method separates the sites in two groups (Figure 3). The first group (G1) contains the sites located on industrial platforms from Timisoara (i.e., THM1-THM4), whereas the second group (G2) includes the sites located within 100 m away from roads with different intensity of vehicular traffic (i.e., THM5-THM7, THR).</p><p>The exploratory PLS analysis for all sites extracted two significant latent factors (Table 2a), which explained 58.11% of the variance of response variables and 52.41% of the variance of predictor variables, respectively. The weights of predictor variables determine VIP (Variable Importance in the Projection), which shows the statistical contribution of the variable in fitting the PLS model. Variables for which the VIP scores are less than 0.8 are regarded to be small [33], and therefore, one can conclude that copper and zinc concentrations in soil and nettle leaves are contributing the most to trace metal accumulation pattern in land snails (Figure 4a). These findings are consistent with land snail physiology, wherein copper is a key player in metabolic activities [34].</p><!><p>Results of the exploratory PLS analysis for all sites (a), the G1 sites(b) and the G2 sites (c)</p><p>Abbreviations: R2X–percentage of X variance explained by each PLS factor; R2X(Cumul.)–cumulative modeled variation in X; R2Y - percentage of Y variance explained by each PLS factor; R2Y(Cumul.) - cumulative modeled variation in Y.</p><p>VIP values for the exploratory PLS model for all sites (a), the G1 sites(b) and the G2 sites (c).</p><!><p>We have reran the PLS analysis with all predictor variables on each of the two groups that were obtained by applying Block clustering. The physiological metals (i.e., Cu, Zn, Mn, Fe, Co) were removed from our analysis because the levels to which these microelements accumulate in soils were generally within NC values at all sites (Table 1). Among the toxic metals (e.g., Ni, Cd, Pb), priority was given to those elements which showed high VIP scores (VIP > 0.8) for concentrations in both the soil and nettle leaves. Reducing the number of independent variables implies that fewer terms are needed in the expansion to find the metals with the highest/lowest risk on environmental and human health in the study areas.</p><p>The PLS model for the G1 sites had five significant factors, which explained 87.12% variance of response variables and 95.05% of the variance of predictor variables, respectively (Table 2b). The importance of most predictor variables was high (VIP > 0.8), excepting Mn and Ni levels in the soil, and Co content in the soil and U.dioica leaves (Figure 4b). It can be seen from the Figure 4b that Cd and Pb concentrations, in contrast with Ni levels, have high VIP scores in both the soil and nettle leaves. Therefore, the G1 sites should be closely monitored in the future with respect to Cd and Pb accumulation along terrestrial food chains. Our results are consistent with recent studies, which found that Cd and Pb are the main determinants of pollution pattern in the Timisoara area [29,35].</p><p>For the G2 sites, the PLS analysis extracted five significant factors, which accounted for 86.76% of the variation of response variables and 96.04% of the variation of predictor variables, respectively (Table 2c). All physiological metals showed high VIP scores in the soil and nettle leaves (Figure 4c). Among toxic metals, Pb was the only element for which the concentrations in soils and nettle leaves displayed high VIP scores (Figure 4c). These findings are not surprising since Pb is among the most common heavy metals (together with Cu and Zn) released from vehicular traffic [36].</p><!><p>The legacy of metals released to the environment from human activities puts increasing pressure on terrestrial ecosystems from anthropized areas. To this end, not only finding novel analytical methods for determining the degree of environmental contamination, but also employing new statistical approaches for analyzing environmental data provide scientists with powerful tools in environmental monitoring and assessment.</p><p>In the present study, we show that applying Block clustering with PLS analysis is simple and intuitive procedure that allows researchers to:</p><p>– overcome the drawbacks imposed by the graphical representation of environmental variables. Although such charts are useful in environmental monitoring, they hide what the data tell us when too many variables are illustrated within the same chart;</p><p>– group sites with similar patterns of metal accumulation at different trophic levels, thus allowing environmental researchers to separate the study areas depending on the type of contamination and to understand the underlying similarities among them;</p><p>– select the most significant latent factors (based on VIP values) which explain metal accumulation in biological end-points;</p><p>– optimize the analytical procedures by selecting for future investigations only the toxic metals with high VIP scores, thus reducing the analytical costs; in this case, the emphasis of contamination with Cd and Pb on industrial platforms near Timisoara and with Pb near roads;</p><p>– provide a benchmark for building exploratory models with potential applications in environmental monitoring surveys when researchers have to deal with data sets having a large number of variables, but a small sample size.</p><!><p>Detailed description concerning the location of sampling sites, the preparation of samples, and the analysis of metals are provided in our previous work [19]. Briefly, the samples were collected in triplicate for each trophic level (soil, nettle, snail) from eight sites located in the western part of Romania, in the Banat area (Timis and Caras-Severin counties). All locations have been exposed to long-term industrial pollution (> 30 years), and lie at most 10 km away from former and/or actual sources of anthropic contamination. The reference area (site THR), the vilage of Salbagelu Nou (Caras-Severin county), is located in a non-polluted area, with less industry [31]. The sites THM1-THM5 are located around the city of Timisoara, the most populated and industrialized city from the Banat area. The sites THM6 and THM7 are located along trafficked roads, in an area which was exposed for more than two centuries to the impact of metallurgical industry [37]. For each sampling plot at least 60 newly matured Helix pomatia specimens were collected, fasted for 48 h and sacrificed by freezing (at −20°C). To provide homogeneous samples the snails were calibrated based on the shell height, which was shown to serve as a more accurate predictor of snail size as compared to the shell width [19]. The measurements were performed with a digital caliper to the nearest 0.01 mm. After defrosting, the whole soft body was removed from the shell and the viscera and the foot were separated. Only the snail hepatopancreas was considered in the present study because this organ was found to serve as the main end-point of metal accumulation for H. pomatia[19]. The samples were analysed in triplicate for each location, and 20 snails were used for each batch.</p><p>The selected food chain included nettle as the main food source of Roman snail (Helix pomatia) based on observations of snail feeding habits in investigated areas. For each location three samples from the top leaves were collected, rinsed in distilled water to wash off potential air pollutants, and then oven dried at 105°C to constant weight. The samples were crushed with a mortar, passed through a 2 mm sieve, and preserved in self-sealing sterile paper pouches at room temperature (t = 22°C). The soil samples were collected (25 g/sample in triplicate) from the top 15 cm layer after removal of vegetation (grass). After removing roots and litter, they were dried (t = 22°C, 7 days), disaggregated, homogenized before being sieved to 2 mm (soil metal concentration analysis), and then stored at ambient temperature (t = 22°C) for further analysis [19].</p><p>The extraction of metals was performed by wet extraction (in HNO3 0.5N) for the soil samples, and by ash digestion (with HNO3 0.5N) for the nettle and snail samples. The ash was obtained by burning the nettle and snail samples for 8 h, at 550°C, in the muffle furnace (Nabertherm B150, Lilienthal, Germany). The metals were determined by flame atomic absorption spectrometry (FAAS) with high resolution continuum source (Model ContrAA 300, Analytik Jena, Germany), in the Environmental Research Test Laboratory, Banat's University of Agricultural Sciences and Veterinary Medicine from Timisoara, Romania. This spectroscopic method was chosen because it is a fast and easy technique with an extremely high sensitivity for elements like Pb, Cd, Cu and Cr. NCS Certified Reference Material-DC 85104a and 85105a (China National Analysis Center for Iron&Steel), were analyzed for quality assurance. Percent recovery means were: Fe (92%), Mn (95%), Zn (102%), Cu (105%), Ni (99%), Pb (94%), Cd (105%), Co (98%). The variation coefficients were below 10%. Detection limits (μg/g) were determined by the calibration curve method: Fe (0.15), Mn (0.19), Zn (0.43), Cu (0.13), Ni (0.14), Cd (0.01), Pb (0.05), Co (0.07).</p><!><p>Because the metal concentrations differed by several degrees of magnitude, the measured values were standardized as follows:</p><p>STV=RV−MV/STD</p><p>where STV defines the standardized value, RV the raw value, MV the mean value, and STD the standard deviation. As a result, the data were displayed on scale from −1 to +1.</p><p>The subsequent data were then checked for normality using both Anderson-Darling test (for comparing distribution functions) and Jaques-Bera test (for comparing between kurtosis and skewness of a function). We performed a cluster analysis (Block clustering) to identify the sites with similar patterns of metal accumulation at different trophic levels and to explore relationships between these variables. Exploratory PLS analysis was used for assessing the principal latent variables (factors) underlying metal accumulation in the snail hepatopanceas. The analysis was carried out for all sites taken together, as well as separately for each cluster of sites obtained by applying the Block clustering method. Metal concentrations in the soil and nettle leaves were considered as independent/predictor variables (X of the PLS matrix), whereas their levels in the snail hepatopancreas were taken into account as dependent/outcome variables (Y of the PLS matrix). To validate the PLS model, the number of extracted factors was chosen through 10-fold cross validation, i.e. fitting the model to part of the data and minimizing the prediction error for the unfitted part [17]. Statistical analyses were performed by using the Statistica 10 software package [38]. All data are presented as the mean ± SD for the absolute measured values.</p><!><p>PLS: Partial least square analysis; VIP: Variable importance in the projection; THR: Reference sampling site; THM1-THM7: Sampling sites 1-7; NC: Normal content; ATV: The alert threshold level.</p><!><p>The authors declare that they have no competing interests.</p><!><p>DVN, DMB, IP, EP, SA, and IG have contributed mainly to the study design, collection of data, sampling of soil, vegetation, and snails, chemical analyses, interpretation of results and preparation of paper. All authors read and approved the final manuscript.</p>

PubMed Open Access

Synthesis of Stable NAD+ Mimics as Inhibitors for the Legionella pneumophila Phosphoribosyl Ubiquitylating Enzyme SdeC

AbstractStable NAD+ analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD+‐consuming enzymes. To investigate the potential of such compounds to inhibit the adenosine diphosphate ribosyl (ADPr) transferase activity of the Legionella SdeC enzyme, we prepared three NAD+ analogues, namely carbanicotinamide adenosine dinucleotide (c‐NAD+), thionicotinamide adenosine dinucleotide (S‐NAD+) and benzamide adenosine dinucleotide (BAD). We optimized the chemical synthesis of thionicotinamide riboside and for the first time used an enzymatic approach to convert all three ribosides into the corresponding NAD+ mimics. We thus expanded the known scope of substrates for the NRK1/NMNAT1 enzyme combination by turning all three modified ribosides into NAD+ analogues in a scalable manner. We then compared the three NAD+ mimics side‐by‐side in a single assay for enzyme inhibition on Legionella effector enzyme SdeC. The class of SidE enzymes to which SdeC belongs was recently identified to be important in bacterial virulence, and we found SdeC to be inhibited by S‐NAD+ and BAD with IC50 values of 28 and 39 μM, respectively.

synthesis_of_stable_nad+_mimics_as_inhibitors_for_the_legionella_pneumophila_phosphoribosyl_ubiquity

<!>Introduction<!><!>Results<!><!>Results<!><!>Results<!><!>Conclusion<!>Conflict of interest<!>

<p>J. M. Madern, R. Q. Kim, M. Misra, I. Dikic, Y. Zhang, H. Ovaa, J. D. C. Codée, D. V. Filippov, G. J. van der Heden van Noort, ChemBioChem 2020, 21, 2903.</p><!><p>Many cellular proteins are decorated with post‐translational modifications (PTMs) during their life‐time that control their function, cellular localization and activity. One of these PTMs is adenosine‐diphosphate‐ribosylation (ADP‐ribosylation), in which a monoadenosine diphosphate ribosyl transferase enzyme (mART) transfers an ADPr fragment from an NAD+ molecule to the nucleophilic side chain of an amino acid residue of a target protein, releasing nicotinamide in the process. This mono ADPr‐modification can be elongated in a linear or branched fashion, giving rise to linear poly‐ and branched poly‐ADPr chains. Enzymes generating both the mono‐ADPr and poly‐ADPr modification play crucial roles in distinct biological processes.1 Another essential PTM that regulates many key cellular processes is ubiquitylation, in which a small 8.5 kDa protein called ubiquitin (Ub) is attached to the target protein.2 Installment of Ub is regulated by a cascade of three enzymes known as E1‐activing enzyme, E2‐conjugating enzyme and E3‐ligase. In this ATP‐dependent process the combination of specific E1‐E2‐E3 enzymes dictates the choice of substrate and site of ubiquitylation. Prokaryotes do not contain the Ub system, but a variety of bacterial pathogens do have the capability to use or hijack the host's Ub system.3 Recently, it was discovered that the Legionella pneumophila bacterium uses a family of so‐called SidE effector enzymes to mediate the ADP‐ribosylation of Ub as a crucial first step in ubiquitylating host proteins of their own choice, finally leading to Legionnaires disease in immunocompromised individuals. These SidE effectors contain multiple domains with different catalytic activities, including a mART and a phosphodiesterase (PDE) domain. Initially, the mART domain catalyzes the transfer of ADPr to arginine 42 (R42) of Ub, releasing nicotinamide. Subsequently the PDE domain will activate the phosphodiester bond, facilitating the covalent attachment to a serine residue of a host substrate protein while displacing adenosine mono‐phosphate (AMP; Figure 1).4, 5, 6, 7, 8 In this way, the enzyme is able to ubiquitylate the substrate, via a unique phosphoribosyl (Pr) linkage without the need for either E1‐E2‐E3 enzymes or ATP. Recent studies link this phenomenon of Pr ubiquitylation to the development and maintenance of a vacuole in which Legionella replicates, by modifying host proteins involved in ER fragmentation and membrane recruitment.9, 10 The unusual Pr ubiquitylation, with ADPribosylation of Ub as first step, is an interesting target for drug development, since this pathway seems to be Legionella specific as it has not been identified in mammals so far. Knocking down the SidE effectors severely effects bacterial virulence and hence these proteins are bona‐fide drug targets.11</p><!><p>Schematic representation of mART and PDE action of SidE‐family enzymes on ubiquitin.</p><!><p>There are two distinct classes of ART enzymes, referred to as diphtheria‐toxin‐like or cholera‐toxin‐like, which have a HYE or RSE catalytic triad, respectively.12 Both types transfer ADPr in a reaction that proceeds through a transition state having a ribosyl oxocarbenium ion character (Figure 2A). Several NAD+ mimics that are unable to form such an oxocarbenium intermediate, such as carbanicotinamide adenosine dinucleotide (c‐NAD+, 1), thionicotinamide adenosine dinucleotide (S‐NAD+, 2) and benzamide adenosine dinucleotide (BAD, 3; Figure 2B), have been developed as inhibitors of NAD+ consuming enzymes and have shown moderate to good inhibition.13, 14, 15 c‐NAD+ has been shown to inhibit activity of the cholera toxin A ART enzyme16 and NAD+‐consuming enzymes Sirt3/5,17 but was ineffective towards NAD+‐glycohydrolase. c‐NAD+ is also ineffective in inhibiting PARP1, while BAD is a low micromolar PARP1 inhibitor.18 S‐NAD+ was developed more recently and shows inhibition of NAD+‐dependent enzyme CD38.19</p><!><p>A) Reaction mechanism of ART mediated ADP‐ribosylation, B) NAD+‐based ART inhibitors 1, 2 and 3.</p><!><p>In this light, we decided to explore these three promising NAD+ analogues, c‐NAD+ (1), S‐NAD+ (2) and BAD (3), for their inhibitory potential on the ART function of the Legionella SidE family member SdeC. Fully synthetic approaches towards BAD and c‐NAD+ have been reported, but formation of the dinucleotide, through coupling of adenosine monophosphate and the phosphorylated ribose analogues remains troublesome. Therefore we decided to adopt a chemo‐enzymatic procedure, recently used to prepare S‐NAD+, to form the difficult pyrophosphate linkages in c‐NAD+ and BAD.19 We synthesized the carbanicotinamide riboside and benzamide riboside using optimized procedures from literature and developed a novel synthesis for the thioriboside. Subsequent chemo‐enzymatic conversion using nicotinate riboside kinase (NRK1) to produce the nicotinamide mononucleotides (NMNs) and subsequent action of nicotinamide mononucleotide adenylyl transferase (NMNAT1) would yield us the three NAD analogues c‐NAD+ (1), S‐NAD+ (2) and BAD (3).</p><p>The known carbanicotinamide riboside (4)17 and benzamide riboside (6;14, 20 Scheme 1B) were prepared following the optimized reported procedures. We devised a new synthetic route for the third analogue, thionicotinamide riboside (S‐NR, 5) that was recently described.19 We opted to employ the synthesis depicted in Scheme 1A that is based on our prior studies on thioribosides.21 First, we synthesized protected thioribitol 13 from ribose, essentially as described by us and others.21, 22 In brief, the synthesis started with readily accessible and inexpensive D‐ribose that was first converted into lactol 9, the aldehyde functionality of which was next reduced to give ribitol 10. Mesylation and double bromide substitution then provided dibromide 12, which was treated with sodium sulfide to give thioribitol 13. After removal of the three p‐methoxybenzyl ethers by acidolysis, we selectively protected the primary alcohol with a TBDPS group and the secondary alcohols with acetyls to obtain 16. The thioether was then oxidized to a sulfoxide using m‐CPBA at low temperature and with controlled amounts of the oxidizing agent to avoid an overoxidation to the corresponding sulfone. The subsequent Pummerer‐rearrangement was performed by dissolving the sulfoxide obtained from 16 in acetic anhydride and heating the reaction mixture to 120 °C. The rearrangement proceeded regio‐ and stereoselectively to provide thioribosyl donor 18 as a mixture of anomers. We observed that the Pummerer rearrangement of a per‐O‐acetylated thioribitol led to the formation of 4‐O‐acetyl thioribitol by oxidation at C‐4, and hence the electron donating properties of the silyl protection at C5 was essential in our synthesis. Donor 18 was next glycosylated with nicotinamide in a Vorbrüggen‐type glycosylation, using TMSOTf in acetonitrile, yielding a mixture of α/β‐anomers in 75 % yield. Finally, the silyl ether was removed using HF in pyridine, and the acetyls were removed using ammonia in methanol at 0 °C, at which stage the anomers could be separated by RP‐HPLC to provide thionicotinamide riboside 5. Thus, S‐NR 5 was obtained in 16 steps in an overall yield of 3.6 %, which compares favorably with the method previously reported for the synthesis of this compound.19 With nicotinamides 4 and 5 and benzamide 6 in hand, we next focused on the enzymatic phosphorylation of the 5'‐hydroxy function using NRK1, followed by introduction of the pyrophosphate using NMNAT1 (Scheme 1B). The riboside was dissolved in buffer containing ATP, MgCl2 and DTT after which NRK1 was added to install the phosphate group on the 5'‐hydroxy function selectively. After one hour, complete formation of the nicotinamide mononucleotides was observed, and NMNAT1 was added to the reaction mixture to install AMP and form the respective NAD+ analogues. After reacting for 16 hours LCMS analysis revealed consumption of the mono‐nucleotides and formation of the NAD+‐analogues, that where purified using RP‐HPLC to yield us compounds 1, 2 and 3 in multi‐milligram amounts (36, 26 and 25 % yield, respectively) as off‐white powders.</p><!><p>A) Synthesis of thionicotinamide riboside (5) a) AllOH, AcCl, 0 °C; b) PMBCl, NaH, DMF; c) KOtBu, DMF, 110 °C; d) THF, NaHCO3 (sat. aq.), I2; e) NaBH4, MeOH; f) MsCl, Et3N, CH2Cl2; g) LiBr, MEK, 80 °C; h) Na2S, DMF, 100 °C; i) TFA, CH2Cl2; j) TBDPSCl, Im., CH2Cl2; k) Ac2O, pyr.; l) m‐CPBA, CH2Cl2, −40 °C; m) Ac2O, 100 °C; n) nicotinamide, BSTFA, TMSOTf, ACN, 80 °C; o) HF/pyr.; p) NH3/MeOH, 0 °C. B) enzymatic conversion of ribosides 4–6 towards NAD+ analogues 1–3. PMB=para‐methoxybenzyl, TBDPS=tert‐butyl‐di‐phenyl silyl, NRK1=nicotinate riboside kinase 1, NMNAT1=nicotinamide mononucleotide adenylyl transferase 1.</p><!><p>Next we investigated inhibition of SdeC activity by c‐NAD+ (1), S‐NAD+ (2) and BAD (3) using an ϵ‐NAD+ hydrolysis assay.23 In ϵ‐NAD+, the fluorescence of etheno‐adenosine is quenched by nicotinamide and the formation of ADPr−Ub as a result of ART activity of SdeC releases nicotinamide, resulting in a fluorescent signal. The increase in fluorescence is therefore a direct measure of substrate ϵ‐NAD+‐consumption and enzyme activity. In this assay, Ub and ϵ‐NAD+ are incubated with SdeC and increase in fluorescence is measured over time. We first assessed the affinity of unmodified NAD+ by incubating SdeC with both ϵ‐NAD+ and NAD+. Such substrate competition between the two NAD+ species led to a concentration‐dependent decrease in fluorescent signal upon increasing the amount of NAD+, allowing us to determine the IC50 value of NAD+ for SdeC to be 27.7±1.9 μM (see extended data in Figure 3). When performing a similar experiment where c‐NAD+, S‐NAD+ or BAD were incubated at increasing concentrations with SdeC, Ub and ϵ‐NAD+, we observed concentration‐dependent inhibition of ART‐activity for S‐NAD+ and BAD. However, only a marginal effect for c‐NAD+ was detected in the used concentration range up to 200 μM (Figure 3A). IC50 values for BAD and S‐NAD+ were determined to be 27.9±6.5 and 38.9±3.9 μM, respectively (Figure 3B). S‐NAD+ thus is an inhibitor of SdeC with a comparable affinity as native substrate NAD+, whereas BAD is a good inhibitor with a slightly higher IC50.</p><!><p>A) ϵ‐NAD+ hydrolysis assays showing nicotinamide displacement by SdeC ART activity and its concentration‐dependent inhibition by BAD, S‐NAD+ and c‐NAD+, B) IC50‐curves of S‐NAD+ and BAD.</p><!><p>In conclusion, we have shown the improved chemical synthesis of thionicotinamide riboside and converted it alongside with carbanicotinamide riboside and benzamide riboside to the respective NAD+ analogues by using the enzyme combination NRK1 and NMNAT1. The adaptability of this chemoenzymatic approach, previously demonstrated only for S‐NAD+, allowed the fast and scalable (∼5 mg) construction of all three NAD+ analogues, including c‐NAD+ and BAD. Thus, we reveal that minimal structural variation in the furanose ring as well as modification of the nicotinamide part does not interfere with the activity of the NRK1/NMNAT1 enzymes. We then tested the three compounds on their inhibitory capacity towards pathogenic Legionella enzyme SdeC. S‐NAD+ and BAD, showed micromolar inhibition of the Legionella enzyme, with an affinity for the enzyme in the same order of magnitude as the native substrate NAD+. The carba analogue only minimally inhibited the ART activity of SdeC, indicating that the substitution of the oxygen atom in the furanose ring with methylene was not tolerated, probably due to its rigidity enhancing effect on the five‐membered ring. When comparing the IC50 values of S‐NAD+ (27.9 μM) and BAD (38.9 μM) with NAD+ (27.7 μM), the S‐NAD+ seems to be most comparable to the native substrate. To the best of our knowledge these are the first NAD+‐mimics that inhibit a member of the Legionella SidE family. Although the compounds tested here will most likely not be specific for the Legionella SidE family, as they also inhibit other NAD+‐consuming enzymes, they do form a starting point for obtaining selective inhibitors. A recent report shows that relatively minor modifications at the 3′‐OH of a modified NAD+ analogue greatly enhances selectivity towards specific enzymes.24 Based on the combined insight that both the thioribofuranose and benzamide scaffold in NAD+ mimics inhibits SdeC activity, and that 3'‐OH modification can tune specificity of ADPr transferases, potential selective inhibitors might be developed in the near future giving rise to a new class of antibiotic candidates against Legionella infection.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Life lessons

Reminiscing about his younger self: “I mean I can’t very well just 86 [in American slang, to “86” is to eject, remove, or discard someone or something, J.R.N.] this guy from my life. On the other hand, if through some as yet undeveloped technology I were to run into him today, how comfortable would I feel about lending him money, or for that matter even stepping down the street to have a beer and talk over old times?” ― Thomas Pynchon, Slow Learner

life_lessons

<!>Review<!><!>Review<!><!>Review<!><!>Review<!><!>Review

<p>This article is part of the Thematic Series "Supramolecular chemistry at the interface of biology, materials and medicine".</p><!><p>I was raised in Syracuse, New York (USA), and went to a series of state schools of varying quality. Only the faces of the bad teachers stick with me, but I remember the names, too, of a few of the good ones – Karen Curry (High School Biology), Michelle Grosnick (Earth Science). Following my parents' divorce I moved to Gainesville, Florida when I was 16, where I enrolled in the International Baccalaureate program of Eastside High School. I had been drawn to chemistry for a few years by then. My chemistry teacher, Susan Zoltewicz (wife of Professor John Zoltewicsz, University of Florida), recognized my interest, and very kindly arranged for me to do a weekly afterschool apprenticeship with a lab technician named Charlie, whom I helped to set up all manner of chemical demonstrations and experiments at the University of Florida.</p><p>I went on to study at Williams College in Massachusetts. I knew chemistry was going to be my major subject, but my interests were broad, leading me to think that a liberal arts education might be a good fit. I remember greatly enjoying courses on art history and 20th century German history, along with a tutorial-style course on heterocyclic chemistry by J. Hodge Markgraf. Two summers stand out in my memory, when I carried out internships at Nanoptics, a company in Gainesville that makes optical fiber and various devices that incorporate it. The company was small enough at the time for me to enjoy considerable interaction with its founder and CEO, Jim Walker, who had been in charge of internally-initiated physics experiments at Fermilab. I had several challenging and very engaging jobs to do, including putting a disassembled fiber-spinning machine back together, and programming a microcontroller to run a stepper motor. I'm pretty sure that Jim's good word got me in to Berkeley for Ph.D. studies, despite mediocre results for my senior project (with Lee Park at Williams) and during a summer internship at SRI in Menlo Park, California.</p><p>I arrived at Berkeley with the impression of having gotten in by the skin of my teeth – that I would have to work twice as hard as anyone else just to scrape through. The first year of the program was a trial by fire, involving coursework – an excellent Physical Organic Chemistry course taught by Bob Bergman stood out – teaching a laboratory section, choosing a supervisor, and racing to get work done for the crucial first-year report. I remember waking up at 4 am during my first year, deeply concerned that I couldn't get a reaction to work consistently, and so heading in to lab to have another go. Although this was an incredibly poor idea from a safety perspective, the underlying attitude served me well. I loved the environment at Berkeley – the place was fizzing with intellectual energy, manifested both as world-class scholarship and as Hunter S. Thompson-style craziness.</p><p>Under T. Don Tilley's supervision, my Ph.D. work involved the development of zirconocene-mediated macrocyclization reactions to make a series of new structures under thermodynamic control, two examples of which are shown in Scheme 1 [1–2].</p><!><p>Zirconocene-coupled dimeric (right) and trimeric (left) macrocycles [1–2].</p><!><p>This Ph.D. work gave me a taste for organic chemistry involving transition metals, and an interest in structures that form under conditions of thermodynamic equilibration.</p><p>Towards the end of my Ph.D. work, Jean-Marie Lehn gave a talk at Berkeley, a highlight of which was some of Bernie Hasenknopf's latest work on circular helicates [3–4]. I remember being greatly impressed by the intricacy and beauty of these assemblies, and struck by the relative simplicity of their precursors. I spent considerable time over the following weeks digging through the literature, which involved quite a bit more physical activity in those pre-digitization days, in pulling great volumes down from high shelves, reading the work, photocopying the most interesting papers, and tracing back through the references for other papers, to gain context and depth. I came away convinced that was the kind of chemistry that I wanted to do for a postdoc.</p><p>So I wrote to Jean-Marie, and ultimately was offered a place in his labs in Strasbourg. I asked two Berkeley alumni who had recently finished postdocs there about their experiences, and was advised quite strongly against going! It's too difficult to get work done there, they said, and the French don't like Americans. A bit of back-and-forth led me to conclude that I might make it through OK if I polished up my high-school French, and tried to tone down some of the American cultural characteristics that seemed least compatible with the French worldview – the 'in your face' attitude that can sometimes compel action in the US, I reckoned, would likely backfire in France.</p><p>After a slow start, I ended up getting some good results in a project related to some new dynamic-covalent grid complexes [5]. This work didn't come to fruition before I went onto the academic job market, however! I thus had few US interviews, and only one offer from Case Western by the end of my time at Strasbourg. I had also applied for a 'maître-assistant' position at the University of Geneva in Switzerland, thinking of it mostly as practice for the 'real' US interviews. The interview went well; I was promised full scientific independence and the opportunity to supervise two Ph.D. students. In the end I chose Geneva because everything I needed was there, the teaching load was relatively light, and it seemed that I might stand a better chance of recruiting good students there. The senior members of the Department and School also impressed me; it seemed that advice and mentoring would be available for the asking. These first impressions held up well with time.</p><p>The group's first two Ph.D. students were hired from the University of Strasbourg, where there was no shortage of very well qualified M.Sc. students in search of opportunity. I interviewed several and extended offers to the two best, David Schultz and Marie Hutin, who did not let me down. Their intelligence and hard work laid the foundations of the group's work to the present day. Just after concluding the interviews, I was called to Berne, where a senior chemistry professor interviewed me on the Swiss National Science Foundation proposal that I had submitted. I did not make out so well as Marie and David, being informed that the work that I proposed was much too ambitious – it would require the mastery of techniques and concepts to which I had never been exposed. "Write a new proposal," came the advice, "convince us that you can get good work done quickly with limited means."</p><p>Although this rejection was devastating, time has told that it was the best advice that I could have gotten at that point in my career, delivered in a context that I could not ignore. My senior colleagues at Geneva came through with bridge funding so that I could still give Marie and David their promised places – a kindness for which I remain grateful – and the next proposal I wrote obtained modest funding.</p><p>This proposal, and the research programme that followed, involved the use of old chemistry – Daryle Busch's metal-templated imine-bond forming reaction [6] – in new ways. We called it subcomponent self-assembly to emphasize the use of simple precursors to build complex products. The preparations and crystal structures of three of these products are shown in Figure 1.</p><!><p>Complex structures prepared from simple subcomponents: a) CuI4 helicate [7]; b) CuI4 grid [8]; c) FeII4 tetrahedral cage [9–10].</p><!><p>Each of these products was prepared simply by mixing the precursors shown in water, and each represents a different way to arrange four metal ions in space, going from a one-dimensional linear array (Figure 1a) [7] to a two-dimensional grid (Figure 1b) [8] to a three-dimensional tetrahedron (Figure 1c) [9], which turned out to be capable of binding white phosphorus (P4) and rendering it air-stable [10]!</p><p>The work went well at Geneva – we were publishing in good journals, and I managed to win a Swiss National Science Foundation Assistant Professorship, which would have allowed me to modestly expand the group. My senior colleagues praised my accomplishments, but advised caution: I was not on a tenure track, and no future at Geneva could be guaranteed.</p><p>I thus set about looking for new opportunities, and I applied for an open lectureship at Cambridge. I was delighted to get the job, although it meant leaving behind the funding I had just won in Switzerland.</p><p>I hadn't anticipated the psychological gulf between the relatively rosy Swiss funding situation and the harsh headwinds of UK academia – much less research funding to go round meant for much sharper competition! Proposal ideas that had been funded with great scores in Switzerland were mercilessly skewered when submitted to the Engineering and Physical Sciences Research Council (EPSRC), our primary UK science funder. Each failed proposal hurt badly, but I made common cause with others – banding together with Richard Layfield and Paul Lusby to protest our first grants' rejection to the head of EPSRC, for example. And I slowly got a sense of what a good grant proposal looked like by UK standards, and how to write one. We kept going in lab, seeking to generate new results of the kind that could underpin a successful UK grant proposal.</p><p>The story of P4 encapsulation [10] garnered substantial positive attention, and an invited Nature Q&A piece on systems chemistry [11] probably helped, too. Ultimate success in attracting funding from the European Research Council (ERC) and EPSRC was the sweeter for the many failures that had preceded it. It has also been a delight to see others making use of subcomponent self-assembly to solve new puzzles, citing our development of the technique [12–25].</p><p>Over the past few years, we have developed a series of new functional structures. A leitmotiv of my group's work is the integration of a new class of hollow container molecules, invented by my group, into complex and dynamically-responsive systems and materials. Three of these containers are shown below: Figure 2a depicts an FeII8L6 cubic cage with walls constructed from porphyrins that binds guests such as fullerenes and coronene [26]; Figure 2b shows a CoII10L15 pentagonal prism that embeds five anions, such as PF6− (shown) or ClO4− in its walls, and a sixth – usually chloride – in its center [27]; Figure 2c illustrates a FeII12L12 pseudo-icosahedron with mer stereochemistry to its FeII centers, shown encapsulating B12F122− [28].</p><!><p>a) A cubic cage [26]; b) a pentagonal prism [27]; c) a pseudo-icosahedron [28].</p><!><p>We also have a flourishing line of enquiry into conjugated polymers that are held together by metal-ion templation – the structure shown in Figure 1a was a key precursor to this work. In collaboration with Richard Friend in the Physics Department at Cambridge, we have built these polymers into devices that emit white light [29], or that show blue-shifted emission at higher voltages [30], intriguingly.</p><p>Key questions that my group and I hope to address over the next few years include:</p><!><p>How can we design a system of chemical assemblies to work together in a network, to accomplish a function collectively?</p><p>Given that increasingly fine-grained control over self-assembled structure is being achieved, how can we design a self-assembling process with a target function in mind, such as light emission or the catalytic transformation of a substrate?</p><!><p>Both of these questions are predicated upon the idea of shifting intellectual effort away from designing and synthesizing complex molecules, and towards understanding and controlling the processes and systems of self-assembly.</p>

PubMed Open Access

Hepatitis C Virus NS3/4A Protease Inhibitors Incorporating Flexible P2 Quinoxalines Target Drug Resistant Viral Variants

A substrate envelope-guided design strategy is reported for improving the resistance profile of HCV NS3/4A protease inhibitors. Analogues of 5172-mcP1P3 were designed by incorporating diverse quinoxalines at the P2 position that predominantly interact with the invariant catalytic triad of the protease. Exploration of structure-activity relationships showed that inhibitors with small hydrophobic substituents at the 3-position of P2 quinoxaline maintain better potency against drug resistant variants, likely due to reduced interactions with residues in the S2 subsite. In contrast, inhibitors with larger groups at this position were highly susceptible to mutations at Arg155, Ala156 and Asp168. Excitingly, several inhibitors exhibited exceptional potency profiles with EC50 values \xe2\x89\xa4 5 nM against major drug resistant HCV variants. These findings support that inhibitors designed to interact with evolutionarily constrained regions of the protease, while avoiding interactions with residues not essential for substrate recognition, are less likely to be susceptible to drug resistance.

hepatitis_c_virus_ns3/4a_protease_inhibitors_incorporating_flexible_p2_quinoxalines_target_drug_resi

INTRODUCTION<!>CHEMISTRY<!>RESULTS AND DISCUSSION<!>Modifications of P1\xe2\x80\xb2 and P4 Capping Groups<!>SAR Exploration of P2 Quinoxaline<!>Effects of P2 Substituent Size and Flexibility<!>CONCLUSIONS<!>General<!>1-(tert-Butyl) 2-methyl (2S,4R)-4-((3-ethyl-7-methoxyquinoxalin-2-yl)oxy)pyrrolidine-1,2-dicarboxylate (9a)<!>1-(tert-Butyl) 2-methyl (2S,4R)-4-((7-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)oxy)pyrrolidine-1,2-dicarboxylate (9d)<!>Methyl (2S,4R)-1-((S)-2-((tert-butoxycarbonyl)amino)non-8-enoyl)-4-((3-ethyl-7-methoxyquinoxalin-2-yl)oxy)pyrrolidine-2-carboxylate (12a)<!>Methyl (2S,4R)-1-((S)-2-((tert-butoxycarbonyl)amino)non-8-enoyl)-4-((7-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)oxy)pyrrolidine-2-carboxylate (12d)<!>tert-Butyl ((S)-1-((2S,4R)-2-(((1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)-4-((3-ethyl-7-methoxyquinoxalin-2-yl)oxy)pyrrolidin-1-yl)-1-oxonon-8-en-2-yl)carbamate (16a)<!>tert-Butyl ((S)-1-((2S,4R)-4-((3-ethyl-7-methoxyquinoxalin-2-yl)oxy)-2-(((1R,2S)-1-(((1-methylcyclopropyl)sulfonyl)carbamoyl)-2-vinylcyclopropyl)carbamoyl)pyrrolidin-1-yl)-1-oxonon-8-en-2-yl)carbamate (17a)<!>tert-Butyl ((2R,6S,13aS,14aR,16aS,Z)-14a-((cyclopropylsulfonyl)carbamoyl)-2-((3-ethyl-7-methoxyquinoxalin-2-yl)oxy)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[ e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate (2)<!>tert-Butyl ((2R,6S,13aS,14aR,16aS,Z)-2-((3-ethyl-7-methoxyquinoxalin-2-yl)oxy)-14a-(((1-methylcyclopropyl)sulfonyl)carbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate (19a)<!>Cyclopentyl ((2R,6S,13aS,14aR,16aS,Z)-14a-((cyclopropylsulfonyl)carbamoyl)-2-((3-ethyl-7-methoxyquinoxalin-2-yl)oxy)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[ e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate (22a)<!>Cyclopentyl ((2R,6S,13aS,14aR,16aS,Z)-2-((3-ethyl-7-methoxyquinoxalin-2-yl)oxy)-14a-(((1-methylcyclopropyl)sulfonyl)carbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate (23a)<!>Ethyl (1R,2S)-1-((2S,4R)-1-((S)-2-((tert-butoxycarbonyl)amino)non-8-enoyl)-4-((7-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)oxy)pyrrolidine-2-carboxamido)-2-vinylcyclopropane-1-carboxylate (25d)<!>Ethyl (2R,6S,13aS,14aR,16aS,Z)-6-((tert-butoxycarbonyl)amino)-2-((7-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)oxy)-5,16-dioxo-1,2,3,6,7,8,9,10,11,13a,14,15,16,16atetradecahydrocyclopropa[ e]pyrrolo[1,2-a][1,4]diazacyclopentadecine-14a(5H)-carboxylate (26d)<!>tert-Butyl ((2R,6S,13aS,14aR,16aS,Z)-14a-((cyclopropylsulfonyl)carbamoyl)-2-((7-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)oxy)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate (18d)<!>tert-Butyl ((2R,6S,13aS,14aR,16aS,Z)-2-((7-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)oxy)-14a-(((1-methylcyclopropyl)sulfonyl)carbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate (19d)<!>Cyclopentyl ((2R,6S,13aS,14aR,16aS,Z)-14a-((cyclopropylsulfonyl)carbamoyl)-2-((7-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)oxy)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate (22d)<!>Cyclopentyl ((2R,6S,13aS,14aR,16aS,Z)-2-((7-methoxy-3-(trifluoromethyl)quinoxalin-2-yl)oxy)-14a-(((1-methylcyclopropyl)sulfonyl)carbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-6-yl)carbamate (23d)<!>Enzyme Inhibition Assays<!>Cell-Based Drug Susceptibility Assays<!>Crystallization and structure determination<!>Molecular Modeling

<p>Hepatitis C virus (HCV) infects over 130 million people globally and is the leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma.1 HCV is known as a "silent killer" as a majority of affected patients remain unaware of their infection, and over time the acute infection progresses to chronic liver disease.2 The rate of cirrhosis is estimated to increase from 16% to 32% by the year 2020 due to the high number of untreated patients.3 Thus, there is an urgent need to ensure that patients infected with HCV receive proper treatment. However, HCV infection is difficult to treat, as the virus is genetically diverse with six known genotypes (genotype 1–6), each of which is further sub-divided into numerous subtypes.4 Genotype 1 (GT1) and genotype 3 (GT3) are the most prevalent accounting for 46% and 30% of global infections, respectively.4,5 Therapeutic regimen and viral response are largely genotype dependent with most treatments being efficacious only against GT1.6</p><p>The recent advent of direct-acting antivirals (DAAs) targeting essential viral proteins NS3/4A, NS5A, and NS5B has remarkably improved therapeutic options and treatment outcomes for HCV infected patients.6,7 Four new all-oral combination treatments have been approved by the US FDA: (1) sofosbuvir/ledipasvir,8 (2) ombitasvir/paritaprevir/ritonavir/dasabuvir,9 (3) elbasvir/grazoprevir,10 and (4) sofosbuvir/velpatasvir.11 The DAA-based therapies are highly effective against GT1 with sustained virological response (SVR) rates greater than 90%.6,7 However, most of the FDA approved treatments and those in clinical development are not efficacious against other genotypes, especially GT3.7 Moreover, except for sofosbuvir, all current DAAs are susceptible to drug resistance.12 Therefore, more robust DAAs need to be developed with higher barriers to drug resistance and a broad spectrum of activity against different HCV genotypes.</p><p>The HCV NS3/4A protease is a major therapeutic target for the development of pan-genotypic HCV inhibitors.13,14 The NS3/4A protease inhibitors (PIs) telaprevir15 and boceprevir16 were the first DAAs approved for the treatment of HCV GT1 infection in combination therapy with pegylated-interferon and ribavirin.17,18 Three recently approved PIs, simeprevir,19 paritaprevir20 and grazoprevir,21 (Figure 1) are integral components of various combination therapies currently used as the standard of care for HCV infected patients.6,7,14 Two other NS3/4A PIs, asunaprevir22 and vaniprevir,23 have been approved in Japan. In addition, a number of next generation NS3/4A PIs are in clinical development including glecaprevir24 and voxilaprevir25 (Figure 1).</p><p>All NS3/4A PIs share a common peptidomimetic scaffold and are either linear or macrocyclic; the macrocycle is located either between P1–P3 or P2–P4 moieties.14 In addition, these inhibitors contain a large heterocyclic moiety attached to the P2 proline, which significantly improves inhibitor potency against wild-type (WT) NS3/4A protease.26,27 However, all NS3/4A PIs are susceptible to drug resistance, especially due to single site mutations at protease residues Arg155, Ala156 and Asp168.28,29 Notably, D168A/V mutations are present in nearly all patients who fail treatment with PIs.12 Moreover, natural polymorphisms at this position are responsible for significantly reduced inhibitor potency against GT3.30 We previously determined the molecular mechanisms of drug resistance due to single site mutations by solving high-resolution crystal structures of PIs bound to WT and mutant proteases.31–34 These crystal structures revealed that the large heterocyclic P2 moieties of PIs bind outside the substrate binding region, defined as the substrate envelope, and make extensive interactions with residues Arg155, Ala156 and Asp168.32,33 The inhibitor P2 moiety induces an extended S2 subsite by forcing the Arg155 side chain to rotate nearly 180° relative to its conformation in substrate complexes.31 This altered Arg155 conformation is stabilized by electrostatic interactions with Asp168, providing additional hydrophobic surface that is critical for efficient inhibitor binding. Disruption of electrostatic interactions between Arg155 and Asp168 due to mutations underlies drug resistance against NS3/4A PIs.31–33,35 Moreover, we have shown that structural differences at the P2 moiety largely determine the resistance profile of these inhibitors.36</p><p>Grazoprevir (MK-5172, 1), one of the most potent HCV NS3/4A PIs, has a unique binding mode where the P2 quinoxaline moiety interacts with residues of the catalytic triad, avoiding direct interactions with Arg155 and Asp168 (Figure 2).32 As a result, 1 has an excellent potency profile across different genotypes and relatively low susceptibility to drug resistance due to mutations at Arg155 and Asp168.21,37 However, 1 is highly susceptible to mutations at Ala156, mainly due to steric clashes of larger side chains with the P2–P4 macrocycle. We have shown that the P1–P3 macrocyclic analogue 5172-mcP1P3 (2) avoids this steric clash while still maintaining the unique binding mode of 1 (Figure 2).38 Compound 2, though slightly less potent than 1 against WT HCV, has an excellent potency profile with EC50 values in the single digit nanomolar range against drug resistant variants including A156T. Similar to 1, the P2 quinoxaline moiety in 2 stacks against the catalytic residues His57 and Asp81 and largely avoids direct interactions with residues around the S2 subsite.38 But unlike 1, the flexible P2 quinoxaline moiety in 2 better accommodates mutations at Ala156, resulting in an overall improved resistance profile.36,38 Thus, the P1–P3 macrocyclic analogue 2 is a promising lead compound for structure-activity relationship (SAR) studies to further improve potency against drug resistant variants and other genotypes.</p><p>The substrate envelope model provides a rational approach to design NS3/4A PIs with improved resistance profiles by exploiting interactions with the protease residues essential for function and avoiding direct contacts with residues that can mutate to confer drug resistance.39–41 Another approach applied to design PIs with improved resistant profiles involves incorporation of conformational flexibility that can allow the inhibitor to adapt to structural changes in the protease active site due to mutations.35 Here, we describe a structure-guided strategy that combines these two approaches and, together with our understanding of the mechanisms of drug resistance, led to the design of NS3/4A PIs with exceptional potency profiles against major drug resistant HCV variants. Based on the lead compound 2, a series of analogues were designed and synthesized with diverse substituents at the 3-position of P2 quinoxaline moiety. Investigation of SARs identified P2 quinoxaline derivatives that predominantly interact with the invariant catalytic triad and avoid contacts with the S2 subsite residues. The results indicate that combining the substrate envelope model with optimal conformational flexibility provides a general strategy for the rational design of NS3/4A PIs with improved resistance profiles.</p><!><p>The NS3/4A PIs with diverse P2 quinoxaline moieties were synthesized using the reaction sequence outlined in Scheme 1. A Cs2CO3-mediated SN2 reaction of 3-substituted quinoxalin-2-ones 8a-g with the activated proline derivative 3 provided the key P2 intermediates 9a-g in 75–90% yield. The alternate SNAr reaction between activated quinoxaline derivatives and Boc-protected hydroxy-proline resulted in lower yields, and purification of the resulting P2 acid products was significantly more challenging. The 3-substituted 7-methoxy-quinoxalin-2-ones 8ab and 8d-e were prepared by condensation reactions of 4-methoxybenzene-1,2-diamine with the corresponding ethyl glyoxylates (see Supporting Information for details). The 3-chloro-7-methoxyquinoxalin-2-one 8c was prepared according to a reported method.21</p><p>The P1–P3 macrocyclic PIs were assembled from the P2 intermediates 9a-g using a sequence of deprotection and peptide coupling steps followed by the ring-closing metathesis (RCM) reaction (Method A). Removal of the Boc group in 9a-g using 4 N HCl provided the amine salts 10a-g, which were coupled with the amino acid 11 in the presence of HATU and DIEA to yield the P2–P3 ester intermediates 12a-g. Hydrolysis of these esters with LiOH and reaction of the resulting carboxylic acids 13a-g with the P1–P1′ fragments 1442 and 1543 under HATU/DIEA coupling conditions provided the bis-olefin intermediates 16a-g and 17a-e. Finally, cyclization of the bis-olefin intermediates was accomplished using a highly efficient RCM catalyst Zhan 1B, and provided the inhibitors 18b-g and 19a-e in 45–80% yield. Interestingly, RCM reactions of bis-olefins 17a-e bearing the 1-methylcyclopropylsulfonamide provided higher yield than the corresponding cyclopropylsulfonamide analogues 16a-g. Finally, removal of the Boc group and reaction of the resulting amine salts 20a-g and 21a-e with the N-(cyclopentyloxycarbonyloxy)-succinimide in the presence of DIEA afforded the inhibitors 22a-g and 23a-e with the N-terminal cyclopentyl P4 moiety.</p><p>A subset of inhibitors was synthesized using an alternate reaction sequence that allowed late-stage modification at both the P1′ and P4 positions as illustrated in Scheme 2 (Method B). The P2–P3 acid intermediates 13a-d were reacted with the commercially available amine salt 24 under HATU/DIEA coupling conditions to afford the bis-olefin intermediates 25a-d. RCM reaction in the presence of Zhan 1B catalyst provided the macrocyclic intermediates 26a-d in 75–90% yield, which was better than that obtained in the presence of the P1′ acylsulfonamide. The P1–P3 macrocyclic core intermediates 26a-d can be modified in either direction after removing the C- or N-terminal protecting groups. Thus, hydrolysis of the C-terminal ethyl ester with LiOH provided the acids 27a-d, which were then reacted with either cyclopropylsulfonamide 28 or 1-methylcyclopropylsulfonamide 29 in the presence of CDI and DBU to afford the final inhibitors 18b-d and 19a-d. The N-terminal tert-butyl capping group was replaced with the cyclopentyl moiety as described earlier to provide the target inhibitors 22a-d and 23a-d.</p><!><p>Our goal was to develop a structure-guided design strategy to improve the resistance profile of HCV NS3/4A PIs based on the substrate envelope model.39,40 Compound 2 is an attractive scaffold for exploring this strategy due to the unique structural features: (1) the P2 quinoxaline moiety that predominantly interacts with the highly conserved catalytic residues Asp81 and His57 and (2) the conformational flexibility that allows the inhibitor to efficiently accommodate structural changes in the S2 subsite due to resistance mutations. Despite these promising features, optimization of substituents at the P2 quinoxaline and the N-terminal capping may be key to discovering analogues with improved potency and resistance profiles. Therefore, efforts were focused on exploration of SARs at the P2 quinoxaline moiety in 2, specifically substituting the ethyl group at the 3-position that directly interacts with protease S2 subsite residues Arg155 and Ala156. The SAR strategy was based on insights from detailed structural analysis of 1 and 2 bound to wild-type NS3/4A protease and drug resistant variants.32,38 Based on these insights, we hypothesized that small hydrophobic groups at the 3-position of the quinoxaline would be preferred for retaining inhibitor potency against drug resistant variants, but larger groups that make extensive interactions with Arg155, Ala156 and Asp168 would result in inhibitors highly susceptible to mutations at these positions. To test this hypothesis, a series of inhibitors with diverse substituents at the 3-position of P2 quinoxaline were designed and synthesized.</p><p>The potency and resistance profiles of NS3/4A PIs were assessed using biochemical and replicon assays. The enzyme inhibition constants (Ki) were determined against wild-type GT1a NS3/4A protease, drug-resistant variant D168A, and GT3a NS3/4A protease (Table 1). The cellular antiviral potencies (EC50) were determined using replicon-based antiviral assays against wild-type HCV and drug-resistant variants R155K, A156T, D168A, and D168V (Table 2). Grazoprevir (GZR, 1) was used as a control in all assays. The observed antiviral potencies are generally higher than protease inhibitory potencies, likely because biochemical assays were performed using the protease domain alone rather then the full-length NS3/4A.</p><p>Compound 1 showed sub-nanomolar inhibitory potency against WT NS3/4A protease and maintained nanomolar activity against drug resistant variant D168A and GT3a protease. Similarly, in replicon assays 1 exhibited an excellent potency profile with sub-nanomolar activity against WT HCV (EC50 = 0.14 nM) and low nanomolar activity against drug resistant variants R155K, D168A, and D168V. However, in line with previous reports,36 1 was highly susceptible to the A156T mutation (EC50 = 238 nM), losing over 1000-fold potency against this variant. Compared to 1, the P1–P3 macrocyclic analogue 2 exhibited lower inhibitory potency against WT protease and the D168A variant. Also, the inhibitory activity of 2 against the GT3a protease was considerably lower than that of 1. However, as we have previously shown,36 2 displayed a superior potency profile in replicon assays with sub-nanomolar activity against WT HCV (EC50 = 0.33 nM) and maintained single digit nanomolar potency against all drug-resistant variants tested. Notably, unlike 1, compound 2 maintained low nanomolar potency against the A156T variant (EC50 = 9.65 nM). Thus, with an improved resistance profile compared to 1, the P1–P3 macrocyclic analogue 2 is an attractive lead compound for further optimization.</p><!><p>Initial SAR efforts to optimize lead compound 2 focused on exploring changes at the P1′ position and N-terminal capping group. Recent SAR studies of diverse NS3/4A PIs indicate that replacement of the cyclopropylsulfonamide moiety at the P1′ position with a slightly more hydrophobic 1-methylcyclopropylsulfonamide improves inhibitor potency in replicon assays.43,44 Moreover, changes at the P4 position have been shown to significantly affect inhibitor potency against drug resistant variants, as these groups bind in close proximity to the pivotal drug resistance site Asp168.45 For carbamate-linked P4 capping groups, generally bulky hydrophobic moieties are preferred but the size of the group appears to be dependent on the heterocyclic moiety present at the P2 position.35</p><p>First, replacing the cyclopropylsulfonamide at the P1′ position in 2 with 1-methylcyclopropylsulfonamide provided the analogue 19a. Compared to the parent compound 2, 19a showed slightly better Ki values against WT, D168A and GT3a proteases and exhibited similar or slightly better antiviral potency against WT and drug resistant variants. Next, the tert-butyl P4 capping group in both 2 and 19a was replaced with a larger cyclopentyl moiety, resulting in analogues 22a and 23a. Unlike the change at the P1′ position, the P4 cyclopentyl modification provided mixed results. Compound 22a afforded a 2-fold increase in potency than 2 in biochemical assays against WT protease and a slight improvement against the D168A variant, but was equipotent to 2 against GT3a protease. Similarly, in replicon assays 22a exhibited 2-fold enhanced potency against WT HCV and D168A variant, but showed similar potency as 2 against the R155K and D168V variants. Compound 23a, with a 1-methylcyclopropylsulfonamide moiety at the P1′ position and a cyclopentyl group at the P4 position, exhibited potency profile largely similar to 22a. Surprisingly, a slight loss in potency was observed against the A156T variant for compounds with a cyclopentyl versus tert-butyl capping group. Overall, these minor modifications at the P1′ and N-terminal capping regions of inhibitor 2 were tolerated and provided analogues with improved potency profiles.</p><!><p>Next, SARs at the P2 quinoxaline in compound 2 were explored. Efforts mainly focused on replacing the 3-position ethyl group with diverse functional groups with respect to size and electronic properties. Replacement of the ethyl group in 2 with a smaller methyl group provided analogue 18b. As expected, reducing the size of the hydrophobic group at this position resulted in improved potency profile. Compound 18b showed slightly enhanced potency against drug resistant variants in biochemical and antiviral assays, with a notable ~2-fold improvement against the D168V variant (EC50 = 3.17 nM). The introduction of 1-methylcyclopropylsulfonamide moiety at the P1′ position afforded inhibitor 19b with protease inhibitory activity comparable to the parent compound 18b. However, similar to the 3-ethylquinoxaline analogue (19a), compound 19b demonstrated significant gain in potency in replicon assays. In fact, compared to 2, 19b exhibited 2- to 6-fold enhancement in potency against drug resistant variants R155K (EC50 = 0.80 nM), A156T (EC50 = 1.57 nM), D168A (EC50 = 2.37 nM), and D168V (EC50 = 1.6 nM). Replacement of the tert-butyl P4 capping in 18b and 19b with a cyclopentyl group, providing 22b and 23b, resulted in an increase in WT and D168A inhibitory activity as well as 2- to 3-fold increase in WT replicon potency. Unlike the corresponding 3-ethylquinoxaline analogues (22a and 23a), the 3-methyquinoxaline compounds 22b and 23b maintained the excellent potency profile observed for the corresponding tert-butyl analogues. Remarkably, with the exception of 18b (A156T EC50 = 5.95 nM), all compounds in the 3-methylquinoxaline series display exceptional potency profiles with EC50 values below 5 nM against WT and clinically relevant drug resistant variants.</p><p>To gain insights into the excellent potency profile observed for the 3-methyquinoxaline series, we determined the X-ray crystal structure of inhibitor 19b in complex with the WT NS3/4A protease at a resolution of 1.8 Å (Figure 3, Table S3, PDB code: 5VOJ). The WT-19b complex structure was compared with the previously reported structures of compound 2 in complex with WT protease and the A156T variant (PDB codes: 5EPN and 5EPY).38 The two WT structures overlap very well, with only minor differences in the S1 and S2 subsites because of modifications in the inhibitor structure. In the WT-2 crystal structure, the 3-ethyl group at the P2 quinoxaline makes hydrophobic interactions with the hydrocarbon portion of the Arg155 side-chain, while the methylene portion of this group interacts with the side-chain of Ala156. The smaller methyl group at this position in the WT-19b structure maintains hydrophobic interactions with Ala156, while minimizing chances of steric clash with a larger side-chain, such as in A156T.</p><p>Unlike inhibitor 1, the P1–P3 macrocyclic analogues retain potency against the A156T variant. Comparison of the WT-2 and A156T-2 (PDB code: 5EPY) structures shows subtle changes in inhibitor interactions with the mutant protease.38 In the A156T-2 structure the P2 quinoxaline largely maintains interactions with the catalytic residues, but the ethyl group is shifted away from Arg155 side chain toward A156T. Moreover, to accommodate a larger Thr side-chain, the Asp168 side chain adopts another conformation, moving away from Arg155. These changes underlie reduced inhibitor potency against the A156T variant, but unlike 1, inhibitor 2 is able to better accommodate these changes due to a flexible P2 moiety. The 3-methylquinoxaline analogues are more potent against the A156T variant than the corresponding 3-ethylquinoxaline compounds likely due the reduced interactions of the smaller methyl group with the Thr side-chain. Replacing the methyl group with hydrogen at the 3-position of quinoxaline would further reduce interactions with the S2 subsite residues, but could result in a highly flexible P2 moiety, likely destabilizing interactions with the catalytic residues. Thus, a small hydrophobic group at the 3-position of P2 quinoxaline is preferred to maintain favorable interactions with Ala156 and avoid steric clashes with the Thr side-chain in the A156T variant.</p><p>The improved potency profile of 3-methyquinoxaline compounds led to exploration of bioisosteric replacements of the 3-methyl group with varied size and electronic properties. To that end, analogues 18c and 19c bearing the 3-chloro-7-methoxyquinoxaline at the P2 position were prepared. The protease inhibitory potency profiles of these compounds were excellent and showed improvement against WT, D168A and GT3a over 2. These potency gains were not only maintained in replicon assays but were more significant, with the only exception of A156T variant. Both compounds 18c and 19c were more active than the corresponding 3-methylquinoxaline analogues (18b and 19b) with EC50 values less than 1 nM against WT, R155K and D168V and less than 2 nM against the D168A variant, but experienced about 3- to 6-fold reduction in potency against the A156T variant. However, potency losses against the A156T variant were largely reversed when the P4 tert-butyl group in 18c and 19c was replaced with a larger cyclopentyl moiety to afford 22c and 23c. Similar to the 3-methylquinoxaline compounds, the 3-chloroquinoxaline analogues displayed exceptional potency profiles with EC50 values of less than 5 nM against all drug resistant variants including A156T. These results clearly demonstrate that small hydrophobic groups with weak electron-donating properties at the 3-position of P2 quinoxaline can be replaced with weak electron-withdrawing groups without affecting the overall potency profile.</p><p>Next, a larger and strongly electron-withdrawing trifluoromethyl moiety was explored at the 3-position of P2 quinoxaline, leading to inhibitors 18d and 19d. This modification, however, resulted in significant potency losses in both biochemical and replicon assays. Compound 18d was about 2- to 4-fold less active than 2 against WT protease and variants. Analogue 19d with the 1-methylcyclopropylsulfonamide moiety at the P1′ position showed similar trends when compared to the corresponding 19a. In line with biochemical data, both 18d and 19d suffered 2- to 6-fold decrease in replicon potency against WT and drug resistant variants, though 19d maintained relatively good potency profile. In contrast to the results in previous series, the introduction of the larger cyclopentyl P4 capping group, as in 22d and 23d, was detrimental to replicon potency, particularly against the A156T variant. Moreover, compounds in the 3-(trifluoromethyl)quinoxaline series were among the least active against the GT3a protease in biochemical assays. These results indicate that strong electron-withdrawing groups at the 3-position of the P2 quinoxaline may be detrimental to potency. However, a recent SAR study indicates that PIs incorporating the 3-(trifluoromethyl)quinoxaline can be optimized with modifications at the 7-position of quinoxaline in combination with changes at the P1–P3 macrocycle and P4 capping group.46</p><p>In the absence of a co-crystal structure, the lower inhibitory potencies of compounds in the 3-(trifluoromethyl)quinoxaline series against WT protease could not be explained by molecular modeling, which suggested a similar binding conformation of the P2 quinoxaline in 18d as observed for 2 (Figure 4A). Perhaps there are repulsive interactions between trifluoromethyl moiety and the side chain of Asp168, and/or the strong electron-withdrawing effect may weaken the overall interactions of the P2 quinoxaline with the catalytic residues. Potency losses against resistant variants may also result from the larger size of the trifluoromethyl moiety, which is comparable to that of an ethyl group, though both have different topographical shapes.47</p><p>To isolate the effects of larger size versus electronic properties on potency, inhibitors 18e and 19e with the larger isopropyl group at the 3-position of the P2 quinoxaline were designed and evaluated. These compounds showed WT protease inhibitory activity similar to the corresponding 3-ethylquinoxaline analogues (2 and 19a), but experienced 2- to 4-fold reduced activity against the D168A variant. A broader reduction in potency was observed for both 18e and 19e in replicon assays against WT and drug resistant variants. The cyclopentyl P4 group in analogues 22e and 23e slightly improved biochemical and replicon potency against WT and D168A variants, but was largely unfavorable to replicon potency against R155K and A156T variants. This trend is broadly similar to the results observed with the 3-(trifluoromethyl)quinoxaline series, indicating that both electronic properties and size of the group at the 3-position of P2 quinoxaline are important for maintaining potency against drug resistant variants. Modeling indicated that compared to 2 the P2 quinoxaline moiety in 18e has to shift away from the catalytic triad in order to accommodate the larger isopropyl group thereby weakening critical stacking interactions with His57 (Figure 4B). Overall, SAR data from the 3-isopropyl- and 3-(trifluoromethyl)-quinoxaline series supports the hypothesis that large substituents at the 3-position of P2 quinoxaline have detrimental effect on inhibitor potency against drug resistant variants.</p><p>These findings were further reinforced by the results obtained for the 3-(thiophen-2-yl)quinoxaline analogues 18f and 22f. Based on molecular modeling, the large thiophene moiety in these compounds was expected to make extensive interactions with the residues Arg155 and Ala156, resulting in improved potency against WT protease. However, mutations at these positions as well as at Asp168 would cause significant potency losses, as these residues are crucial for efficient inhibitor binding. As expected, compound 18f (a previously reported NS3/4A PI incorrectly labeled as ABT-450)19,48 showed a 3-fold enhancement in WT biochemical potency but was dramatically less active against the D168A variant, losing over 1800-fold potency. Similarly, in replicon assays analogue 18f showed considerably reduced potency against all drug resistant variants with losses ranging from 20- to 250-fold compared to WT (Table S1 and S2). The cyclopentyl P4 analogue 22f also experienced large potency losses against the variants, albeit to a lesser extent than 18f. Thus inhibitors with large groups at the 3-position of P2 quinoxaline are highly susceptible to mutations at residues Arg155, Ala156 and Asp168, leading to poor resistance profiles.</p><p>The X-ray crystal structure of inhibitor 18f in complex with WT NS3/4A protease was determined at a resolution of 1.9 Å, providing insights into the binding modes of P2 quinoxaline with a larger thiophene substituent at the 3-position (Figure 5, Table S3, PDB code: 5VP9). Comparison of the WT-18f and WT-2 crystal structures showed significant differences in the interactions of quinoxaline moieties with the catalytic triad and S2 subsite residues. As predicted, the quinoxaline moiety in WT-18f structure is shifted toward the active site to accommodate the larger thiophene substituent. The thiophene ring makes extensive interactions with residues in the S2 subsite, including cation-π interactions with Arg155, likely contributing to the improved potency against the WT protease. As this Arg155 conformation is stabilized by electrostatic interactions with Asp168, mutations at either residue would disrupt inhibitor binding by loss of direct interactions as well as indirect structural effects. In addition, the A156T mutation would result in a steric clash with the thiophene ring, as reflected in the antiviral data for this variant. These biochemical and structural findings are in line with previous studies that show inhibitors that are dependent on extensive interactions with the S2 subsite residues for potency are highly susceptible to mutations at residues Arg155, Ala156 and Asp168.</p><p>As compounds 18f and 22f lacked the C-7 substituent at the P2 quinoxaline, analogues 18g and 22g were prepared to investigate the effect of this group on inhibitor potency. Compared to 2, analogue 18g experienced about 2-fold decrease in biochemical potency and only minor loss in replicon potency against WT and drug resistant variants. The P4 cyclopentyl analogue 22g resulted in about 2-fold reduced potency compared to the corresponding compound 22a. Thus removal of the C-7 methoxy group has minimal effect on inhibitor potency. The slightly reduced potency of 18g and 22g is likely due to the reduced hydrophobic interactions with the aromatic ring of Tyr56 and the methylene portion of His57 of the catalytic triad. In contrast, the observed potency losses against resistant variants for the 3-(thiophen-2-yl)quinoxaline compounds most likely result from loss of interactions of the 2-thiophene moiety with the S2 subsite residues of the protease.</p><!><p>Taken together, our SAR results indicate that resistance profiles of compound 2 and analogues are strongly influenced by the substituent at the 3-position of P2 quinoxaline and N-terminal capping group. While all PIs showed reduced potency against drug resistance variants in both enzyme inhibition and replicon assays, fold potency losses varied significantly depending on the substituents at the 3-position of P2 quinoxaline. To evaluate susceptibility to the clinically important D168A variant, to which all current NS3/4A PIs are susceptible, potencies were normalized to WT for PIs with the same P4 capping groups (Figure 6). Fold changes in Ki against the D168A protease variant for PIs with the same P1′ and P4 capping groups largely trended with the size of the substituent at the 3-position of P2 quinoxaline, with the exception of trifluoromethyl compounds. Losses in potency were significantly higher for compounds with the larger 2-thiophene substituent at the P2 quinoxaline. These results strongly support using the substrate envelope model to reduce direct inhibitor interactions in the S2 subsite, thereby reducing inhibitor susceptibility to drug resistance.</p><p>As we and others have shown,31–33,35 the reduced potencies of NS3/4A PIs against drug resistant variants R155K, A156T, and D168A/V mainly result from disruption of the electrostatic interactions between Arg155 and Asp168. Compared to 1, compound 2 and most analogues incorporating flexible P2 quinoxaline showed lower fold-changes in potency against these variants (Table S1 and S2). In these P1–P3 macrocyclic PIs the conformational flexibility of the P2 allows this moiety to adapt to the structural changes caused by mutations at Arg155, Ala156 and Asp168, resulting in better resistance profiles. Potency losses were higher for compound 1 because constraint imposed by the macrocycle does not allow the P2 moiety to adapt to the structural changes resulting from these mutations. Compound 1 and similar P2–P4 macrocyclic PIs, such as voxilaprevir and glecaprevir, are likely to be more susceptible to mutations that cause significant structural changes in the protease active site. However, the P1–P3 macrocyclic compounds reported here, as well as those reported in patent literature that incorporate similar flexible P2 quinoxaline moieties,49 are likely to be more effective against clinically relevant drug resistant variants. More broadly, combining the substrate envelope model with optimal conformational flexibility provides a rational approach to design NS3/4A PIs with improved resistance profiles.</p><!><p>Drug resistance is a major problem across all DAA classes targeting HCV. As new therapies are developed the potential for drug resistance must be minimized at the outset of inhibitor design. The substrate envelope model provides a rational approach to design robust NS3/4A PIs with improved resistance profiles. Our SAR findings support the hypothesis that reducing PI interactions with residues in the S2 subsite leads to inhibitors with exceptional potency and resistance profiles. Specifically, the P1–P3 macrocyclic inhibitors incorporating flexible P2 quinoxaline moieties bearing small hydrophobic groups at the 3-position maintain excellent potency in both enzymatic and antiviral assays against drug resistant variants. While these inhibitors protrude from the substrate envelope, they leverage interactions with the essential catalytic triad residues and avoid direct contacts with residues that can mutate to confer resistance. Moreover, conformational flexibility at the P2 moiety is essential to efficiently accommodate structural changes due to mutations in the S2 pocket in order to avoid resistance. These insights provide strategies for iterative rounds of inhibitor design with the paradigm that designing inhibitors with flexible P2 quinoxalines, leveraging evolutionarily constrained areas in the protease active site and expanding into the substrate envelope may provide inhibitors that are robust against drug resistant variants.</p><!><p>All reactions were performed in oven-dried round bottomed or modified Schlenk flasks fitted with rubber septa under argon atmosphere, unless otherwise noted. All reagents and solvents, including anhydrous solvents, were purchased from commercial sources and used as received. Flash column chromatography was performed using silica gel (230–400 mesh, EMD Millipore). Thin-layer chromatography (TLC) was performed using silica gel (60 F-254) coated aluminum plates (EMD Millipore), and spots were visualized by exposure to ultraviolet light (UV), exposure to iodine adsorbed on silica gel, and/or exposure to an acidic solution of p-anisaldehyde (anisaldehyde) followed by brief heating. 1H NMR and 13C NMR spectra were acquired on Varian Mercury 400 MHz and Bruker Avance III HD 500 MHz NMR instruments. Chemical shifts are reported in ppm (δ scale) with the residual solvent signal used as reference and coupling constant (J) values are reported in hertz (Hz). Data are presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, m = multiplet, br s = broad singlet), coupling constant in Hz, and integration. High-resolution mass spectra (HRMS) were recorded on a Thermo Scientific Orbitrap Velos Pro mass spectrometer coupled with a Thermo Scientific Accela 1250 UPLC and an autosampler using electrospray ionization (ESI) in the positive mode. The purity of final compounds was determined by analytical HPLC and was found to be ≥95% pure. HPLC was performed on a Waters Alliance 2690 system equipped with a Waters 2996 photodiode array detector and an autosampler under the following conditions: column, Phenomenex Luna-2 RP-C18 (5 μm, 4.6 × 250 mm, 120 Å, Torrance, CA); solvent A, H2O containing 0.1% formic acid (FA), solvent B, CH3CN containing 0.1% FA; gradient, 50% B to 100% B over 15 min followed by 100% B over 5 min; injection volume, 10 μL; flow rate, 1 mL/min. Retention times and purity data for each target compound are provided in the experimental section.</p><!><p>A solution of 3-ethyl-7-methoxyquinoxalin-2-one 8a (3.0 g, 14.7 mmol) in anhydrous NMP (45 mL) was treated with Cs2CO3 (7.40 g, 22.7 mmol). After stirring the reaction mixture at room temperature for 15 min, proline derivative 3 (6.20 g, 13.3 mmol) was added in one portion. The reaction mixture was heated to 55 °C, stirred for 4 h, and then another portion of proline derivative 3 (0.48 g, 1.0 mmol) was added. The resulting reaction mixture was stirred at 55 °C for an additional 2 h, cooled to room temperature, quenched with aqueous 1 N HCl solution (150 mL), and extracted with EtOAc (300 mL). The organic fraction was washed successively with saturated aqueous NaHCO3 and NaCl (150 mL each), dried (Na2SO4), filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography using 15–30% EtOAc/hexanes as the eluent to provide 9a (5.50 g, 87%) as a white foamy solid. 1H NMR (400 MHz, CDCl3) (mixture of rotamers, major rotamer) δ 7.85 (d, J = 9.0 Hz, 1 H), 7.18 (m, 1H), 7.11 (d, J = 2.8 Hz, 1 H), 5.73 (br s, 1 H), 4.47 (t, J = 8.0 Hz, 1 H), 3.98–3.86 (m, 5 H), 3.78 (s, 3 H), 2.92 (q, J = 7.2 Hz, 2 H), 2.68–2.60 (m, 1 H), 2.43–2.36 (m, 1 H), 1.43 (s, 9 H), 1.31 (t, J = 7.2 Hz, 3 H) ppm; 13C NMR (100 MHz, CDCl3) δ 173.56, 160.59, 155.38, 154.02, 148.95, 141.26, 134.12, 129.07, 119.02, 106.11, 80.76, 73.81, 58.43, 55.93, 52.73, 52.40, 36.88, 28.47, 26.68, 11.97 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C22H30N3O6, 432.2129; found 432.2135.</p><!><p>The same procedure was used as described above for compound 9a. 7-methoxy-3-(trifluoromethyl)quinoxalin-2(1H)-one 8d (4.76 g, 19.5 mmol) in NMP (65 mL) was treated with Cs2CO3 (9.80 g, 30.0 mmol) and proline derivative 3 (9.0 g, 19.3 mmol) to provide 9d (6.50 g, 71%) as a pale yellow foamy solid. 1H NMR (500 MHz, CDCl3) (mixture of rotamers, major rotamer) δ 7.77 (d, J = 9.0 Hz, 1 H), 7.48–7.43 (m, 2 H), 5.76 (br s, 1 H), 4.50 (t, J = 8.0 Hz, 1 H), 3.97–3.91 (m, 5 H), 3.78 (s, 3 H), 2.69–2.64 (m, 1 H), 2.41–2.34 (m, 1 H), 1.42 (s, 9 H) ppm; 13C NMR (125 MHz, CDCl3) δ 173.43, 159.58, 153.98, 152.11, 138.39, 137.22, 127.99, 125.73, 120.70 (q, J = 273.4 Hz), 107.64, 80.69, 74.62, 58.27, 56.02, 52.32, 52.11, 36.70, 28.34 ppm; 19F NMR (470 MHz, CDCl3); −67.73 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C21H25F3N3O6, 472.1690; found 472.1689.</p><!><p>A solution of ester 9a (4.80 g, 11.1 mmol) in anhydrous CH2Cl2 (30 mL) was treated with a solution of 4 N HCl in 1,4-dioxane (30 mL). After stirring the reaction mixture at room temperature for 3 h, solvents were evaporated under reduced pressure, and the residue was dried under high vacuum. The pale yellow solid was triturated with diethyl ether (3 × 30 mL) and dried under high vacuum to yield the amine salt 10a (4.0 g, 98%) as an off-white powder.</p><p>A mixture of amine salt 10a (4.0 g, 10.9 mmol) and (S)-2-((tert-butoxycarbonyl)amino)non-8-enoic acid 11 (3.0 g, 11.1 mmol) in anhydrous DMF (60 mL) was treated with DIEA (7.30 mL, 44.2 mmol) and HATU (6.35 g, 16.7 mmol). The resulting reaction mixture was stirred at room temperature for 4 h, then diluted with EtOAc (400 mL), and washed successively with aqueous 0.5 N HCl, saturated aqueous NaHCO3, and saturated aqueous NaCl (250 mL each). The organic portion was dried (Na2SO4), filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography using 20–30% EtOAc/hexanes as the eluent to provide 12a (5.50 g, 86%) as a white foamy solid. 1H NMR (400 MHz, CDCl3) (mixture of rotamers, major rotamer) δ 7.86 (d, J = 8.8 Hz, 1 H), 7.20 (dd, J = 9.2, 2.8 Hz, 1 H), 7.12 (d, J = 2.8 Hz, 1 H), 5.87–5.75 (m, 2 H), 5.20 (d, J = 8.4 Hz, 1 H), 5.02–4.92 (m, 2 H), 4.73 (t, J = 8.4 Hz, 1 H), 4.38 (q, J = 7.2 Hz, 1 H), 4.17 (d, J = 12.0 Hz, 1 H), 4.06 (dd, J = 11.6, 4.4 Hz, 1 H), 3.94 (s, 3 H), 3.78 (s, 3 H), 2.90 (q, J = 7.6 Hz, 2 H), 2.69–2.64 (m, 1 H), 2.41–2.34 (m, 1 H), 2.05 (app q, J = 6.8 Hz, 2 H), 1.82–1.74 (m, 1 H), 1.63–1.56 (m, 1 H), 1.45–1.25 (m, 18 H) ppm; 13C NMR (100 MHz, CDCl3) δ 172.34, 171.96, 160.61, 155.61, 155.13, 148.95, 141.08, 139.18, 129.22, 119.08, 114.58, 106.14, 79.84, 74.48, 58.19, 55.91, 52.88, 52.67, 52.05, 35.16, 33.88, 32.88, 29.14, 28.96, 28.46, 26.52, 24.92, 11.86 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C31H45N4O7 585.3283; found 585.3286.</p><!><p>The same procedure was used as described above for compound 12a. Compound 9d (6.0 g, 12.7 mmol) was treated with 4 N HCl (40 mL) to afford amine salt 10d (5.10 g, 12.5 mmol), which was coupled with acid 11 (3.80 g, 14.0 mmol) using DIEA (9.25 mL, 56.0 mmol) and HATU (7.60 g, 20.0 mmol) to provide 12d (6.40 g, 81%) as a pale yellow foamy solid. 1H NMR (500 MHz, CDCl3) (mixture of rotamers, major rotamer) δ 7.78 (d, J = 9.0 Hz, 1 H), 7.48 (dd, J = 9.0, 2.5 Hz, 1 H), 7.44 (d, J = 2.5 Hz, 1 H), 5.86 (br s, 1 H), 5.84–5.78 (m, 1 H), 5.18 (d, J = 9.0 Hz, 1 H), 5.01–4.92 (m, 2 H), 4.75 (t, J = 8.0 Hz, 1 H), 4.35 (q, J = 7.5 Hz, 1 H), 4.19 (d, J = 12.0 Hz, 1 H), 4.08 (dd, J = 11.5, 4.5 Hz, 1 H), 3.95 (s, 3 H), 3.78 (s, 3 H), 2.70–2.65 (m, 1 H), 2.41–2.35 (m, 1 H), 2.04 (app q, J = 7.0 Hz, 2 H), 1.80–1.75 (m, 1 H), 1.60–1.54 (m, 1 H), 1.45–1.28 (m, 15 H) ppm; 13C NMR (125 MHz, CDCl3) δ 172.10, 171.60, 159.99, 155.37, 151.78, 138.98, 138.41, 136.93, 134.40 (q, J = 36.3 Hz), 127.85, 125.66, 120.53 (q, J = 273.4 Hz), 114.33, 107.54, 79.58, 75.05, 57.83, 55.91, 52.44, 52.33, 51.75, 34.77, 33.65, 32.70, 28.91, 28.73, 28.18, 24.70 ppm; 19F NMR (470 MHz, CDCl3); −67.73 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C30H40F3N4O7, 625.2844; found 625.2844.</p><!><p>A solution of ester 12a (5.86 g, 10.0 mmol) in THF-H2O mixture (1:1, 140 mL) was treated with LiOH.H2O (1.40 g, 33.4 mmol). The resulting reaction mixture was stirred at room temperature for 24 h. The reaction mixture was cooled to ~5 °C, acidified to a pH of 2.0 by slow addition of aqueous 0.25 N HCl (~ 200 mL), and extracted with EtOAc (2 × 400 mL). The organic portions were washed separately with saturated aqueous NaCl (200 ml), dried (Na2SO4), filtered, and evaporated under reduced pressure. The gummy residue was dissolved in CHCl3 (50 mL), concentrated under reduced pressure, and the residue was dried under high vacuum overnight to yield the acid 13a (5.70 g, 100%) as a white foamy solid.</p><p>A mixture of acid 13a (2.10 g, 3.7 mmol) and amine salt 1442 (1.20 g, 4.5 mmol) in anhydrous DMF (35 mL) was treated with DIEA (2.43 mL, 14.7 mmol) and HATU (2.1 g, 5.5 mmol). The resulting reaction mixture was stirred at room temperature for 2.5 h, then diluted with EtOAc (300 mL) and washed successively with aqueous 0.5 N HCl, saturated aqueous NaHCO3, and saturated aqueous NaCl (200 mL each). The organic portion was dried (Na2SO4), filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography using 50– 70% EtOAc/hexanes as the eluent to provide the bis-olefin compound 16a (2.50 g, 86%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.24 (s, 1 H), 7.84 (d, J = 8.8 Hz, 1 H), 7.18 (dd, J = 8.8, 2.4 Hz, 1 H), 7.13 (d, J = 2.8 Hz, 1 H), 7.04 (s, 1 H), 5.91 (br s, 1 H), 5.85–5.73 (m, 2 H), 5.32 (d, J = 8.4 Hz, 1 H), 5.27 (d, J = 17.2 Hz, 1 H), 5.14 (d, J = 11.2 Hz, 1 H), 5.01–4.90 (m, 2 H), 4.47 (t, J = 7.6 Hz, 1 H), 4.38–4.33 (m, 1 H), 4.20 (d, J = 11.6 Hz, 1 H), 4.02 (dd, J = 11.2, 4.0 Hz, 1 H), 3.94 (s, 3 H), 2.96–2.84 (m, 3 H), 2.56–2.51 (m, 2 H), 2.11 (q, J = 8.8 Hz, 1 H), 2.05–1.99 (m, 3 H), 1.74–1.54 (m, 2 H), 1.47–1.10 (m, 21 H), 1.08–1.03 (m, 2 H) ppm; 13C NMR (100 MHz, CDCl3) δ 174.09, 172.58, 168.69, 160.54, 155.89, 154.99, 148.88, 140.95, 139.07, 134.69, 132.71, 129.45, 119.02, 118.77, 114.67, 106.13, 80.0, 74.66, 60.61, 55.91, 53.42, 52.62, 41.83, 35.46, 34.47, 33.89, 32.40, 31.39, 28.98, 28.89, 28.47, 26.68, 25.47, 23.83, 11.85, 6.68, 6.26 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C39H55N6O9S, 783.3746; found 783.3734.</p><!><p>The same procedure was used as described above for compound 16a. Acid 13a (1.50 g, 2.6 mmol) was coupled with amine salt 1543 (0.90 g, 3.2 mmol) using DIEA (1.75 mL, 10.6 mmol) and HATU (1.50 g, 3.9 mmol) to provide the bisolefin compound 17a (1.75 g, 84%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.02 (s, 1 H), 7.84 (d, J = 9.2 Hz, 1 H), 7.19 (dd, J = 8.8, 2.8 Hz, 1 H), 7.13 (d, J = 2.8 Hz, 1 H), 7.06 (s, 1 H), 5.90 (br s, 1 H), 5.83–5.73 (m, 2 H), 5.37 (d, J = 9.2 Hz, 1 H), 5.27 (d, J = 17.2 Hz, 1 H), 5.14 (d, J = 10.8 Hz, 1 H), 5.98 (dd, J = 17.2, 1.6 Hz, 1 H), 4.92 (dd, J = 10.4, 1.2 Hz, 1 H), 4.48 (t, J = 8.0 Hz, 1 H), 4.39–4.33 (m, 1 H), 4.16 (d, J = 12.0 Hz, 1 H), 4.02 (dd, J = 11.6, 4.0 Hz, 1 H), 3.94 (s, 3 H), 2.89 (q, J = 7.6 Hz, 2 H), 2.57–2.50 (m, 2 H), 2.12 (q, J = 8.8 Hz, 1 H), 2.05– 1.99 (m, 3 H), 1.75–1.58 (m, 4 H), 1.49 (s, 3 H), 1.45–1.18 (m, 19 H), 0.93–0.79 (m, 2 H) ppm; 13C NMR (100 MHz, CDCl3) δ 173.79, 172.41, 167.51, 160.31, 155.71, 154.76, 148.63, 140.73, 138.85, 134.41, 132.60, 129.18, 118.80, 118.54, 114.41, 105.89, 79.74, 74.42, 60.36, 55.68, 53.17, 52.43, 41.71, 36.56, 35.23, 34.22, 33.64, 32.19, 28.70, 28.67, 28.25, 26.43, 25.35, 23.49, 18.37, 14.27, 13.29, 11.64 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C40H57N6O9S, 797.3902; found 797.3887.</p><!><p>A degassed solution of bis-olefin 16a (1.40 g, 1.8 mmol) in 1,2-DCE (300 mL) was heated to 50 °C under argon, then Zhan 1b catalyst (0.150 g, 0.20 mmol) was added in two portions over 10 min. The resulting reaction mixture was heated to 70 °C and stirred for 6 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by flash chromatography using 50–80% EtOAc/hexanes as the eluent to yield the P1–P3 macrocyclic product 2 (0.72 g, 53%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 10.28 (s, 1 H), 7.84 (d, J = 9.6 Hz, 1 H), 7.20–7.15 (m, 2 H), 6.91 (s, 1 H), 5.90 (br s, 1 H), 5.69 (q, J = 8.8 Hz, 1 H), 5.14 (d, J = 7.6 Hz, 1 H), 4.96 (t, J = 9.2 Hz, 1 H), 4.59 (t, J = 7.6 Hz, 1 H), 4.49 (d, J = 11.6 Hz, 1 H), 4.30–4.24 (m, 1 H), 4.02 (dd, J = 10.8, 4.0 Hz, 1 H), 3.94 (s, 3 H), 2.94– 2.85 (m, 3 H), 2.70–2.51 (m, 3 H), 2.31 (q, J = 8.8 Hz, 1 H), 1.93–1.64 (m, 2 H), 1.60–1.05 (m, 24 H), 0.95–0.89 (m, 1 H) ppm; 13C NMR (100 MHz, CDCl3) δ 177.15, 173.28, 168.02, 160.29, 155.00, 154.90, 148.66, 140.88, 136.31, 134.28, 128.90, 124.47, 118.82, 105.91, 79.84, 74.68, 59.45, 55.72, 53.08, 51.92, 44.57, 34.65, 32.81, 31.01, 29.70, 28.14, 27.11, 27.16, 26.31, 26.06, 22.16, 20.92, 11.56, 6.67, 6.12 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C37H51N6O9S, 755.3433; found 755.3410. Anal. HPLC: tR 14.23 min, purity 97%.</p><!><p>The same procedure was used as described above for compound 2. Bis-olefin 17a (1.45 g, 1.8 mmol) was treated with Zhan 1b catalyst (0.150 g, 0.20 mmol) in 1,2-DCE (300 mL) to afford the P1–P3 macrocyclic product 19a (1.0 g, 71%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 10.16 (s, 1 H), 7.82 (d, J = 10.0 Hz, 1 H), 7.18–7.15 (m, 2 H), 6.94 (s, 1 H), 5.90 (br s, 1 H), 5.69 (q, J = 9.2 Hz, 1 H), 5.16 (d, J = 8.0 Hz, 1 H), 4.99 (t, J = 9.2 Hz, 1 H), 4.59 (t, J = 8.0 Hz, 1 H), 4.49 (d, J = 11.6 Hz, 1 H), 4.30–4.25 (m, 1 H), 4.04 (dd, J = 11.6, 4.0 Hz, 1 H), 3.94 (s, 3 H), 2.87 (q, J = 7.6 Hz, 2 H), 2.70–2.51 (m, 3 H), 2.33 (q, J = 8.0 Hz, 1 H), 1.92–1.68 (m, 4 H), 1.60–1.15 (m, 24 H), 0.85–0.78 (m, 2 H) ppm; 13C NMR (100 MHz, CDCl3) δ 177.19, 173.24, 167.0, 160.23, 154.99, 154.88, 148.73, 140.84, 136.26, 134.25, 129.03, 124.89, 118.72, 105.92, 79.84, 74.67, 59.48, 55.72, 53.11, 51.92, 44.71, 36.43, 34.68, 32.80, 29.62, 28.14, 27.09, 26.38, 26.12, 22.19, 20.93, 18.17, 14.51, 12.50, 11.54 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C38H53N6O9S, 769.3589; found 769.3561. Anal. HPLC: tR 15.01 min, purity 99%.</p><!><p>Compound 2 (0.40 g, 0.53 mmol) was treated with a solution of 4 N HCl in 1,4-dioxane (10 mL). The reaction mixture was stirred at room temperature for 3 h, then concentrated under reduced pressure, and the residue was dried under high vacuum. The off-white solid was triturated with diethyl ether (3 × 10 mL) and dried under high vacuum to yield the amine salt 20a (0.37 g, 100%) as a white powder.</p><p>A solution of the above amine salt 20a (0.37 g, 0.53 mmol) in anhydrous CH3CN (15 mL) was treated with DIEA (0.35 mL, 2.1 mmol) and N-(cyclopentyloxycarbonyloxy)-succinimide (0.15 g, 0.66 mmol). The reaction mixture was stirred at room temperature for 36 h, then concentrated under reduced pressure and dried under high vacuum. The residue was purified by flash chromatography using 50–90% EtOAc/hexanes as the eluent to provide the target compound 22a (0.32 g, 79%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.29 (s, 1 H), 7.83 (d, J = 9.6 Hz, 1 H), 7.21–7.16 (m, 2 H), 6.94 (s, 1 H), 5.93 (br s, 1 H), 5.70 (q, J = 8.8 Hz, 1 H), 5.26 (d, J = 8.0 Hz, 1 H), 4.96 (t, J = 8.4 Hz, 1 H), 4.86–4.82 (m, 1 H), 4.60 (t, J = 7.6 Hz, 1 H), 4.45 (d, J = 11.2 Hz, 1 H), 4.34–4.28 (m, 1 H), 4.03 (dd, J = 11.2, 4.0 Hz, 1 H), 3.94 (s, 3 H), 2.93–2.85 (m, 3 H), 2.70–2.48 (m, 3 H), 2.30 (q, J = 8.8 Hz, 1 H), 1.93–1.23 (m, 23 H), 1.15–1.06 (m, 2 H), 0.96–0.88 (m, 1 H) ppm; 13C NMR (100 MHz, CDCl3) δ 177.18, 173.03, 168.04, 160.28, 155.65, 154.93, 148.78, 140.90, 136.27, 134.20, 128.92, 124.46, 118.80, 105.92, 77.87, 74.55, 59.47, 55.72, 53.01, 52.17, 44.54, 34.58, 32.72, 32.63, 32.59, 31.01, 29.70, 27.14, 27.05, 26.40, 26.05, 23.56, 22.16, 20.90, 11.61, 6.67, 6.12 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C38H51N6O9S, 767.3433; found 767.3408. Anal. HPLC: tR 14.50 min, purity 98%.</p><!><p>The same procedure was used as described above for compound 22a. Compound 19a (0.40 g, 0.52 mmol) was treated with 4 N HCl in 1,4-dioxane (10 mL) to yield the amine salt 21a, which was treated with DIEA (0.35 mL, 2.1 mmol) and N-(cyclopentyloxycarbonyloxy)-succinimide (0.15 g, 0.66 mmol) to provide the target compound 23a (0.30 g, 74%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.17 (s, 1 H), 7.81 (d, J = 9.6 Hz, 1 H), 7.21–7.16 (m, 2 H), 6.93 (s, 1 H), 5.92 (br s, 1 H), 5.70 (q, J = 9.2 Hz, 1 H), 5.26 (d, J = 7.6 Hz, 1 H), 4.99 (t, J = 9.6 Hz, 1 H), 4.86–4.81 (m, 1 H), 4.59 (t, J = 7.6 Hz, 1 H), 4.45 (d, J = 11.2 Hz, 1 H), 4.34–4.28 (m, 1 H), 4.04 (dd, J = 11.6, 4.0 Hz, 1 H), 3.94 (s, 3 H), 2.87 (q, J = 7.2 Hz, 2 H), 2.70–2.48 (m, 3 H), 2.32 (q, J = 8.8 Hz, 1 H), 1.92–1.23 (m, 27 H), 0.85–0.78 (m, 2 H) ppm; 13C NMR (100 MHz, CDCl3) δ 177.21, 172.99, 166.98, 160.22, 155.63, 154.90, 148.84, 140.85, 136.22, 134.36, 129.05, 124.88, 118.70, 105.93, 77.86, 74.54, 59.51, 55.71, 53.05, 52.16, 44.70, 36.43, 34.61, 32.72, 32.64, 32.58, 29.63, 27.13, 27.06, 26.47, 26.12, 23.56, 22.18, 20.94, 18.17, 14.49, 12.50, 11.59 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C39H53N6O9S, 781.3589; found 781.3561. Anal. HPLC: tR 15.25 min, purity 99%.</p><!><p>A solution of ester 12d (6.40 g, 10.25 mmol) in THF-H2O (1:1 mixture, 140 mL) was treated with LiOH.H2O (1.38 g, 32.0 mmol). The resulting reaction mixture was stirred at room temperature for 24 h, then cooled to ~5 °C, acidified to a pH of 2.0 by slow addition of aqueous 0.25 N HCl (~ 200 mL), and extracted with EtOAc (2 × 500 mL). The organic portions were washed separately with saturated aqueous NaCl (250 ml), dried (Na2SO4), filtered, and evaporated under reduced pressure. The gummy residue was dissolved in CHCl3 (50 mL), concentrated under reduced pressure, and the residue was dried under high vacuum to yield the acid 13d (6.12 g, 98%) as a pale yellow foamy solid.</p><p>A solution of acid 13d (6.12 g, 10.0 mmol) and amine salt 24 (2.50 g, 13.0 mmol) in anhydrous CH2Cl2 (100 mL) was treated with DIEA (9.10 mL, 55.0 mmol), HATU (5.30 g, 14.0 mmol) and DMAP (0.60 g, 4.9 mmol). The resulting reaction mixture was stirred at room temperature for 14 h, then diluted with EtOAc (500 mL), and washed successively with aqueous 1.0 N HCl, saturated aqueous NaHCO3, and saturated aqueous NaCl (250 mL each). The organic portion was dried (Na2SO4), filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography using 25–35% EtOAc/hexanes as the eluent to provide the bisolefin compound 25d (6.54 g, 87%) as a pale yellow foamy solid. 1H NMR (500 MHz, CDCl3) (mixture of rotamers, major rotamer) δ 7.78 (d, J = 9.2 Hz, 1 H), 7.53 (br s, 1 H), 7.47 (dd, J = 9.2, 2.8 Hz, 1 H), 7.43 (d, J = 2.4 Hz, 1 H), 5.88 (br s, 1 H), 5.81–5.70 (m, 2 H), 5.30 (dd, J = 16.8, 0.8 Hz, 1 H), 5.14–5.10 (m, 2 H), 5.01–4.89 (m, 2 H), 4.79 (dd, J = 14.0, 5.6 Hz, 1 H), 4.35–4.29 (m, 1 H), 4.21–4.08 (m, 3 H), 3.94 (s, 3 H), 2.90–2.82 (m, 1 H), 2.48–2.38 (m, 1 H), 2.16 (q, J = 9.0 Hz, 1 H), 2.04–1.98 (m, 2 H), 1.86 (dd, J = 8.0, 5.2 Hz, 1 H), 1.66–1.52 (m, 2 H), 1.46 (dd, J = 9.6, 5.6 Hz, 1 H), 1.43–1.21 (m, 19 H) ppm; 13C NMR (125 MHz, CDCl3) δ 173.02, 171.00, 169.87, 159.62, 155.52, 152.03, 138.92, 138.48, 137.16, 133.66, 128.02, 125.73, 120.72 (q, J = 273.6 Hz), 118.08, 114.52, 107.66, 79.98, 75.26, 61.40, 58.41, 56.02, 52.58, 52.43, 40.14, 33.89, 33.77, 32.76, 32.62, 28.97, 28.78, 28.31, 25.18, 23.11, 14.48 ppm; 19F NMR (470 MHz, CDCl3); −67.77 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C37H49F3N5O8, 748.3528; found 748.3514.</p><!><p>A degassed solution of bis-olefin 25d (1.50 g, 2.0 mmol) in 1,2-DCE (300 mL) was heated to 50 °C under argon, then Zhan 1b catalyst (0.150 g, 0.20 mmol) was added in two portions over 10 min. The resulting mixture was heated to 70 °C and stirred for 5 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by flash chromatography using 25–35% EtOAc/hexanes as the eluent to yield the P1–P3 macrocyclic product 26d (1.0 g, 70%) as an off-white foamy solid. 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 8.8 Hz, 1 H), 7.47 (dd, J = 8.8, 2.8 Hz, 1 H), 7.44 (d, J = 2.4 Hz, 1 H), 7.03 (br s, 1 H), 5.84–5.80 (m, 1 H), 5.56–5.49 (m, 1 H), 5.32–5.22 (m, 2 H), 4.92 (q, J = 4.4 Hz, 1 H), 4.49 (t, J = 7.6 Hz, 1 H), 4.24–4.05 (m, 4 H), 3.95 (s, 3 H), 3.05–2.99 (m, 1 H), 2.41–2.35 (m, 1 H), 2.24–2.14 (m, 3 H), 1.93–1.86 (m, 2 H), 1.66–1.60 (m, 1 H), 1.55 (dd, J = 96, 5.2 Hz, 1 H), 1.46–1.20 (m, 18 H) ppm; 13C NMR (100 MHz, CDCl3) δ 172.81, 171.95, 169.74, 159.69, 155.21, 152.20, 138.56, 137.25, 134.50, 128.08, 125.91, 125.84, 120.80 (q, J = 276 Hz), 107.73, 80.04, 75.42, 61.50, 58.08, 56.13, 52.21, 51.39, 41.36, 32.16, 31.77, 28.45, 28.10, 28.02, 26.37, 25.74, 23.70, 22.57, 14.72 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C35H45F3N5O8, 720.3215; found 720.3203.</p><!><p>A solution of ester 26d (1.0 g, 1.4 mmol) in THF-MeOH-H2O (1:1:1 mixture, 20 mL) was treated with LiOH.H2O (0.18 g, 4.2 mmol). The resulting reaction mixture was stirred at room temperature for 24 h, then cooled to ~5 °C, acidified to a pH of 2.0 by slow addition of aqueous 0.25 N HCl, and extracted with EtOAc (2 × 150 mL). The organic portions were washed separately with saturated aqueous NaCl (100 ml), dried (Na2SO4), filtered, and evaporated under reduced pressure. The gummy residue was dissolved in CHCl3 (10 mL), concentrated under reduced pressure, and the residue was dried under high vacuum to yield the acid 27d (0.95 g, 98%) as a pale yellow foamy solid.</p><p>A mixture of acid 27d (0.40 g, 0.58 mmol) and CDI (0.131 g, 0.81 mmol) in anhydrous THF (8 mL) was heated at reflux for 1.5 h. The solution was cooled to room temperature and slowly added to a solution of cyclopropanesulfonamide 28 (0.10 g, 0.82 mmol) in anhydrous THF (4 mL) followed by DBU (0.12 mL, 0.81 mmol). The resulting reaction mixture was stirred at room temperature for 24 h, then quenched with aqueous 0.5 N HCl to pH ~2. Solvents were partially evaporated under reduced pressure, and the residue was extracted with EtOAc (2 × 100 mL). The combined organic portions were washed with saturated aqueous NaCl (100 mL), dried (Na2SO4), filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography using 40–70% EtOAc/hexanes as the eluent to afford the title compound 18d (0.28 g, 60%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.28 (s, 1 H), 7.83 (d, J = 9.2 Hz, 1 H), 7.49 (dd, J = 8.8, 2.8 Hz, 1 H), 7.42 (d, J = 2.8 Hz, 1 H), 6.87 (s, 1 H), 5.92 (br s, 1 H), 5.70 (q, J = 8.8 Hz, 1 H), 5.13 (d, J = 7.6 Hz, 1 H), 4.97 (t, J = 8.4 Hz, 1 H), 4.62–4.56 (m, 2 H), 4.23–4.17 (m, 1 H), 4.01 (dd, J = 11.6, 3.2 Hz, 1 H), 3.94 (s, 3 H), 2.93–2.87 (m, 1 H), 2.68–2.50 (m, 3 H), 2.31 (q, J = 8.8 Hz, 1 H), 1.95–1.54 (m, 2 H), 1.53–1.02 (m, 21 H), 0.96–0.88 (m, 1 H) ppm; 13C NMR (100 MHz, CDCl3) δ 176.99, 173.31, 167.91, 159.45, 154.93, 151.76, 138.27, 136.99, 136.32, 134.56 (q, J = 36.2 Hz), 127.99, 125.57, 124.53, 120.8 (q, J = 274.0 Hz), 107.40, 79.76, 75.54, 59.44, 55.89, 52.72, 51.86, 44.65, 34.61, 32.82, 31.02, 29.61, 28.02, 27.04, 25.99, 22.21, 20.93, 6.67, 6.12 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C36H46F3N6O9S, 795.2994; found 795.2974. Anal. HPLC: tR 14.59 min, purity 100%.</p><!><p>The same procedure was used as described above for compound 18d. Acid 27d (0.43 g, 0.62 mmol) was treated with CDI (0.141 g, 0.87 mmol), 1-methylcyclopropanesulfonamide 29 (0.118 g, 0.87 mmol) and DBU (0.13 mL, 0.87 mmol) to afford the title compound 19d (0.34 g, 68%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.15 (s, 1 H), 7.83 (d, J = 9.2 Hz, 1 H), 7.48 (dd, J = 9.2, 2.8 Hz, 1 H), 7.42 (d, J = 2.8 Hz, 1 H), 6.90 (s, 1 H), 5.91 (s, 1 H), 5.70 (q, J = 9.2 Hz, 1 H), 5.14 (d, J = 7.6 Hz, 1 H), 5.00 (t, J = 9.2 Hz, 1 H), 4.62–4.55 (m, 2 H), 4.24–4.18 (m, 1 H), 4.02 (dd, J = 11.6, 3.6 Hz, 1 H), 3.94 (s, 3 H), 2.71–2.51 (m, 3 H), 2.33 (q, J = 8.4 Hz, 1 H), 1.93–1.75 (m, 4 H), 1.56–1.18 (m, 21 H), 0.85–0.78 (m, 2 H) ppm; 13C NMR (100 MHz, CDCl3) δ 177.30, 173.46, 167.15, 159.68, 155.16, 152.01, 138.50, 137.23, 136.50, 134.60 (q, J = 36.0 Hz), 128.23, 125.79, 125.19, 120.83 (d, J = 274.0 Hz), 107.65, 80.01, 75.79, 59.70, 56.12, 52.97, 52.08, 45.03, 36.65, 34.86, 33.06, 29.81, 28.26, 27.31, 27.24, 26.32, 22.47, 21.21, 18.42, 14.73, 12.77 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C37H48F3N6O9S, 809.3150; found 809.3129. Anal. HPLC: tR 15.23 min, purity 99%.</p><!><p>Compound 18d (0.40 g, 0.52 mmol) was treated with a solution of 4 N HCl in 1,4-dioxane (10 mL). The reaction mixture was stirred at room temperature for 3 h, concentrated under reduced pressure, and the residue was dried under high vacuum. The pale yellow solid was triturated with diethyl ether (3 × 10 mL) and dried under high vacuum to yield the amine salt 20d (0.37 g, 100%) as a white powder.</p><p>A solution of the above amine salt 20d (0.37 g, 0.52 mmol) in anhydrous CH3CN (15 mL) was treated with DIEA (0.35 mL, 2.1 mmol) and N-(cyclopentyloxycarbonyloxy)-succinimide (0.15 g, 0.66 mmol). The reaction mixture was stirred at room temperature for 24 h, then concentrated under reduced pressure and dried under high vacuum. The residue was purified by flash chromatography using 50–90% EtOAc/hexanes as the eluent to provide the target compound 22d (0.30 g, 74%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.27 (s, 1 H), 7.82 (d, J = 9.2 Hz, 1 H), 7.48 (dd, J = 9.2, 2.8 Hz, 1 H), 7.42 (d, J = 2.8 Hz, 1 H), 6.78 (s, 1 H), 5.95 (s, 1 H), 5.70 (q, J = 9.6 Hz, 1 H), 5.23 (d, J = 8.0 Hz, 1 H), 4.98 (t, J = 8.8 Hz, 1 H), 4.74–4.69 (m, 1 H), 4.60 (t, J = 7.6, 1 H), 4.54 (d, J = 11.6, 1 H), 4.25–4.19 (m, 1 H), 3.99 (dd, J = 11.6, 4.0 Hz, 1 H), 3.94 (s, 3 H), 2.94–2.88 (m, 1 H), 2.68–2.50 (m, 3 H), 2.31 (q, J = 8.8 Hz, 1 H), 1.94–1.24 (m, 21 H), 1.20–1.07 (m, 2 H), 0.96–0.89 (m, 1 H) ppm; 13C NMR (100 MHz, CDCl3) δ 177.33, 173.29, 168.27, 159.68, 155.87, 152.08, 138.56, 137.24, 136.47, 134.74 (q, J = 36.0 Hz), 128.24, 125.75, 124.79, 120.87 (d, J = 273.2 Hz), 107.62, 78.02, 75.70, 59.68, 56.11, 52.90, 52.35, 44.83, 34.71, 32.92, 32.81, 32.64, 31.26, 29.87, 27.27, 26.24, 23.81, 23.75, 22.49, 21.11, 6.89, 6.34 ppm; HRMS (ESI) m/z: [M + H]+ calcd for C37H46F3N6O9S, 807.2994; found 807.2976. Anal. HPLC: tR 14.98 min, purity 99%.</p><!><p>The same procedure was used as described above for compound 22d. Compound 19d (0.40 g, 0.52 mmol) was treated with 4 N HCl in 1,4-dioxane (10 mL) to yield the amine salt 21d, which was treated with DIEA (0.35 mL, 2.1 mmol) and N-(cyclopentyloxycarbonyloxy)-succinimide (0.15 g, 0.66 mmol) to provide the target compound 23d (0.30 g, 74%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.18 (s, 1 H), 7.83 (d, J = 9.6 Hz, 1 H), 7.48 (dd, J = 8.8, 2.8 Hz, 1 H), 7.41 (d, J = 2.8 Hz, 1 H), 6.94 (s, 1 H), 5.94 (s, 1 H), 5.70 (q, J = 8.8 Hz, 1 H), 5.28 (d, J = 7.6 Hz, 1 H), 5.00 (t, J = 8.8 Hz, 1 H), 4.74– 4.69 (m, 1 H), 4.60 (t, J = 7.6, 1 H), 4.54 (d, J = 12.0, 1 H), 4.25–4.19 (m, 1 H), 4.00 (dd, J = 11.6, 3.6 Hz, 1 H), 3.94 (s, 3 H), 2.68–2.50 (m, 3 H), 2.31 (q, J = 8.4 Hz, 1 H), 1.92–1.20 (m, 24 H), 0.85–0.78 (m, 2 H) ppm; 13C NMR (100 MHz, CDCl3) δ 177.33, 173.25, 167.13, 159.68, 155.84, 152.08, 138.56, 137.24, 136.44, 134.75 (q, J = 35.2 Hz), 128.25, 125.74, 125.21, 120.86 (d, J = 274.0 Hz), 107.62, 78.02, 75.71, 59.73, 56.12, 52.89, 52.34, 45.03, 36.65, 34.73, 32.93, 32.82, 32.64, 29.83, 27.26, 27.21, 26.29, 23.81, 23.75, 22.52, 21.23, 18.42, 14.73, 12.76 ppm. HRMS (ESI) m/z: [M + H]+ calcd for C38H48F3N6O9S, 821.3150; found 821.3133. Anal. HPLC: tR 15.65 min, purity 97%.</p><!><p>For each assay, 2 nM of NS3/4A protease (GT1a, D168A and GT3a) was pre-incubated at room temperature for 1 h with increasing concentration of inhibitors in assay buffer [50 mM Tris, 5% glycerol, 10 mM DTT, 0.6 mM LDAO, and 4% dimethyl sulfoxide]. Inhibition assays were performed in nonbinding surface 96-well black half-area plates (Corning) in a reaction volume of 60 μL. The proteolytic reaction was initiated by the injection of 5 μL of HCV NS3/4A protease substrate (AnaSpec), to a final concentration of 200 nM and kinetically monitored using a Perkin Elmer EnVision plate reader (excitation at 485 nm, emission at 530 nm). Three independent data sets were collected for each inhibitor with each protease construct. Each inhibitor titration included at least 12 inhibitor concentration points, which were globally fit to the Morrison equation to obtain the Ki value.</p><!><p>Mutations (R155K, A516T, D168A and D168V) were constructed by site-directed mutagenesis using a Con1 (genotype 1b) luciferase reporter replicon containing the H77 (genotype 1a) NS3 sequence.50 Replicon RNA of each protease variant was introduced into Huh7 cells by electroporation. Replication was then assessed in the presence of increasing concentrations of protease inhibitors by measuring luciferase activity (relative light units) 96 h after electroporation. The drug concentrations required to inhibit replicon replication by 50% (EC50) were calculated directly from the drug inhibition curves.</p><!><p>Protein expression and purification were carried out as previously described (see Supporting Information for details).32 The Ni-NTA purified WT1a protein was thawed, concentrated to 3 mg/mL, and loaded on a HiLoad Superdex75 16/60 column equilibrated with gel filtration buffer (25 mM MES, 500 mM NaCl, 10% glycerol, and 2 mM DTT, pH 6.5). The protease fractions were pooled and concentrated to 25 mg/mL with an Amicon Ultra-15 10 kDa filter unit (Millipore). The concentrated samples were incubated for 1 h with 3:1 molar excess of inhibitor. Diffraction-quality crystals were obtained overnight by mixing equal volumes of concentrated protein solution with precipitant solution (20–26% PEG-3350, 0.1 M sodium MES buffer, 4% ammonium sulfate, pH 6.5) at RT or 15 °C in 24-well VDX hanging drop trays. Crystals were harvested and data was collected at 100 K. Cryogenic conditions contained the precipitant solution supplemented with 15% glycerol or ethylene glycol.</p><p>Diffraction data were collected using an in-house Rigaku X-ray system with a Saturn 944 detector. All datasets were processed using HKL-3000.51 Structures were solved by molecular replacement using PHASER.52 The WT-2 complex structure (PDB code: 5EPN)38 was used as the starting structure for all structure solutions. Model building and refinement were performed using Coot53 and PHENIX,54 respectively. The final structures were evaluated with MolProbity55 prior to deposition in the PDB. To limit the possibility of model bias throughout the refinement process, 5% of the data were reserved for the free R-value calculation.56 Structure analysis, superposition and figure generation were done using PyMOL.57 X-ray data collection and crystallographic refinement statistics are presented in the Supporting Information (Table S3).</p><!><p>Molecular modeling was carried out using MacroModel (Schrödinger, LLC, New York, NY).58 Briefly, inhibitors were modeled into the active site of WT1a and A156T proteases using the WT-2 and A156T-2 co-complex structures (PDB code: 5EPN and 5EPY).38 Structures were prepared using the Protein Preparation tool in Maestro 11. 2D chemical structures were modified with the appropriate changes using the Build tool in Maestro. Once modeled, molecular energy minimizations were performed for each inhibitor–protease complex using the PRCG method with 2500 maximum iterations and 0.05 gradient convergence threshold. PDB files of modeled complexes were generated in Maestro for structural analysis.</p>

PubMed Author Manuscript

Hybrid coordination-network-engineering for bridging cascaded channels to activate long persistent phosphorescence in the second biological window

We present a novel "Top-down" strategy to design the long phosphorescent phosphors in the second biological transparency window via energy transfer. Inherence in this approach to material design involves an ingenious engineering for hybridizing the coordination networks of hosts, tailoring the topochemical configuration of dopants, and bridging a cascaded tunnel for transferring the persistent energy from traps, to sensitizers and then to acceptors. Another significance of this endeavour is to highlight a rational scheme for functionally important hosts and dopants, Cr/Nd co-doped Zn 1−x Ca x Ga 2 O 4 solid solutions. Such solid-solution is employed as an optimized host to take advantage of its characteristic trap site level to establish an electron reservoir and network parameters for the precipitation of activators Nd 3+ and Cr 3+ . The results reveal that the strategy employed here has the great potential, as well as opens new opportunities for future new-wavelength, NIR phosphorescent phosphors fabrication with many potential multifunctional bio-imaging applications.There is an increasing interest in the use of long persistent phosphorescence in the biologically transparent window to drive the photonic bioprobe for tracing the cancer cells 1 . Long phosphorescent phosphors (LPPs) can help avoiding the challenging requirement of high-intensity illumination during the signal collection, which often leads to decreased signal-to-noise ratio and photon-induced deterioration of analytes 2 . This emerging research trend, which incorporates various fields of materials science, biology, chemistry, engineering, physics and pharmaceuticals, follows two main directions: operation waveband and persistent duration, with many relevant crossing points in between 3,4 . As we know, there are two biologically transparent windows: first one at 650-950 nm and second one at 1000-1350 nm 5 ; Near-infrared (NIR) light in the first transparency window can penetrate biological tissues such as skin and blood more efficiently than visible light 6 , yet the second region has even lower absorption and scattering therefore offers more efficient tissue penetration 7 . However, the main researches about the operational waveband of NIR LPPs mainly focus on the short wavelength region, i.e. first NIR window.In addition to altering the emission center and tailoring the crystal field surrounding the activator, another useful strategy to extend the operational waveband, is to transfer the persistent energy of sensitizers to acceptors 8 . In fact, although the afterglow properties are predominantly controlled by the active traps, more subtle effects, such as topochemical coordination-configuration of dopant ions, can also have a profound role to the spectroscopic features of LPPs, which has long been recognized as a significant issue lying at the heart of doping chemistry and photoluminescent theory 9 . Considering the advanced engineering of cascaded tunnel of energy transfer

hybrid_coordination-network-engineering_for_bridging_cascaded_channels_to_activate_long_persistent_p

<!>Results and Discussion<!>71<!>Methods

<p>(traps → activator(A) → activator(B)) and going into the details of it, one has at one's disposal several decades worth of well-established principles in the coincident matching of macroscopical and microscopic features in spectroscopy, coordination chemistry and network connectivity relating to activators and hosts 10,11 . Traditionally, materials scientists view such network-engineering design accessed via active impurities with a practical eye intent on describing integral architectures in terms of ion types, valency and radius, local coordination geometries, as well as their concomitant implications for electronegativity and chemical bonding 12 . However, due to the complex attribute of topological network, there are still remaining grand challenges: to gain better modulation for the local coordination configuration of dopants, to understand the principle linking the indispensable transfer channel of independent individual, and to realize true predictability to the arrangements of traps and dopants (sensitizers, activators, or co-dopants) in coordinated network.</p><p>In this work we present a new "Top-down" approach to design and synthesize the long phosphorescent phosphors in the second biological transparent window. The material design approach employed here involves an ingenious engineering for hybridizing the coordination networks of hosts, tailoring the topochemical configuration of dopants, and bridging cascaded channels for transferring the persistent energy from traps, to sensitizers, to acceptors. We present a closed energy transfer channel from Cr 3+ to Nd 3+ in ZnGa 2 O 4 phosphor and invalid electronic reservoir in CaGa 2 O 4 phosphor, respectively. Persistent energy-transfer could occur in Zn 1-x Ca x-Ga 2 O 4 solid-solution because two dopants were successfully locked in a cage via the efficient crystal packing at an appropriate distance, in addition to the preservation of native electron traps. The hybrid network topologies and structural motifs, thus far will be outlined with particular emphasis on how specific route of energy transfer can be prepared via premeditatedly designing a material system. Such design strategy will notably open a vista of potential avenues for the design of new optical functional materials for the future.</p><!><p>Our strategy was inspired by the fundamental spectroscopic theory of energy transfer and local intercalation reaction in inorganic polycrystals (Fig. 1) 13 . In our view, a typical long phosphorescent phosphor (MSI, in Fig. 1a) features a prominent electron reservoir (C, in Fig. 1) with the distinct ability of storing and releasing the captured electrons, as well as a notable photon-emitter (A, in Fig. 1) with higher quantum efficiency under the condition of accurately matching lattice-coordination network and atomic radius 14 . A pre-established electronic transfer channel (AG, traps (C) → activator (A), in Fig. 1) ensures the long persistent phosphorescence. However, the topological network does not provide an opportunity for another activator (B, in Fig. 1) to embed itself into the suitable lattice site. Such structural constraint thus, closes the possible channel of energy transfer (ET, in Fig. 1) between (A) and (B), leading to the luminescent and phosphorescent quenching. Fortunately, the existing chemical and spectroscopic knowledge offer a far-sighted technique to select another material system (MSII, in Fig. 1b), which allows a synchronous precipitation of activator (A) and (B), as well as engineers a theoretically existent energy-transfer channel (activator (A) → activator (B)). But to our surprise, this scheme misses the necessary electron reservoir so as to completely decrease the probability of electrons trapping-detrapping (Fig. 1b).</p><p>The use of solid-solution complexes to engineer predictable, multi-dimensional infinite networks has received ever-increasing attention in the area of chemistry and materials science 15 . Solid-solutions have already proven their superiorities in the areas of optical, optoelectronic, electrical and magnetic properties than the single component 16 a state-of-the-art Li x FePO 4 solid-solution technology, opening the door for lithium ion batteries to take their place in large-scale applications 17 . In addition, a series of solid-solution, such as AgGa 1−x Al x O 2 , Zn 1−x Cu x S, (SrTiO 3 ) 1−x (LaTiO 2 N) x , also have been developed and used as the advanced photocatalysts to enhance the photocatalytic activity of a given semiconductor photocatalyst 18 . Therefore, solid-solution highlights hybrid coordination network of host, and is expected to open up a possibility in the visualization of the structural and functional binding process of traps and all activators into an independent system 19 . By rationally deploying an indirect intercalation complex comprised by polyhedron ligands of materials (MSI) and (MSII), hybrid coordination-network of novel solid-solution (MSIII, in Fig. 1c) is engineered to steady the activators (A) and (B), modulate the topochemical configuration of activators, and realize the cascaded energy transfers, traps(C) → activator(A) → activator(B) (Fig. 1c). Such novel structural motif is anticipated to adopt a disturbance to native unit cell and bridging a predictable periodic coordination network.</p><p>To validate research idea, a typical NIR long phosphorescent phosphor ZnGa 2 O 4 : Cr was pursued as preferential material system, which has been proven capable of supporting high defect densities, thought to be associated primarily with Zn vacancies (V Zn ) and O vacancies (V O ), as well as some antisite deficiencies (Zn Ga ) 20 . Making use of its defect capacity, ZnGa 2 O 4 : Cr has been demonstrated as a NIR photo-emitter with surprisingly long persistent phosphorescence in first NIR window (Supplementary Fig. S1). Here, we target the operating waveband in the second NIR window by transferring the persistent energy of Cr 3+ to Nd 3+ in Cr/Nd-codoped ZnGa 2 O 4 LPPs. Nd 3+ ion is chosen as the emission center in order to take advantage of the appropriate energy level characteristic, i.e. NIR-absorption (680, 750 and 800 nm) and NIR emission (1064 nm) 21 . The various sharp transitions of Nd 3+ [ 4 I 9/2 → 4 F 5/2 ], [ 4 I 9/2 → 4 F 7/2 ], [ 4 I 9/2 → 4 F 9/2 ], just overlap the electron transition from metastable state ( 4 T 2 ) to ground state ( 4 A 2 ) of Cr 3+ , allowing the potential energy transfer from Cr to Nd 22 . However, no any NIR phosphorescence in the second NIR window can be observed in Cr/Nd-codoped ZnGa 2 O 4 LPPs (Fig. 2a). In fact, the desired phosphorescence is still absent in Nd 3+ singly doped ZnGa 2 O 4 phosphor after ceasing the excitation (Supplementary Fig. S2). It is notable that the diffuse reflection spectrum consists of the characteristic transition bands centered at 530, 588, 688, 748 and 808 nm, respectively, corresponding to Nd 3+ f-f transition,</p><p>in Nd 3+ doped ZnGa 2 O 4 phosphor (Supplementary Fig. S3) 21 ; yet under the excitation at 748 nm, emission peak at 1064 nm, attributed to Nd 3+ [ 4 F 3/2 → 4 I 11/2 ] transition is not identifiable (Supplementary Fig. S4). This attractive optical quenching-phenomenon of luminescence and phosphorescence may be not concerned with the trap distribution, but the microcosmic network architecture.</p><p>In ZnGa 2 O 4 , a majority of [Ga VI ] cations occupy octahedral sites, whereas all of the [Zn IV ] cations occupy tetrahedral sites 23 . As a preliminary conjecture, Cr 3+ has proven its strong ability to substitute for Ga 3+ in distorted octahedral coordination, whereas Nd 3+ cannot be effectively introduced into this specific network configuration (inset of Fig. 2a). In order to identify this possibility of ion doping, we focus on the intricate topochemical coordination geometry of Cr and Nd ions in zinc gallate spinel. The elucidation is performed in detail by a combination of XRD data and 71 Ga solid state nuclear magnetic resonance (NMR) studies. XRD patterns of ZnGa 2 O 4 : xCr (x = 0.5%, 5%, 10% and 20%) and ZnGa 2 O 4 : xNd (x = 0.5%, 5%, 10% and 20%) phosphors were measured and shown in Fig. 2b, Supplementary Fig. S5, S6. The peaks in XRD patterns of all Cr-doped samples are well indexed to pure ZnGa 2 O 4 spinel structure (JCPDS 86-0848). In stark contrast, the higher doping content (up to 5%) of Nd ion gives rise to an impure phase, NdGaO 3 (JCPDS, 70-3810) in Nd-doped samples. Another interesting phenomenon, i.e. XRD dominated peak (PI in Fig. 2b) shifting towards to higher 2θ value with the increment of Cr content, reveals a small linear variation in ZnGa 2 O 4 unit cell lattice parameter with Cr 3+ substitution, whereas no any shift of same peak is observed in Nd-doped samples, further ensuring the distinct phase splitting (Fig. 2c). In addition, a decline of the peak intensity in Fig. 2c also is present. Nevertheless, the causes of this decline may be different and rooted from either the substitution or the phase splitting.</p><p>NMR allows the observation of specific quantum mechanical and magnetic properties of atomic nucleus, as well as provides the detailed information about the structure, dynamics, reaction state, and chemical environment of molecules 24 . Many scientific techniques exploit NMR phenomena to cover the interplay between the ligands and geometric centers, as well as study the topological network motif in crystals, microcrystalline powders, or anisotropic solutions, etc 25 . 71 Ga solid-state NMR is famous for the permission of quantitative analyses to different Ga 3+ central coordination state in inorganic solids 26 . Figure 2d,e shows the systematical physical investigations of Ga coordination geometry in Cr and Nd singly doped ZnGa 2 O 4 , respectively. For the undoped ZnGa 2 O 4 samples, 71 Ga NMR spectra exhibit two well-resolved resonances. The relative higher intensive signal at about 31 ppm is characteristic of sixfold coordinated Ga atoms, and the other weaker one ~at 170 ppm corresponds to Ga atoms in the tetrahedral sites of the spinel structure 26 . It is necessary to mention that with increasing Cr content (from 0.5% to 10%), 71 Ga NMR spectra present a significant broadening of spectral lines (Fig. 2d). In prominent contrast, scarcely any distinct influence on NMR spectral lines can be found by varying the Nd doping content in solid NMR spectra of ZnGa 2 O 4 : xNd (x = 0.5% and 10%) phosphors (Fig. 2e). The clear separation of NMR chemical shift at ~31 ppm between the two samples implies the precipitation of Cr 3+ into the octahedral lattice site and the excludability of local configuration to Nd 3+ ions. The NMR results are in accordance with XRD data, offering a powerful structural evidence to explain the interesting phenomena of phase splitting and luminescence quenching.</p><p>Actually, rare-earth elements generally form complexes which have high coordination numbers (CNs) and weak metal-ligand bonds, because of their large ionic radii and relatively low oxidation states 27 . Typically transition-metal and main-group elements have coordination numbers 2-6, while rare-earth metals have CNs > 6 28 . The resulting coordination polyhedra include trigonal prisms (CN = 6) or its variation by stepwise capping of the prism face up to CN = 9, in addition to square antiprisms (CN = 8); Coordination number 3 is realized only under extreme conditions 28 . Therefore, to supply an ideal dwelling for Nd 3+ , a suitable material system should be proposed. Alkaline-earth metals have large ionic radii and various coordination-numbers 3-8 in different hosts, which ensure the selection of alkaline-earth gallates 29 . CaGa 2 O 4 has a similar spinel crystal structure with ZnGa 2 O 4 . In CaGa 2 O 4 , [Ca VI ] cations occupy octahedral sites 29 . This configuration thus features a path of easy doping ion precipitation into the octahedral [Ca VI ] under the condition of matching geometrical lattice and atomic radius, which occurs with rare earth ion, Nd.</p><p>As expected, Fig. 3a exhibits the characteristic transitions of Nd 3+ in Nd singly doped CaGa 2 O 4 phosphor. However, the idealistic and aspirational long persistent phosphorescence is still absence in Cr singly, Nd singly and Cr/Nd doped CaGa 2 O 4 phosphors, respectively (Supplementary Fig. S7). A possible cause of this problem is due to the lack of effective traps (Fig. 3b). In sharp contrast to Cr/Nd codoping ZnGa 2 O 4 , photoluminescence excitation (PLE) spectrum monitored at 1064 nm of Cr/Nd codoping CaGa 2 O 4 sample consists of two specific excitation bands centered at ~410 and ~620 nm, in addition to Nd 3+ characteristic f-f transitions (Fig. 3c), indicating an energy transfer from Cr 3+ to Nd 3+ . Obviously, the strong one is attributed to the Cr 3+ [ 4 A 2 → 4 T 1 ], while the weak one corresponds to Cr 3+ [ 4 A 2 → 4 T 2 ] 30 . Further verification of energy transfer between Cr 3+ and Nd 3+ is supplied by emission spectrum and decay curve monitored at 1064 nm under the excitation wavelength at 410 nm (Fig. 3c and Supplementary Fig. S8). A possible channel of energy transfer from Cr 3+ to Nd 3+ is Cr 3+ [ 4 T 2 → 4 A 2 ]: Nd 3+ [ 4 I 9/2 → 4 F 5/2 ], [ 4 I 9/2 → 4 F 7/2 ], or [ 4 I 9/2 → 4 F 9/2 ], depending on the overlap between Cr 3+ emission band and Nd 3+ absorption band (Fig. 3d) 31 . As discussed above, due to the similar atomic radius and geometric configurations, Nd ions can easily precipitate on Ca lattice site in CaGa 2 O 4 , enabling the distinct photoluminescence (PL). To probe the lattice configuration and substitution progress in CaGa 2 O 4 , we performed XRD and solid state NMR experiments. X-ray diffraction pattern first confirms the crystallization of Nd-doped calcium gallate (Fig. 3e). In contrast to Nd-doped ZnGa 2 O 4 , all Nd-doped CaGa 2 O 4 samples can be indexed as standard phase CaGa 2 O 4 (JCPDS 16-0593). There is no any apparent observation of phase splitting from XRD data, even under a higher doping content of Nd 3+ , firmly supporting the rational inclusion of Nd 3+ into an inert matrix, CaGa 2 O 4 . This result is also supported by 71 Ga solid state NMR spectra. In contrast to ZnGa 2 O 4 host, the undoped CaGa 2 O 4 sample has a dominant chemical shift at 170 ppm (Fig. 3f). With increasing dopants content, CaGa 2 O 4 : Nd also has the same effect of NMR resonances' line broadening and the linear increase of NMR resonances integrated intensity, strongly suggesting the successful substitution in substantial amounts of Nd into Ca lattice site.</p><p>Seemingly, as the individual backbone, MGa 2 O 4 (Zn and Ca) polymorph is chosen as the prototypical coordination network for its respective ability to engineer the functionally independent tunnel, traps(C) → activator(A), or activator(A) → activator(B), used to transfer the required energy. The only regret is the fundamentally missing connection of traps(C) → activator(A) → activator(B) in a separate material system. To address this issue, we anticipate a novel solid-solution Zn 1-x Ca x Ga 2 O 4 to bridge a new channel for transferring the persistent energy from traps to desired ions, based on the cautious consideration for crystal structure, ion valency and chemical bond relating to hosts and dopants. The desired NIR phosphorescence at 1064 nm is finally present in the afterglow spectra of Zn 1-x Ca x Ga 2 O 4 (x = 0.1, 0.3, 0.4 and 0.5) solid-solution (Fig. 4a). Significantly, we also observe a strong dependence (i.e. rising first followed by a decline) of phosphorescent peak intensity and decay dynamics on Ca concentration in Fig. 4a. We attribute this special spectral change of Nd 3+ to the successful persistent energy transfer from Cr 3+ to Nd 3+ , which is supported by the meticulous spectral studies of Nd 3+ in an optimal Zn 0.6 Ca 0.4 Ga 2 O 4 : 0.5Cr/0.5Nd solid-solution: PLE band at 410 nm should be assigned to Cr 3+ transition [ 4 A 2 − 4 T 2 ], while a distinct NIR PL peak at 1064 nm is observed under the excitation at 410 and 600 nm (Fig. 4a and Supplementary Fig. S9). The additional support for the formation of an unrestricted energy tunnel, traps → Cr 3+ → Nd 3+ , is the analysis of kinetic processes in Z 0.6 C 0.4 GO: 0.5%Cr/xNd (x = 0, 0.5%, 1% and 2%) samples (Fig. 4b). PL decay dynamics study of Cr 3+ shows a notable shortening in decay lifetime from 7.8 (Z 0.6 C 0.4 GO− 0.5Cr), to 7.59 (Z 0.6 C 0.4 GO− 0.5Cr0.5Nd), to 7.32 ms (Z 0.6 C 0.4 GO− 0.5Cr2Nd), giving clear evidence of successfully simultaneous precipitation of two activators into the corresponding lattice along with the effective energy transfer from Cr 3+ to Nd 3+ .</p><p>It should be noted that, to the best of our knowledge, this type of NIR long-persistence phosphorescence has not been previously reported to occur in hybrid coordination networks by engineering cascaded energy transfer channels. Such substantial progress is strongly influenced by two key attributes; one is trap distribution and another is network architecture. Apparently, the variation of trap distribution may be not a crucial factor in exploring the nature of transfer channel, because the indispensable electron reservoir is still steadily embedded in all the Zn 1-x Ca x Ga 2 O 4 solid-solutions (Fig. 4c). To probe the evolution of topological network-dependent topochemical coordination, the systematic characterization, such as, XRD, solid NMR, EDX mapping and Raman spectra should be conducted 32 . XRD peaks in Z 1-x C x GO (x = 0.1, 0.4, 0.5 and 0.7) samples indicate their ZnGa 2 O 4 spinel solid-solution nature, while the superimposed peaks in samples Z 0.5 C 0.5 GO and Z 0.3 C 0.7 GO can be well indexed by the diffraction peaks of ZnGa 2 O 4 and CaGa 2 O 4 (Fig. 4d, and Supplementary Fig. S10). EDX mapping analysis reveals the solid-solutions have uniform distribution of Ca elements in all of the spinel solid-solutions (Supplementary Fig. S11, S12 and Fig. 4e). EDX experimental composition approximating the theoretical value supports the successful inclusion of Ca elements in spinel crystals (Supplementary Table S2).</p><!><p>Ga NMR spectra have provided some insights into the coordination variation of Ga center in ZnGa 2 O 4 and CaGa 2 O 4 phosphors, due to the incorporation of Cr and Nd. it is also expected to manifest its ability in resolving the question of topochemical configuration's evolution process, as the addition of Ca element. As shown in Fig. 4f, with increasing Ca content (0, 0.2, 0.4 and 0.7), two resonances at 170 and 31 ppm in 71 Ga NMR spectra increasingly present the linear broadening. In these solid solutions, Zn-O and Ga-O tetrahedron could suppress the intrusion of Ca element due to the mismatch of coordination configuration. In fact, to steady Ca ion, parts of Ga-O octahedron must reorient to form the new polyhedron network Ca-O octahedron along with the transformation from Ga-O octahedron to Ga-O tetrahedron due to the decrease of Zn-O tetrahedron. In detail, for the samples Z 1-x C x GO (x = 0, 0.2 ,0.4), the motion of local hybrid coordination-networks evolution include: (1) the precipitation of Ca on the lattice site of octahedron Ga, giving rise to the broadening of NMR resonance at 164 ppm; (2) the conversion from Ga-O octahedron to Ga-O tetrahedron, resulting in the enhancement of NMR resonance at 65 ppm. This interesting redeployment of network configuration thus permits the modification of topochemical state of dopants, as well as opens the possibility of bridging cascaded channels to transfer the persistent energy. To further validate the research idea aiming at the network configuration, Raman spectra of the fabricated samples also can be selected as the pertinent tool to further analyze the evolution of network architecture (Fig. 4g and Supplementary Fig. S13) 33 . In stark contrast to samples ZGO-0.5Nd and ZGO-5Nd, normalized Raman spectra of samples CGO-0.5Nd and CGO-5Nd do not exhibit the notable Raman peak shift and variation of Raman peak intensity, indicating a strong constraint of topological network to the migration of Nd ions in CaGa 2 O 4 . In fact, only two distinct Raman bands at ~1358 and 1434 cm −1 are present in the Raman spectrum of CGO-0.5Nd, while the Raman spectrum of ZGO-0.5Nd includes three identifiable Raman peaks at ~1341, 1389 and 1425 cm −1 . Thus, Raman spectra of Z 1-x C x GO (x = 0.1, 0.4 and 0.7) solid-solutions consequentially show a unit number decrease of Raman peaks with the increment of Ca content (inset of Fig. 4g). The variation of middle peak at 1401 cm −1 as a function of Ca doping content ensures the strong signature of the hybrid network structure, which is in accordance with the XRD and solid-state NMR data.</p><p>In summary, we report a principle of bridging cascaded energy transfer channels to activate long persistent phosphorescence in the second biological window and fabrication of novel near-infrared phosphorescent phosphor Cr/Nd codoped Zn 1-x Ca x Ga 2 O 4 solid-solutions. Structural studies offer the powerfully fundamental evidences to explain the closed energy transfer channel from Cr 3+ to Nd 3+ in ZnGa 2 O 4 phosphor and invalidation of electronic reservoir in CaGa 2 O 4 phosphor. We believe that the ingenious solid-solution technology featuring the superiority of engineering a hybrid coordination-network opens new paths for advanced dynamic management of activation energy and gives the inspiration to design future new-wavelength, NIR phosphorescent phosphors by energy transfer.</p><!><p>Materials. 4N pure CaCO 3 , Ga 2 O 3 , ZnO, Nd 2 O 3 and Cr 2 O 3 were selected as the raw materials.</p><p>Preparation of ZnGa 2 O 4 : xCr/yNd. Phosphors with molar compositions of ZnGa 2 O 4 : xCr/yNd (x = 0, 0.5%, 5%, 10%, 20%; y = 0, 0.5%, 5%, 10%, 20%), (Supplementary Table S1) were prepared by the solid state reaction method. The reaction included a two-step thermal treatment (i.e., initial calcination at 800 °C for 5 h, secondary calcination at 1350 °C for 3 h).</p><p>Preparation of CaGa 2 O 4 : xCr/yNd. Phosphors with molar compositions of CaGa 2 O 4 : xCr/yNd (x = 0, 0.5%; y = 0, 0.5%, 5%, 10%), (Supplementary Table S1) were prepared by the solid state reaction method. The reaction included a two-step thermal treatment (i.e., initial calcination at 800 °C for 5 h, secondary calcination at 1200 °C for 3 h).</p><p>Preparation of Zn 1-x Ca x Ga 2 O 4 : 0.5Cr/yNd. Phosphors with molar compositions of Zn 1-x Ca x Ga 2 O 4 : 0.5Cr/yNd (y = 0, 0.5%, 1%, 2%; x = 0.1, 0.2, 0.3, 0.4, 0.5, 0.7), (Supplementary Table S1) were prepared by the solid state reaction method. The reaction included a two-step thermal treatment (i.e., initial calcination at 800 °C for 5 h, secondary calcination at 1350, 1350, 1300, 1300, 1270, 1250 °C for 3 h as a function of x, respectively).</p><p>Characterization. The prepared materials were analyzed by X-ray diffraction (Cu/Kα ) to confirm the sole crystalline phase. Room-temperature photoluminescence (PL), photoluminescence excitation (PLE) spectra, afterglow spectra and decay curves were measured with a high-resolution spectrofluorometer (UK, Edinburgh Instruments, FLS920) equipped with a 500 W Xenon lamp as an excitation source, with a Hamamatsu R928P visible photomultiplier (PMT) (250-850 nm) and a liquid nitrogen-cooled Hamamatsu R5509-72 NIR PMT as the detectors. TL glow curves and TL excitation (TLE) spectra were measured with a FJ-427A TL meter (China, Beijing) to characterize defect properties. Unless otherwise mentioned, the samples were pre-annealed at 600 K before testing, and some measurements were taken after pre-irradiating the samples for 10 min by using a xenon lamp. EDX images are characterized by a field emission scanning electron microscopy (FE-SEM), Nova NanoSEM 430. 71 Ga Hahn echo NMR experiments were performed on Bruker Avance III spectrometers operating at magnetic fields of 111.4 T corresponding to 71 Ga Larmor frequencies of 152.54 MHz) using Bruker 2.5 mm triple and double resonance probe heads. The 90 0 degree pulse length is 1.25 μ m with a recycle delay of 8s. 71 Ga chemical shifts were referenced relative to a 1.0 M aqueous solution of Ga(NO 3 ) 3 . All 71 Ga spectra were fitted using the Dmfit software. Raman spectra were collected with a Renishaw inVia Raman microscope irradiated by a visible laser at 532 nm.</p>

Scientific Reports - Nature

Vesicle tubulation with self-assembling DNA nanosprings

A major goal of nanotechnology and bioengineering is to build artificial nanomachines capable of generating specific membrane curvatures on demand. Inspired by natural membrane-deforming proteins, here we design DNA-origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA-coated membrane tubules emerge from spherical vesicles when DNA-origami polymerization or high membrane-surface coverage occurs. Unlike many previous methods, the DNA self-assembly-mediated membrane tubulation eliminates the need for detergent or top-down manipulation. The DNA-origami design and deformation conditions have substantial influence on the tubulation efficiency and tube morphology, underscoring the intricate interplay between lipid bilayers and vesicle-deforming DNA structures.

vesicle_tubulation_with_self-assembling_dna_nanosprings

<p>Complex and dynamic membrane shapes are hallmarks of living cells.[1] Tubular shape is one of the most common membrane architectures that exists during cellular events including endocytosis, viral budding, and cytokinesis. Various membrane-deforming proteins that sense and generate membrane curvatures contribute to the formation and stabilization of membrane tubules in cells.[2] While many existing techniques can recapitulate membrane tubulation in vitro,[3] experimental restraints (e.g., dependence on lipid composition, detergents, or mechanical force) pose limitations on a method's programmability and applications.</p><p>Stemming from the simple idea of combining branched DNA motif with complementary "sticky-ends",[4] the field of structural DNA nanotechnology has matured into a stage where one can easily generate three-dimensional nanostructures with programmable geometry, surface chemistry, and dynamics.[5] In recent years, the field has seen considerable progress towards using DNA nanostructures for membrane engineering in order to better control synthetic membrane properties as well as to manipulate biological membranes.[6] For example, supported lipid-bilayer membranes have been coated with DNA "rafts" and promoted DNA-tile association;[7] DNA nanochannels labeled with hydrophobic moieties have been developed for membrane penetration;[8] DNA templates have been engineered to guide the assembly of size- and shape-defined liposomes and to induce membrane fusion and bending.[3e, 9] Particularly relevant to this work, the lateral association of cholesterol-modified DNA nanoblocks on membrane have led to vesicle flattening, presumably imposed by a large flat DNA surface.[10] Although inserting DNA nanochannels at high concentration can result in tubule-like structures on giant unilamellar vesicles (GUVs), such deformation is likely driven by surface crowding; the exact mechanism remains unclear.[8b] Therefore, engineering DNA nanostructures capable of programmable vesicle tubulation remains challenging.</p><p>While existing membrane-deforming DNA structures mostly mimic BAR-family proteins,[10] we took our design inspiration from dynamins[2a] and ESCRT machineries[2c], two major classes of proteins that polymerize into helical structures coating lipid tubules. Specifically, we set out to generate DNA curls that would self-assemble into helical structures similar to the Snf7 filament, which form spiral-like assemblies on membrane with outer diameter of 50–100 nm and filament thickness of 5–10 nm.[11] To achieve this, we bent and twisted a ~100-nm long, 14-nm thick DNA 24-helix-bundle rod a −55° of bend per 77-bp was achieved by inserting and deleting equal number of base-pairs on the opposite sides of the DNA rod[12] and the twist was implemented by changing the bending axis by 30° every 77-bp. The result of such a design is a 'C'-shaped structure with an out-of-plane twist (Fig. 1a, S1, and S2). To render the DNA structure with stronger membrane affinity, we reserved 24 single-stranded DNA (ssDNA) extensions along the inner surface of the DNA-origami curl for attaching amphipathic peptides as membrane anchors, which we designed to mimic the N-terminal ANCHR helix of Snf7.[13] The distance between neighboring peptides is 4–7 nm, close to their ~3 nm spacing in snf7 filaments.[14] Gratifyingly, upon conjugation with Cy5-labeled ssDNA, this peptide dissolved readily in detergent-free aqueous buffer solutions, bound well to the attachment sites on the DNA curl, and had minimal tendency to aggregate DNA nanostructures (Fig. 1b and S3). We further designed a set of "linker strands" that would bridge the front and rear ends of the DNA curls, causing monomeric DNA curls to polymerize and form "nanosprings" with an expected inner diameter of 27 nm and a helical pitch of 53 nm. Indeed, adding linkers to the peptide-labeled DNA curls triggered polymerization; when examined by negative-stain transmission electron microscopy (TEM), the resulting nanosprings measured a length of 330±190 nm with an inner diameter of 26±5 nm and a helical rise of 80±9 nm (Fig. 1c). The discrepancy between designed and measured nanospring dimensions is likely due to the structural distortion on TEM grids[15] and the overwound DNA caused by numerically balanced insertions and deletions[16]. A considerable amount of closed DNA circles also emerged after addition of linkers as a result of self-closing dimers. It is worth noting that the DNA-curl polymerization is tunable by changing Mg2+ and K+ concentrations. The polymerization in solution is optimal at 10 mM Mg2+ without K+; higher Mg2+ promoted nanospring aggregation and higher K+ led to shorter polymers (Fig. S4).</p><p>After establishing the folding of DNA curls and their polymerization in solution, we then tested their ability to bind and deform vesicles. The experimental procedure is summarized in Fig. 2a. Briefly, purified DNA-curl monomers (4 nM) labeled by ANCHR-mimicking peptide were incubated with extruded liposomes (99.2 μM 1,2-dioleoyl-sn-glycero-3-phosphocholine or DOPC, 0.8 μM 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); mean diameter: 134 nm) at 4°C for 1 hour to allow for binding before mixed with linker strands and further incubated at 30°C for up to 32 hours; the osmolarity of the solution was kept constant throughout the entire process. TEM analyses (Fig. 2b) on the mixture of DNA curls and liposomes showed their binding depended on the presence of membrane anchors. To better analyze the liposome binding affinity, we subjected the mixture of DNA-curl monomers and liposomes to isopycnic centrifugation. A fluorescence scan of the recovered fractions confirmed the membrane-anchor dependent binding between Cy5-labeled DNA curls and rhodamine-labeled vesicles (Fig. 2c), which co-migrated with amphipathic peptides decorating DNA curls but parted ways in the absence of these peptides. However, in addition to DNA-curl-bound liposomes, TEM revealed free DNA curls and naked liposomes in the fractions where the two coexisted (Fig. S5), suggesting their binding is transient. Interestingly, individual ANCHR-mimicking-peptide-DNA conjugate molecules showed no detectable affinity towards vesicles (Fig. S5), highlighting the DNA curl's role in facilitating membrane binding, possibly by both bringing together multiple (a maximum of 24) membrane anchors and providing a negatively charged surface to interact with the zwitterionic lipid head group in the presence of magnesium cations. Adding linker strands after the initial binding step resulted in the formation of membrane tubules wrapped by DNA nanosprings (Fig. 2d and S6). Among the deformed liposomes, some retained a spherical body with a tubule protruding outwards while others turned entirely into a tubular shape. A time-course study showed that the tubule abundance and length reached maximum after the first two hours of polymerization and stayed stable for 16 hours; another 16-hour incubation at 30°C led to fewer and shorter tubules, likely because of degradation (Fig. 2e and S6). We note that vesicle tubulation occurred in a solution with 10 mM Mg2+ and 100–800 mM K+, despite the fact that the DNA-curl polymerization in solution was most efficient without K+. This is because monovalent cations weaken the electrostatic interactions between DNA and lipid, hence mitigating DNA-curl induced vesicle aggregation (Fig. S7 and S8). Neither polymerizing membrane-anchor-free DNA curls in the presence of vesicles (Fig. S9) nor incubating pre-polymerized membrane-anchor-labeled DNA nanosprings with vesicles (Fig. S10) could generate membrane tubules, indicating that vesicle tubulation necessitates membrane-binding DNA-curl monomers. Despite the low deformation yield (<4% of vesicles were deformed after 16 hours), the membrane tubules unambiguously formed as a result of DNA-nanostructure coating. In fact, nuclease digestion of the DNA coat after tubulation resulted in the disappearance of the vast majority of membrane protrusions and tubular vesicles (Fig. S11).</p><p>A closer inspection of the apparent membrane tubule width and the associated nanospring periodicity identified two types of tubes (Fig. 3a). The first type features a tubule diameter of 15±2 nm and an apparent helical pitch of 35±6 nm (peak-to-peak distance of the DNA coat). The second type of tubule is considerably wider (diameter: 29±4 nm) with a more densely packed DNA-curl coating (apparent pitch: 16±2 nm). In rare cases, a continuous membrane tubule exhibits both morphologies in different segments. One possible explanation is that the wider tubes resulted from the dehydration and flattening of the thinner tubes on TEM grids. An alternative hypothesis is that both types of membrane tubules existed in solution and the wider ones formed because of higher membrane coverage by DNA curls. To test this hypothesis, we increased the concentration of DNA-curl monomers from 4 nM to a maximum of 20 nM, which translates to an increase of the maximal vesicle-surface coverage from 16% to 80%. The higher surface coverage led to two significant changes (Fig. 3b, 3c, and S12). First, the overall deformation yield (the portion of vesicles that undergo notable tubulation) increased with a higher initial concentration of DNA-curl monomers, up to ~13% with 20 nM DNA curls. Second, the occurrence of wider tubules increased with greater membrane coverage and dominated the tubule population with DNA-curl concentration greater than 10 nM (or roughly 40% maximal surface coverage). Reducing the DNA curl and lipid concentration proportionally resulted in a similar trend, confirming that surface coverage is the main determinant of deformation efficiency and tubule morphology (Fig. 3c and S13). Based on these data, our interpretation is that the wider tubules' diameters are likely defined by the multiple, moderately compressed DNA springs, whereas the narrower tubes, surrounded by only 1–2 slightly compressed springs are free to adjust their diameters to approach the minimum allowed by the membrane bending energy (see Technical Notes in SI for more discussion). The compressed DNA springs have to adhere to membrane with sufficient strength (through both electrostatic and hydrophobic interactions) to prevent the retraction of lipid tubules, which exposes membrane anchors. Intriguingly, an appreciable amount of tubules (width: 24±8 nm, max. yield≈5%) was produced at high membrane coverage conditions (Fig. 3d and S14) without linker strands, where the tubules were covered by DNA curls, but with less regular patterns. This linker-independent tubulation suggests a different, although less efficient, deformation mechanism driven by surface crowding and membrane-anchor insertion, consistent with the notion of coupling between protein/lipid shape and membrane curvature.[1, 17]</p><p>Having confirmed the DNA nanospring's capability in deforming organelle-size large unilamellar vesicles (LUVs), we attempted to test their tubulation efficacy on GUVs with sizes approximating eukaryotic cells. We first incubated Cy5-labeled DNA-origami curls with nitrobenzoxadiazole (NBD)-labeled GUVs (lipid concentration ≈ 10 μM) at 4°C for 1 hour in an iso-osmotic buffer solution containing 10 mM Mg2+ and 100 mM K+. We then held this mixture (with or without additional linker strands) at 30°C for 16 hours and monitored vesicles using a confocal fluorescence microscope. The DNA curls bound to GUVs (Fig. 4a) in a similar way as they bound to LUVs: the surface coverage of DNA curls depended on their initial concentration and membrane-anchor density (Fig. S15); the bound and unbound DNA curls were in a dynamic equilibrium with slow off-rate (Fig. S16). With 20 nM DNA curls, noticeable tubulation occurred after at least 1.5 hours of incubation at 30°C. After 16 hours, these outward protrusions grew long (some longer than 250 μm) and abundant. The tube-like structures carried both Cy5 and NBD fluorescence (Fig. 4a), suggesting they were coated by DNA curls, as confirmed by negative-stain TEM studies. Under TEM, a deformed GUV usually featured multiple emanating protrusions (Fig. 4b). Comparing to the membrane tubules originated from LUVs, these GUV protrusions were much wider (116±50 nm) with extremely dense DNA coating. The DNA curls lay closely against one another in parallel at the edges of the protrusions but showed no apparent order in the central areas, albeit the high surface density (Fig. 4c). We found that the linker strands, but not the membrane-binding DNA curls, were dispensable for tubulation (Fig. S17); the vesicles were deformed rather similarly with or without linkers (Fig. S18 and S19). Further, DNA curls with cholesterol modification and DNA rings (outer diameter: 60 nm) with ANCHR-mimicking peptide modification on the inside deformed vesicles (GUVs and LUVs) in a similar manner and without linker induced DNA-origami oligomerization (Fig. S20–S23). Changing peptide anchors to cholesterol increased the aggregation of DNA curls, but did not reduce their tubulation capability in general. Although the influence of membrane-anchor type and DNA nanostructure geometry on vesicle deformation warrants further study, the phenomena observed here, combined with the linker-independent LUV deformation, strongly support that DNA-origami structures can drive vesicle tubulation solely by introducing a large number of membrane anchors and covering the vesicle surface with DNA structures at high density.</p><p>In summary, we have built bioinspired nanostructures that drive tubular deformations from spherical vesicles. Such a membrane-sculpting structure comprises three modules: a structural module (the curved DNA-origami body) that provides a negatively charged interior with dozens of "open seats" (ssDNA extensions) for guest molecules, a membrane-anchoring module (DNA-peptide or DNA-cholesterol conjugate) that decorates the DNA curl's inner surface and confers hydrophobicity, and a connector module (linker strands) that join the ends of monomeric structures to form a long helical filament. Both membrane incorporation and DNA-origami polymerization provide energy to overcome membrane tension and intrinsic curvature for vesicle deformation. Embedding amphipathic anchors into lipid bilayers expands the outer leaflet and introduces higher membrane curvature; the elongation of DNA springs "pulls" on the lipid and "holds" the shape of the membrane tubules (see Technical Notes in SI for more discussion). The vesicle deformation takes two different forms that both require the membrane-anchoring module and DNA structures surrounding the membrane tubules, but result in distinct tubule morphology and DNA-coating patterns. This process can be modulated by changing the surface density of DNA nanostructures (systematically tested) and the membrane tension of vesicles (uncontrolled in this study). With sparse DNA coating and high membrane tension, the connector module is essential for making tubules wrapped by nanosprings. In contrast, when a low-tension membrane surface is saturated with membrane-anchor-labeled DNA structures, low energy cost, polymerization-independent tubulation becomes favorable. This may explain the dispensable linker strands in GUV deformation, where membrane tension is likely lowered due to evaporation and the DNA curls are in ~8-fold excess for complete membrane coverage. Additionally, higher surface coverage and lower membrane tension can both increase tubulation efficiency. Tension-dependent vesicle tubulation has been demonstrated using motor proteins[3c] and concentration-dependent self-assembly on membrane has been observed with curvature sensing/inducing proteins (e.g., amphiphysin[17b] and FtsZ[18]). Deforming membranes with engineered synthetic constructs in a way that resembles the work of naturally existing proteins is not only a major feat in synthetic biology,[19] but also provides a tool to study the membrane remodeling mechanisms. The modular self-assembling nanosprings extend our membrane manipulating capability and pave the way for future development of more sophisticated, multistep membrane-sculpting machineries (e.g., with pre-inhibited components that are activated upon membrane binding) that can sort cargo molecules, drive vesicle budding, alter deformation topology (i.e., tubulation toward vesicle lumen), and act on cell membranes.</p>

PubMed Author Manuscript

Electronic differentiation competes with transition state sensitivity in palladium-catalyzed allylic substitutions

Electronic differentiations in Pd-catalyzed allylic substitutions are assessed computationally from transition structure models with electronically modified phospha-benzene-pyridine ligands. Although donor/acceptor substitutions at P and N ligand sites were expected to increase the site selectivity, i.e. the preference for "trans to P" attack at the allylic intermediate, acceptor/acceptor substitution yields the highest selectivity. Energetic and geometrical analyses of transition structures show that the sensitivity for electronic differentiation is crucial for this site selectivity. Early transition structures with acceptor substituted ligands give rise to more intensive Pd-allyl interactions, which transfer electronic P,N differentiation of the ligand more efficiently to the allyl termini and hence yield higher site selectivities.

electronic_differentiation_competes_with_transition_state_sensitivity_in_palladium-catalyzed_allylic

Introduction<!>Table 1:<!>Results and Discussion<!>Conclusion<!>Computational details

<p>Palladium-catalyzed allylic substitutions allow very selective and mild allylations of C-,N-and O-nucleophiles. [1][2][3][4][5][6][7][8][9][10][11][12][13] The selectivity derives from steric and electronic properties of substrate and catalyst structures. "Side arm guidance" of nucleophiles with multifunctional phosphinoferrocenes [14][15][16][17][18] or "chiral pockets" in C 2 -symmetric diphosphanes based on 2-(diphenyl-phosphino)benzoic acid amides [19][20][21][22] were applied especially successfully. Chiral P,N-ligands (e.g. phosphinooxazolines, phox) [23][24][25][26][27] provide in addition to steric control the possibility for "electronic differentiation", originating from the trans-influence [28] of different donor atoms. Nucleophiles (e.g. dimethylmalonate) normally favour addition to the "trans to phosphorus" position at the Pd-η 3 -allylic inter-mediate (Scheme 1). [29][30][31][32][33][34][35][36][37][38][39][40][41][42] This "trans to P" rule is supported by X-ray and computational analyses of Pd-η 3 -allylic intermediates, which exhibit longer and hence weaker Pd-C allyl bonds trans to P (i.e. the stronger π-acceptor vs. N) and hence are more susceptible to nucleophilic attack (Scheme 1). [29][30][31][32][33][34][35][36][37][38][39][40][41] This electronic differentiation contributes to the high selectivity in Pd-catalyzed asymmetric allylic substitutions [19] and provides also an explanation for α-memory effects. [42,43] Computational model systems for P,N-ligands, i.e. PH 3 and para-substituted pyridines, have shown that cis-trans differentiations, i.e. the electronic site selectivity, of nucleophilic additions to Pd-η 3 -allylic intermediates is highest for electron poor pyridine ligands. [45] Scheme 1: Electronic and steric differentiations provide the basis for the high selectivity of P,N-ligands in Pd-catalyzed allylic substitutions. Effects are studied with P-N-model ligands with para-substituted, coplanar phosphabenzene and pyridine moieties.</p><!><p>Activation (E a ) and reaction energies (E r ) reflecting electronic differentiations in transition structures (ΔE a cis-trans ) and Pd-ene products relative to Pd-allyl and NH 3 reactands (pb = phosphabenzene; py = pyridine moieties) [a] To further explore origins of site selectivities based on electronic differentiations in Pd-catalyzed allylic substitutions, we here employ a more advanced model system with phosphabenzene, [45][46][47][48] and pyridine moieties for the crucial step of Pd-catalyzed allylic substitutions. Both P-and N-coordination sites are tuned electronically with para-substituents to reveal energetic and geometrical effects on cis-vs. trans-additions of nucleophiles to the Pd-η 3 -allylic intermediates (Scheme 1).</p><!><p>Electron donating or withdrawing groups (i. e. X, Y = HNMe, H, NO 2 ) in para-positions of phosphabenzene (X) and pyridine (Y) units tune electronic characteristics of P,N-ligand models in Pd-catalyzed allylic substitutions (Scheme 1). The phosphabenzene and pyridine moieties are linked via C ar -C ar bonds and a methylene bridge retains planarity and limits conformational flexibility. NHMe rather than higher substituted NMe 2 was employed as donor group, to retain lp-aryl conjugation. Ammonia serves as model nucleophile and attacks the Pd-η 3allylic intermediate cis or trans to phosphorus. This cis vs. trans site selectivity is employed as measure for electronic differentiation induced by the ligand system (Scheme 2).</p><p>The lowest activation energies (E a , Table 1) for ammonia addition to the Pd-η3-allylic intermediate are apparent for strong electron withdrawing para-substituted phosphabenzene and pyridine units, i.e. X, Y = NO 2 (Figure 1 and Figure 2, E a trans = 2.19, E a cis = 2.52 kcal mol -1 , Table 1). The highest activation energies result from electron donating amino groups X, Y = NHMe (Figure 3 and Figure 4, E a trans = 10.67, E a cis = 10.47 kcal mol -1 , Table 1, Scheme 2). Such electronic tunings of the ligands strongly affect the reactivity and give rise to increased or decreased electrophilicity of Pd-allyl intermediates.</p><p>The reaction energies (E r ) for ammonia addition to the Pd-η3allylic intermediate show a similar preference: Pd-ene-adduct formation is favoured most for X, Y = NO 2 (E r trans = 0.29, E r cis Scheme 2: Activation (ΔE a ) and reaction (ΔE r ) energies (kcal mol -1 ), computed for the P,N-ligand model with tuneable electronic differentiation. = -0.25 kcal mol -1 ) and becomes most unfavourable (i.e. endothermic) for X, Y = NHMe (E r trans = 10.98, E r cis = 10.33 kcal mol -1 , Table 1, Scheme 2). This points to a more π-donating character of the ene product relative to the allyl-cation reactant. In agreement with the "trans to phosphorus" rule, [23][24][25][26][27][28] attack of ammonia is preferred for most X, Y combinations trans to P, due to the stronger π*/σ* acidity at P in phosphabenzene relative to N in pyridine (Table 1).</p><p>[44] Surprisingly however, this electronic site selectivity, as it is measured from relative energies of the transition structures (ΔE a TS ), is not largest for different X, Y donor-acceptor combinations (Figure 5, Figure 6, Figure 7 and Figure 8), but is highest for X and Y = NO 2 (ΔE a TS = 0.33 kcal mol -1 , Table 1). Likewise, the smallest electronic site "trans to P" selectivity is not found for X, Y donoracceptor combinations, but for strong donating X and Y = NHMe. Here, the selectivity is so low, that it even inverts to "cis to P" (ΔE a TS = -0.20 kcal mol -1 , Table 1).</p><p>For each phosphabenzene moiety with X = H or NHMe or NO 2 , the "trans to P" site selectivity ΔE a TS increases for pyridine substituents Y in the order NHMe < H < NO 2 (Figure 9, Table 1). Hence, there is apparently an additional effect, which controls the site selectivity ΔE a TS besides the electronic donor vs. acceptor properties of different ligand atoms, i.e. P vs. N. Via this effect; electron withdrawing groups (e.g. NO 2 ) give rise to the highest site-selectivities.</p><p>NO 2 -substituted ligands give rise to earlier transition structures with longer (forming) H 3 N-C α bonds (Table 2, Figure 1 to Figure 8), e.g. trans-TS with X = Y = NO 2 : H 3 N-C α = 2.04 Å (Figure 1). In contrast, amino-donor substitution leads to later transition structures with shorter H 3 N-C α distances, e.g. trans-TS with X = Y = NHMe: H 3 N-C α = 1.866 Å (Figure 3). This agrees with the more electrophilic properties of cationic Pd-allyl intermediates induced by electron withdrawing ligands.</p><p>These positions on the reaction coordinate indeed correspond to the site selectivity of the transition structures, i.e. ΔE a TS : earlier transition structures have higher, later transition structures exhibit lower "trans to P" selectivities (Figure 10).</p><p>The distance between Pd and the allylic systems decreases from early (allyl cation like) to late (ene like) positions on the reaction coordinate. A closer, more intense Pd-C α contact (e.g. 2.674 Å, Figure 2, Table 2) stronger delivers electronic differentiation of the ligand, and hence "trans to P" selectivity. Hence, higher electronic site selectivity closely corresponds to intense Pd-allyl interactions with short Pd-C α distances (Figure 11).</p><p>Apparently, the positions on the reaction coordinate influence the site selectivity even stronger than the electronic differentiation between P and N ligand atoms: No substitution (X = Y = H) gives rise to even higher ΔE a TS than more pronounced electronic differentiations with X, Y = NO 2 or NHMe (Figure 11), due to higher TS-sensitivity originating from closer Pd-allyl contact.</p><!><p>In Pd-catalyzed allylic substitutions, the electronic site selectivity, i.e. the preference for "trans to P" addition, is affected by the intrinsic electronic differentiation of the ligand atoms, e.g. P vs. N. However, the sensitivity for this electronic differentiation depends on the intensity of the Pd-allyl interaction. A close Pd-allyl distance in an early, allyl cation like transition structure delivers the electronic differentiation of the ligand system more efficiently to the allylic termini (C α ) than a more distant Pd-allyl (more ene like) unit of a late transition structure. Electron withdrawing (e.g. NO 2 ) substituents in the ligand system generate earlier transition structures with more intense Pd-allyl interactions and higher sensitivity for electronic differentiations. Hence, both intrinsic electronic differentiation in the ligand and high TS-sensitivity appear to be crucial for high site-selectivity in Pd-catalyzed allylic substitutions.</p><!><p>All structures were fully optimized and characterized by frequency computations as minima or transition structures using Gaussian 03 [49] with standard basis sets [50,51] and the B3LYP [52][53][54][55] hybrid-DFT method. Zero point energies and thermochemical analysis were scaled by 0.9806. [56]</p>

Beilstein

Formal Synthesis of (\xe2\x88\x92)-Englerin A and Cytotoxicity Studies of Truncated Englerins

An efficient formal synthesis of (\xe2\x88\x92)-englerin A (1) is reported. The target molecule is a recently isolated guaiane sesquiterpene that possesses highly potent and selective activity against renal cancer cell lines. The developed strategy proceeds in an enantioselective manner by constructing the BC ring system of 1 via a Rh(II)-catalyzed [4+3] cycloaddition and subsequently attaching the A ring via an intramolecular aldol condensation reaction. As such, this strategy allows the synthesis of truncated englerins. Evaluation of these analogues in A498 renal cancer cell line suggests that the A ring of englerin is crucial to its antiproliferative activity. Moreover, evaluation of these analogues led to the identification of potent growth inhibitors of CEM cells with GI50 values ranging from 1\xe2\x80\x933 \xce\xbcM.

formal_synthesis_of_(\xe2\x88\x92)-englerin_a_and_cytotoxicity_studies_of_truncated_englerins

Introduction<!>Retrosynthetic analysis<!>[4+3] Rh-catalyzed cycloadditions<!>Formation of the A ring via olefin metathesis<!>Formation of the 5-membered ring (A-ring) via an intra-molecular aldol condensation and completion of the synthesis<!>Cytotoxicity studies of truncated englerins<!>Conclusion<!>(1R,5S)-(R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl3-[(tert-butyldimethylsilyl)oxy] -5-isopropyl-1-methyl-8-oxabicyclo[3.2.1]octa-2,6-diene-2-carboxylate (8)<!>(1R,5S)-5-isopropyl-1-methyl-2-methylene-8-oxabicyclo[3.2.1]oct-6-en-3-one (17)<!>(1S,2S,5R)-2-hydroxy-1-isopropyl-5-methyl-4-methylene-8-oxabicyclo[3.2.1]oct-6-en-3-one (19)<!>(1S,2S,5R)-1-isopropyl-5-methyl-4-methylene-3-oxo-8-oxabicyclo[3.2.1]oct-6-en-2-yl acetate (37)<!>(1S,2S,5R)-1-isopropyl-5-methyl-4-methylene-3-oxo-8-oxabicyclo[3.2.1]oct-6-en-2-yl benzoate (38)<!>(1S,2S,5R)-1-isopropyl-5-methyl-4-methylene-3-oxo-8-oxabicyclo[3.2.1]oct-6-en-2-yl cinnamate (39)<!>(1R,5S,6S)-1-isopropyl-5-methyl-2-oxo-8-oxabicyclo[3.2.1]oct-3-ene-6-carbonitrile (42)<!>3H-Thymidine incorporation assay

<p>The need to identify new chemical motifs as potential drug leads has spurred the screening of plant extracts used in traditional African, Ayurvedic (Indian) and Chinese medicines.[i] In particular, South Africa has a remarkable botanical diversity with over 30,000 flowering species, from which more than 3,000 are used for medicinal purposes throughout the country.[ii] Among them, plants of the genus Phyllanthus (Euphorbiaceae) are widely distributed and have long been used in African folk medicine to treat kidney and urinary tract infections.[iii] With this in mind, the Beutler laboratory has been screening extracts of the Tanzanian plant Plyllanthus engleri against renal cell carcinoma (RCC) and has recently reported the isolation of two novel bioactive sesquiterpenes, named englerin A (1) and englerin B (2) (Figure 1).[iv]</p><p>Preliminary biological investigations[4] have shown that 1 possesses very potent growth inhibitory activity (GI50 = 1–87 nM) against RCC with approximately 1,000-fold tissue selectivity as compared to other carcinomas. These findings are of particular significance since RCC: (a) is among one of the ten leading cancer types in the US;[v] (b) is characterized by a lack of early warning signs, has diverse clinical manifestations and shows resistance to radiation;[vi] and (c) cannot be effectively treated with current chemotherapeutic agents, leaving surgical procedures as the only treatment option.[vii]</p><p>Biogenetically, the englerins belong to the family of guaiane natural products in which a common bicyclic sesquiterpene skeleton has undergone a sequence of oxygenations and oxo-cyclizations.[viii]</p><p>Further decoration at the periphery of this core produces the structures of 1 and 2. Due to the combination of uncommon structural architecture, potent and selective cytotoxicity against RCC and promising pharmacology, the englerins have received the attention of the chemical community. Since their isolation in 2009, five total syntheses[ix] and many strategies and studies[x] have been reported. These studies also led to the identification of synthetic englerin analogues, such as 3, 4 and 5, products of various esterifications of the common tricyclic core. Several recent reviews nicely summarize the current status of englerins.[xi] Our continuous interest in exploring natural products from ethnomedicine as medicinal leads[xii] has prompted us to design an enantioselective strategy toward englerins.[xiii] In this account we describe in detail the results of our efforts and the biological evaluation of structurally simplified englerin analogues.</p><!><p>Our retrosynthetic strategy is shown in Scheme 1. We anticipated that a sequence of reactions including: inversion of the C6 stereocenter, hydrogenation of the C4-C5 alkene (englerin numbering) and selective esterifications at C6 and C9 hydroxyl groups would form englerin (1) from diol 6. Compound 6 could be derived from enone 7 following a C3 deoxygenation and a regioselective hydroboration of the C8–C9 double bond. We theorized that the cyclopentene moiety of 7 could be constructed from an intramolecular aldol condensation, while an α-hydroxylation would produce the C6 hydroxyl moiety. Disconnection along these lines led us to target the construction of bicyclic motif 8 possessing the BC ring scaffold of englerins. We hypothesized that compound 8 could be formed in enantiomerically pure form by exploring a Rh(II)-catalyzed [4+3] cycloaddition between readily available furan 9, representing the C ring of 1, and chiral pantolactone-derived diazoester 10.</p><!><p>Rhodium-triggered cyclization reactions have been widely applied in synthetic chemistry.[xiv] In 1996, Davies et al. reported an elegant enantioselective Rh(II)-catalyzed [4+3] cycloaddition between furans and chiral diazoesters.[xv] Selection of the appropriate pantolactone enantiomer, as the chiral auxiliary, allows diastereo-control of the produced oxy-bridged adduct. The transfer of chirality, observed in this reaction, can be rationalized by considering that the carbonyl moiety of pantolactone interacts with the Rh-carbenoid as shown in Scheme 2. This interaction blocks one of the two faces of the carbene 10 leading to a selective si-face attack by the 2-methyl-5-isopropyl-furan (9). The stereochemical outcome is consistent with a tandem cyclopropanation/Cope rearrangement (see intermediate [11]). Cyclopropanation of the achiral furan 9 was expected to occur in a regioselective manner at the less substituted and thus less hindered C9-C10 double bond. The potential of this cycloaddition to construct bicyclic structure 8 was of great interest to our strategy, as it provided an efficient, rapid and enantioselective access to the englerin core. Notably, both of starting materials 9[xvi] and 10[15a,xvii] can be readily prepared in more than 50 gram-scale from commercially available materials in 3 steps.</p><p>To the best of our knowledge, 2,5-disubstituted furans have not been evaluated as substrates in the Rh(II)-catalyzed [4+3] cycloaddition. With this in mind, we initially evaluated the regioselectivity of this reaction using achiral diazoester 14[xviii] as a model system (Scheme 3). The synthesis of furan 9 is highlighted in Scheme 3. Commercially available 2-methyl furan (12) was treated with n-BuLi and acetone followed by the dehydration (Ac2O/KOAc) to afford alkene 13. A regioselective hydrogenation of 13 proceeded smoothly by controlling the reaction time to 1 hour and the sequential distillation delivered the desired 2,5-disubstituted furan 9. Notably, this 3-step preparation of 9 can be done in more than 50 gram-scale and only one distillation is needed for purification. Refluxing of 9 and diazoester 14 in hexane in the presence of catalytic amounts of rhodium(II) octanoate (2 mol%), afforded oxacyclic product (±)-15 as the only regio-isomer in excellent yields (95%). Cleavage of the ester auxiliary proved to be more challenging than anticipated. In fact, the reported LiAlH4 reductive cleavage led only to decomposition.[15a] However, treatment of ester 15 with DIBAL-H yielded the labile β-hydroxy silyl enol ether 16 that, upon immediate treatment with stoichiometric amounts of BF3•Et2O, underwent rearrangement[xix] to afford exocyclic enone (±)-17 in good yield (81%).</p><p>Encouraged by these results, we synthesized chiral diazoester 10 departing from (R)-pantolactone.[15a,17] The Rh(II)-catalyzed [4+3] cycloaddition of 10 with 9 proceeded efficiently with Rh2(OOct)4 to yield 8 in good yield (90%) albeit in moderate diastereoselectivity (dr 3:1, calculated via 1H-NMR) (Scheme 4). It should be noted that, under the same conditions, use of Rh2(OAc)2 led only to decomposition of the starting materials. Moreover, attempts to increase the d.r. by performing this reaction at lower temperature or by using less catalyst loading led to a significant decrease in the yield without any significant enhancement of diastereoselectivity. Table 1 highlights our efforts to optimize this cycloaddition. The diastereomeric mixture of 8 could be separated via a silica gel column chromatography. Our previously established reductive cleavage conditions were applied to 8 to produce optically active enone (−)-17, although the yield was significantly lowered (59%). This decrease of yield may be due to the presence of the two reactive carbonyl moieties in 8 that require extended reaction times and excess of DIBAL-H (7.5 equiv) as compared to reduction of (±)-15 (2.5 equiv), leading to partial decomposition of the sensitive silyl enol ether moiety.</p><p>The next attempts of installing a hydroxyl group on the C6 carbon of (−)-17 with Davis oxaziridines[xx] proved unsuccessful. However, Rubottom oxidation[xxi] provided the desired hydroxy enone 19 in good yield (36%; 87% brsm), although with the inverse stereochemistry as compared to the englerin C6 hydroxyl moiety. The absolute stereochemistry of hydroxyl enone 19 was established by a single crystal X-ray analysis,[xxii] which simultaneously confirmed the absolute stereochemistry of oxy-bridged ester 8. We were pleased to observe that the Rubottom oxidation proceeded regioselectively at the more electronically rich TMS enol ether without affecting any other alkenes in this molecule. The stereochemical outcome of this reaction was also satisfactory since it proceeded exclusively from the top face of the intermediate TMS-enolate [18] suggesting that this face is less hindered. We predicted that this selectivity would allow us to have complete substrate control during the following steps and invert the C6 stereochemistry at a later stage in our synthesis.</p><!><p>The next stage of the synthesis involved constructing the tricyclic core of englerin from compound 19. We envisioned to accomplish this cyclization by the means of a Grubbs ring closing metathesis (RCM)[xxiii] of precursor 21 (Scheme 5) that after oxidation of C6 hydroxyl group would reveal a conjugate system for 1,4-addition of methyl nucleophile at the C4 site. To this end, a Lewis acid-promoted conjugate allylation with allyltrimethylsilane afforded the corresponding ketone 20 in moderate yield (68%) as a single diastereomer. The ensuing olefination of C5, however, proved to be unexpectedly challenging. After attempting many different methods including Wittig reaction, Peterson olefination,[xxiv] Petasis[xxv] and Tebbe olefination[xxvi] etc., only the TiCl4-assisted Nysted olefination[xxvii] gave low yields (15-36%) of trialkene 21. Despite the inefficient formation of 21 we attempted the RCM. We were pleased to see that this reaction proceeded smoothly in almost quantitative yields to give allylic alcohol, which after TPAP/NMO oxidation,[xxviii] formed α,β-unsaturated ketone 22. Unfortunately, all efforts to add methyl nucleophiles to 22 via conjugate addition were unsuccessful. To overcome this issue, we sought to perform the RCM on substrate 24 that contains the challenging C11 methyl group. Consequently, we allylated 19 with methylallyltrimethyl silane. While we were successful in producing 24, olefination of this precursor at C5 resulted in irreproducible results. This prompted us to abandon this approach and seek an alternative strategy for the formation of the 5-membered ring from 19.</p><!><p>Based on the above results, we pursued an alternative strategy for the construction of the tricyclic core of englerin based on an intramolecular aldol condensation. Initially, we explored the feasibility of this reaction using non-hydroxylated enone 17 as a model system. To this end, treatment of propanal with thiazolium salt 26 and enone 17 under Stetter conditions[xxix] produced diketone 27 (Scheme 6). To our delight, the intramolecular aldol reaction of 27 proceeded under mild conditions (KOH/EtOH, rt, 24 hours) to afford 28 in good yield (76%).</p><p>Motivated by this result, we converted 19 to the C6 silyl ether 29 and treated this product with propanal under the previously established Stetter conditions. This reaction proceeded efficiently under basic conditions to furnish diketone 30 in good yield (75% over two steps) as a single diastereomer. However, application of previously successful KOH condensation procedure only resulted in deprotection of the TBS ether with no further reaction. This prompted an extensive investigation on this intramolecular condensation, with different C6 protecting groups (H-, MOM-, TES-, etc.), various bases (t-BuOK, KOH, NaOMe, LDA, etc.) and several reaction conditions. Many failed attempts of the aldol condensation to provide the tricyclic motif proved this reaction to be unexpectedly difficult. Eventually, treatment of diketone 30 with NaHMDS afforded the corresponding aldol addition product, which underwent sequential dehydration process (NaOMe/MeOH, heat) to produce the key tricyclic core 7 in an acceptable yield (36%; 43% brsm). NaBH4 reduction of enone 7 yielded allylic alcohol 31 (99%) as a single diastereomer that, without further purification, was protected as the benzyl ether to furnish 32 in good yield (71%). Treatment of 32 with BH3•THF followed by H2O2 oxidation afforded regio- and stereoselectively alcohol 33 (60%). Silylation of 33 followed by deprotection of the C3 benzyl ether yielded 35 via 34 in 99% overall yield. (Scheme 7). Our efforts to hydrogenate the tetra-substituted alkene of 35 using different catalysts and H2 pressure were unsuccessful presumably due to the steric hindrance of the double bond (C4-C5). This prompted us to deoxygenate the C3 hydroxyl group under Barton-McCombie conditions[xxx] (40% yield). In our efforts to improve this transformation, we discovered that dehydration of 35 with Burgess reagent[xxxi] followed by standard hydrogenation improved significantly the yield to 90% over the two steps. Deprotection of the di-TBS ether 36 with TBAF gave poor results, however, we were pleased to find that a microwave accelerated reaction under similar conditions (TBAF/THF, 80 °C) made diol 6 in a quick and high yielding conversion (45 min, 93%).</p><p>To access the natural product from diol 6, we would need to achieve stereoselective saturation of the tetra-substituted bond followed by esterification of the C9 hydroxyl group, inversion of C6 stereocenter by oxidation/reduction sequence and finally esterification in the presence of cinnamic acid. An alternative synthesis of (+)-6 was reported by Ma et. al.[9b] Our synthetic route to (+)-6 provides another novel and facile method for the construction of englerin A and related compounds. More significantly, the Rh-catalyzed [4+3] annulation/ intramolecular aldol condensation sequence presents a very useful, practical and general method for the preparation of the guaiane sesquiterpene core present in englerin related compounds.</p><p>The intriguing bioactivity of englerin has prompted several groups to perform SAR studies.[9f,10e] These studies have focused exclusively on modifications of the C6 and C9 side chains. An advantage of our strategy is that it can produce in an efficient and stereoselective manner compound 19, which represents the BC ring system of englerins. In other words, this approach could provide information on the biological significance of the A ring of englerin. With this in mind, we synthesized truncated englerins 37, 38 and 39 (Scheme 8). To further expand such SAR studies, we designed a new approach towards compound 46 representing the nor-A-ring englerin A. The synthesis of 46 is highlighted in Scheme 9 and is inspired by Wender's pioneering [5+2] cycloaddition reactions.[xxxii,9d] 2-Methyl-5-furfural (40) was first alkylated under Grignard conditions and the resulting alcohol was converted to hemiacetal 41 in 75% overall yield. Treatment of 41 with excess acrylonitrile (iPr2NEt/MsCl, 100 °C, 14 h) under gave us the desired bicyclic product 42 in 32% yield. Moreover, we found that this reaction could be accelerated under microwave conditions with improved yield (iPr2NEt/MsCl, 150 °C, 4 h, 45%). To the best of our knowledge, this is the first example of microwave accelerated this type of [5+2] cycloaddition reaction. The Luche reduction[xxxiii] followed by hydrogenation provided compound 43[xxxiv] in almost quantitative yield. The transformation from 43 to 44 was accomplished in a 3-step sequence that included: (a) oxidative cleavage of the cyanide group to the corresponding ketone; (b) esterification with cinnamic acid under Yamaguchi condition;[xxxv] (c) hydride reduction of C9 carbonyl moiety. The last two steps were performed following the reported method[9b] to furnish 46. Moreover, the acetate analogue 45 was readily prepared from acetylation of 44.</p><!><p>The cytotoxicity of the truncated englerins was evaluated in both A498 renal cancer cells and CEM T-cell acute lymphoblastic leukemia (T-ALL) cells using a 3H-thymidine incorporation assay. In our initial study, we compared the growth inhibitory activity of englerin A to that of truncated englerins 44 and 46. We found that englerin A inhibited the growth of A498 renal cancer cells with a GI50 of 45 nM which is in agreement with previous findings (Figure 2).[4,9d,9f,10e] However, compounds 44 and 46 did not have any effect on the growth of A498 cells even at concentrations of 100 nM or greater (Figure 2). These results suggest that the A ring is essential for the growth inhibitory activity of englerin A in renal cancer cells.</p><p>We then evaluated the antiproliferative activity of englerin analogues in CEM cells, a cell line in which englerin A has little activity with a reported GI50 of 20.4 μM.[4] Of all analogues tested, 17, (±)-17, and 19 had significant cytotoxicity with GI50 values of 3.3, 1.8, and 2.4 μM, respectively (Figure 3, Table 2). It is likely that these cytotoxicities are due to the exocyclic enone moiety that acts as a conjugate electrophile with bionucleophiles.[xxxvi] In contrast, englerin A and analogues containing an additional ring, such as 7, had little or no cytotoxicity at concentrations as high as 20 μM. These results suggest that the single ring analogues can target leukemia cells effectively and the addition of structural complexity may result in a loss of cytotoxicity to leukemia cells.</p><!><p>We have accomplished an efficient and enantioselective formal synthesis of englerin A (1), a potent and selective growth inhibitor of renal cancer cells. The synthetic approach to intermediate 6 proceeds in 15 steps from readily available compounds 9 and 10 in 5% overall yield. Key to our strategy is the enantioselective formation of the BC ring of 1 via a Rh(II)-induced enantioselective [4+3] cycloaddition, followed by the construction of the A ring via an intramolecular aldol condensation. Inspired by this sequence, we have also synthesized a small family of truncated englerins and have evaluated their growth inhibitory activities against certain renal cancer and leukemia cell lines. These studies suggest that the A-ring of englerin A plays an important role in its bioactivity and tissue selectivity. Interestingly, compounds (−)-17, (±)-17 and (−)-19 have shown significant growth inhibitory activity against CEM cell lines at low micromolar concentrations (GI50 = 1-3 μM). Consequently, these compounds may represent new lead structures for the development of small molecule therapeutics against leukemia.</p><!><p>A solution of 10 (11.2 g, 31.6 mmol) in dry hexanes (750 mL) was added dropwise over 5.5 hours to a refluxing solution of 9 (7.85 g, 8.8 mL, 63.2 mmol) and rhodium(II) octanoate dimer (492 mg, 0.63 mmol) in anhydrous hexanes (750 mL). The reaction mixture was stirred for an additional 30 minutes, at which time TLC showed no starting material remaining. The reaction mixture was then allowed to cooled down to room temperature, filtered through a silica plug and concentrated. Crude product purification using flash column chromatography (100:1 to 9:1, slow gradient Hexanes: EtOAc) afforded 8.1 g of bicyclic ester 8 as colorless thick oil (57%). [α]D23 = + 36.40 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 6.38 (dd, J = 5.7, 0.5 Hz, 1H), 5.72 (d, J = 5.7 Hz, 1H), 5.40 (s, 1H), 4.04 (q, J = 8.9 Hz, 2H), 2.41 (d, J = 17.4 Hz, 1H), 1.95 - 1.83 (m, 2H), 1.62 (s, 3H), 1.24 (s, 3H), 1.17 (s, 3H), 1.00 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.92 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 172.4, 164.2, 157.1, 141.6, 126.8, 117.9, 88.5, 82.8, 76.3, 75.0, 40.2, 37.1, 34.3, 25.8, 23.3, 21.5, 20.6, 18.4, 17.2, 17.1, −3.4, −3.5. HRMS (FAB) m/z: cacld for C24H38O6SiNa(M+Na+) 473.2330, found: 473.2331.</p><!><p>To a solution of 8 (20.7 g, 45.9 mmol) in dry CH2Cl2 (459 mL) was added quickly dropwise via addition funnel a 1.0 M solution of DIBAL-H in heptanes (344.3 mmol, 344.3 mL) at −78°C. After completion of addition, the reaction was stirred for 15 min at which time TLC showed no starting material. The reaction mixture was quenched with saturated Rochelle salt solution (300 mL), was allowed to reach room temperature and was stirred for 1 hour. The reaction mixture was filtered through Celite and washed with CH2Cl2 until TLC showed no more crude product remaining in the filter cake. The filtered mixture was separated, extracted with CH2Cl2 (2 × 150 mL), washed with brine (300 mL) dried over Na2SO4, filtered, and concentrated under reduced pressure to 500 mL of CH2Cl2. This solution was flushed with Argon and treated directly with BF3•Et2O (68.9 mmol, 8.7 mL) dropwise at −30 °C. 5 min after addition TLC showed no starting material. The reaction mixture was further diluted with CH2Cl2 (250 mL), quenched with saturated NaHCO3 solution (350 mL) and extracted with CH2Cl2 (2 × 200 mL). The combined organic layers were washed with brine (300 mL), dried over Na2SO4, filtered, and concentrated under vacuum. Crude product purification using flash column chromatography afforded 5.2 g of ketone 17 as a yellow oil (59%). [g=a]D23 = + 103.01 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 6.05 (d, J = 5.8 Hz, 1H), 5.96 (s, 1H), 5.92 (d, J = 5.7 Hz, 1H), 5.24 (s, 1H), 2.56 (d, J = 17.7 Hz, 1H), 2.46 (d, J = 17.7 Hz, 1H), 2.00 – 1.89 (m, 1H), 1.61 (s, 3H), 0.99 (d, J = 4.9 Hz, 3H), 0.98 (d, J = 4.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 197.8, 147.3, 136.3, 133.4, 115.3, 89.8, 84.9, 45.9, 33.7, 19.8, 17.3, 17.3. HRMS (FAB) m/z: cacld for C12H16O2Na (M+Na+) 215.1043, found: 215.1045.</p><!><p>n-Butyllithium (77.6 mL, 124.2 mmol, 1.6 M in hexane) was added dropwise to a solution of dry diisopropylamine (19 mL, 135.0 mmol) in dry THF (800 mL) at −78°C. The reaction mixture was stirred for 15 minutes, and then a solution of 17 (5.2 g, 27.0 mmol) in dry THF (500 mL) was added quickly dropwise to the reaction mixture. The reaction mixture temperature was raised from −78 °C to 0 °C over 45 minutes and stirred for 1 hour at 0 °C. The reaction mixture was cooled down to −78 °C and TMSCl (15.8 mL, 124.2 mmol) was added dropwise. The reaction mixture temperature was raised from −78 °C to 0 °C over 45 minutes and stirred for 1 hour at 0°C at which time TLC showed no starting material. The reaction mixture was diluted with hexane (650 mL) quenched with 5% NaHCO3 solution (500 mL), washed with brine (300 mL), dried over Na2SO4, filtered, and concentrated under vacuum. To a solution of the crude enol ether product in CH2Cl2 (250 mL) a solution of NaHCO3 (250 mL, 10%) was added all at once at 0°C. Then a solution of mCPBA (5.2 g, 30.0 mmol) in CH2Cl2 (60 mL) was added slowly while vigorous stirring of the reaction mixture. The reaction was closely monitored by TLC. When traces of enol ether (<5%) was observed the reaction was further diluted in CH2Cl2 (200 mL), quenched with saturated NaHSO3 solution (300 mL), allowed to reach room temperature, extracted with CH2Cl2 (2 × 200 mL), washed with brine (300 mL), dried over Na2SO4, filtered, and concentrated under vacuum to approximately 250 mL. To this solution of crude epoxide product was added a solution of (COOH)2 (15.6 g, 124.2 mmol) in MeOH (150 mL). After 30 min of stirring at room temperature TLC showed no epoxide product remaining. The reaction mixture was slowly quenched with saturated K2CO3 solution until neutral pH was achieved and extracted with CH2Cl2 (2 × 200 mL). The combined organic layers were washed with brine (300 mL), dried over Na2SO4, filtered, and concentrated under vacuum. Crude product purification using flash column chromatography (100:1 to 4:1, hexanes: EtOAc) afforded 17 (2.7 g) and α-hydroxy ketone 19 as a crystalline solid (2.2 g, 36%; 87% b.r.s.m). Recrystallization from hexanes afforded high purity crystals for X-Ray characterization. [g=a]D23 = + 101.00 (c 1.3, CHCl3). 1H NMR (500 MHz, CDCl3) δ 6.08 (dd, J = 5.8, 0.9 Hz, 1H), 6.00 (d, J = 5.9 Hz, 1H), 5.97 (s, 1H), 5.28 (s, 1H), 3.81 (d, J = 6.4 Hz, 1H), 2.37 – 2.28 (m, 1H), 1.62 (s, 3H), 1.05 (d, J = 6.9 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 199.0, 146.9, 140.2, 130.1, 116.5, 93.4, 85.5, 73.3, 28.4, 19.4, 17.7, 17.0. HRMS (ESI) m/z: cacld for C12H16O3Na (M+Na+) 231.0992, found: 231.0993.</p><!><p>To a solution of 19 (5 mg, 0.024 mmol) in pyridine (0.25 mL) was added 4-DMAP (0.6 mg, 0.005 mmol) and Ac2O (0.011 mL, 0.12 mmol). The reaction was heated to 60 °C for 3 hours. The reaction was cooled down to room temperature quenched with saturated NaHCO3 and extracted 3 times with ethyl acetate. The combined organic layers were dried over MgSO4, concentrated and the residue was purified by preparatory plate chromatography to yield 37 as a white foam (5.7 mg, 95%). 1H NMR (400 MHz, CDCl3) δ: 6.16 (d, J = 5.9 Hz, 1 H), 6.02 (d, J = 5.9 Hz, 1 H), 6.00 (s, 1H), 5.30 (s, 1H), 5.27 (s, 1H), 2.16 (s, 3H), 2.16 (m, 1H), 1.65 (s, 3H), 0.95 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ: 194.1, 170.0, 146.6, 141.2, 129.3, 116.9, 92.6, 85.7, 71.7, 29.9, 28.8, 19.5, 17.4, 17.1. HRMS (ESI) m/z: cacld for C14H18O4 Na (M+Na+) 273.1097, found: 273.1098.</p><!><p>To a stirred mixture of benzoic acid (7.3 mg, 0.06 mol) and 19 (6.5 mg, 0.026 mol) in dry toluene (0.55 mL) were added successively NEt3 (15 μL, 0.11mol) and 2,4,6-trichlorobenzoyl chloride (9 μL, 0.075 mol). The reaction mixture was stirred for 10 min, then catalytic amount of 4-DMAP (1 crystal) was added. After 16 h of stirring at room temperature, the reaction was diluted with ethyl acetate, and the organic phase was washed successively with aqueous HCl solution (1M), saturated sodium bicarbonate solution, and brine. The organic phase was dried over MgSO4 and concentrated in vacuum after filtration. The residue was purified by flash chromatography to afford the benzoic keto-ester 38 (9.1 mg, 90%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 8.10 (m, 2H), 7.57 (m, 1H), 7.24 (m, 2H), 6.21 (dd, J = 5.8, 0.9 Hz, 1H), 6.09 (d, J = 5.8 Hz, 1H), 6.02 (s, 1H), 5.51 (s, 1H), 5.32 (s, 1H), 2.25 (m, 1H), 1.69 (s, 3H), 0.96 (d, J = 6.9 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 193.8, 165.5, 146.7, 141.1, 133.4, 130.1, 129.4, 128.4, 92.8, 85.6, 72.0, 28.9, 19.6, 17.4, 17.1. HRMS (ESI) m/z: cacld for C19H20O4 Na (M+Na+) 335.1254, found: 335.1256.</p><!><p>To a stirred mixture of cinnamic acid (8 mg, 0.053 mol) and 19 (5.5 mg, 0.026 mol) in dry toluene (0.5 mL) were added successively NEt3 (11 μL, 0.08 mol) and 2,4,6-trichlorobenzoyl chloride (7 μL, 0.07 mol). The reaction mixture was stirred for 10 min, then catalytic amount of 4-DMAP (1 crystal) was added. After 16 h of stirring at room temperature, the reaction was diluted with ethyl acetate, and the organic phase was washed successively with aqueous HCl solution (1M), saturated sodium bicarbonate solution, and brine. The organic phase was dried over MgSO4 and concentrated in vacuum after filtration. The residue was purified by flash chromatography to afford compound 39 (7.8 mg, 88%) as a colorless oil: 1H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 15.9 Hz, 1H), 7.52 (m, 2H), 7.39 (m, 3H), 6.52 (d, J = 16.0 Hz, 1H), 6.19 (d, J = 5.8 Hz, 1H), 6.06 (d, J = 5.8 Hz, 1H), 6.03 (s, 1H), 5.42 (s, 1H), 5.33 (s, 1H), 2.22 (m, 1H), 1.68 (s, 3H), 0.96 (t, J = 6.9 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ 194.1, 166.0, 146.5, 141.2, 134.3, 130.7, 129.4, 129.0, 128.4, 117.2, 116.9, 92.8, 85.7, 71.7, 28.8, 19.6, 17.5, 17.1. HRMS (ESI) m/z: cacld for C21H22O4Na(M+Na+) 361.1416, found: 361.1417.</p><!><p>To a solution of 41 (1.6 g, 11.0 mmol) in acrylonitrile (60 mL) was added diisopropylethylamine (2.3 mL, 13 mmol), followed by methanesulfonyl chloride (1.0 mL, 13 mmol). The resulting solution was microwave heated at 150°C for 4 hours. After cooling to room temperature, the reaction mixture was filtered through a plug of Celite, and the filtrate was concentrated in vacuo. Flash chromatography of the residue (20% ethyl acetate in hexanes) provided the cycloadduct 42 (880 mg, 45%) as colorless oil. 1H NMR (500 MHz, CDCl3) δ: 6.93 (d, J = 9.7 Hz, 1H), 6.01 (d, J = 9.8 Hz, 1H), 3.06 (dd, J = 9.2, 2.9 Hz, 1H), 2.52 (dd, J = 14.3, 2.9 Hz, 1H), 2.30 (p, J = 7.0 Hz, 1H), 2.19 (dd, J = 14.3, 9.3 Hz, 1H), 1.74 (s, 3H), 1.08 (d, J = 6.9 Hz, 3H), 1.04 (d, J = 6.9 Hz, 3H) 13C NMR (126 MHz, CDCl3) δ: 196.7, 151.8, 128.1, 118.8, 100.0, 90.4, 37.4, 35.2, 30.0, 21.6, 17.6, 16.5. HRMS (ESI) m/z: calcd for C12H15NNaO2 (M+Na+) 228.0995, found: 228.0996.</p><!><p>CEM cells were plated at 10-20 × 103 cells/well and A498 cells at 3,500 cells/well in 96-well plates in RPMI supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 u/ml penicillin/streptomycin (complete medium). Englerin A or its analogues were added to the cells at increasing concentrations and 0.1% DMSO was added to control cells. Cells were incubated for 48 hours and then pulsed with 3H-thymidine for 6 hours. Incorporation of 3H-thymidine was determined in a scintillation counter (Beckman Coulter Inc., Fullerton, CA) after cells were washed and deposited onto glass microfiber filters using a cell harvester M-24 (Brandel, Gaithersbur, MD).</p>

PubMed Author Manuscript

Binding Between G-quadruplexes at the Homodimer Interface of the Corn\nRNA Aptamer Strongly Activates Thioflavin T Fluorescence

SUMMARY Thioflavin T (ThT) is widely used for the detection of amyloids. Many unrelated DNAs and RNAs that contain G-quadruplex motifs also bind ThT, and strongly activate its fluorescence. To elucidate the structural basis of ThT binding to G-quadruplexes and its fluorescence turn-on, we determined its co-crystal structure with the homodimeric RNA \'Corn\', which contains two G-quadruplexes. We find that two ThT molecules bind in the dimer interface, constrained by a G-quartet from each protomer into a maximally fluorescent planar conformation. The unliganded Corn homodimer crystal structure reveals a collapsed fluorophore-binding site. In solution, Corn must fluctuate between this and an open, binding-competent conformation. A co-crystal structure with another benzothiazole derivate, thiazole orange (TO) also shows binding at the Corn homodimer interface. As the bound ThT and TO make no interactions with the RNA backbone, their Corn co-crystal structures likely explain their fluorescence activation upon sequence-independent DNA and RNA G-quadruplex binding.

binding_between_g-quadruplexes_at_the_homodimer_interface_of_the_corn\nrna_aptamer_strongly_activate

INTRODUCTION<!>ThT Activates Corn Fluorescence More Strongly than the Cognate Ligand<!>ThT is Bound at the Corn Homodimer Interface in a Planar Conformation<!>The Fluorophore-binding Pocket Collapses in Unliganded Corn<!>The Asymmetric Non-specific Fluorophore Thiazole Orange Binds Symmetric\nCorn<!>Interfacial Adenines Modulate Corn Dimerization<!>DISCUSSION<!>Chemicals<!>RNA Preparation<!>Fluorescence Spectroscopy<!>Quantum Yield Determination<!>Circular Dichroism (CD)<!>Analytical Ultracentrifugation (AUC)<!>Isothermal Titration Calorimetry (ITC)<!>Van\'t Hoff Analysis<!>Crystallization and Diffraction Data Collection<!>Structure Determination<!>Fluorescence Lifetime Measurements<!>Native Polyacrylamide Gel Electrophoresis<!>QUANTIFICATION and STATISTICAL ANALYSIS<!>DATA and SOFTWARE AVAILABILITY<!>CONTACT and REAGENT RESOURCE SHARING<!>EXPERIMENTAL MODEL and SUBJECT DETAILS:

<p>Corn is a 28 nucleotide (nt) in vitro selected RNA aptamer that binds and turns on by over 1,000-fold the fluorescence of DFHO (Figure 1A, 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxyme, 1), a small molecule analog of the intrinsic fluorophore of red fluorescent protein (RFP). DFHO is cell-permeable and non-cytotoxic, and exhibits minimal non-specific binding to cellular nucleic acids. By soaking cells in DFHO, Corn was successfully employed as a genetically encoded fluorescent tag to visualize biological RNAs in live cells (Song et al., 2017). Crystallographic structure determination revealed that Corn homodimerizes and binds one molecule of DFHO at its interprotomer interface (Warner et al., 2017). Each of the protomers folds as a stem-loop, with a four-tiered mixed-sequence quadruplex distal to the stem; the two most distal tiers are both canonical G-quartets. In the complex, DFHO is held into a planar, maximally fluorescent conformation by stacking between two G-quartets, one from each protomer. Notably, the homodimer interface lacks any base pairs, and the bound DFHO is surrounded by six unpaired adenine residues (three from each protomer) that locally break symmetry. Biochemical analyses demonstrated that Corn activates DFHO fluorescence only as a dimer, and that the dimer is very stable (Kd < 1 nM) even in the absence of the small molecule, suggesting some degree of pre-organization of the DFHO binding site (Song et al., 2017; Warner et al., 2017).</p><p>Thioflavin T [Figure 1A, ThT, 4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline, 2] is a benzothiazole dye widely employed for detection and quantitation of amyloids, upon binding which it exhibits an increase in fluorescence (Biancalana and Koide, 2010; Naiki et al., 1989; Vassar and Culling, 1959). Recently, it was reported that ThT binds to G-quadruplex DNAs and RNAs with micromolar dissociation constants and with little sequence preference. Association with G-quadruplexes gives rise to strong fluorescence enhancement, which is generally much lower or absent when ThT is mixed with single-stranded, duplex or triplex DNA or RNA (Gabelica et al., 2013; Li et al., 2016; Mohanty et al., 2013; Renaud de la Faverie et al., 2014; Warner et al., 2017; Xu et al., 2016). In solution, the benzothiazole and aniline rings of ThT lie predominantly at an angle to each other, which leads to non-radiative decay after photoexcitation. Binding to amyloid, or increase of the viscosity of the medium, enhances fluorescence by populating the conformation in which the two rings are coplanar (Biancalana and Koide, 2010; Stsiapura et al., 2008). Molecular dynamics simulations suggested that ThT would bind preferentially to the exposed guanine bases of the human telomeric G-quadruplex DNA, stacking against which may restrain it to a planar conformation, consistent with increased fluorescence (Mohanty et al., 2013).</p><p>The presence of two G-quadruplexes in the structure of Corn-DFHO, and the likely preorganization of its fluorophore binding site, led us to examine whether this aptamer RNA would bind ThT. We found that Corn binds ThT with Kd ~ 3 μM, and despite the difference in structure between DFHO and ThT, strongly (> 10,000 fold) enhances fluorescence of the latter (Warner et al., 2017). In order to elucidate how Corn binds ThT and activates its fluorescence, and to provide a structural framework for understanding how other G-quadruplex nucleic acids preferentially turn on the fluorescence of ThT, we have now determined the crystal structure of the Corn-ThT complex at 2.9 Å resolution. The structure reveals two molecules of ThT occupying the interprotomer space, and the presence of alternate conformations in the crystal structure is consistent with sequence-independent binding to the planar binding site. We also determined crystal structures of Corn bound to thiazole orange {Figure 1A, TO, 1-methyl-4-[(3-methyl-2(3H)-benzothiozolydiene)methyl]quinolinium, 3}, a second benzothiazole derivative that exhibits preferential binding to G-quadruplex nucleic acids, and of the unliganded Corn dimer, at 2.9 A and 2.8 Å resolution, respectively. The former shows one molecule of TO bound at the Corn homodimer interface in two alternate conformations. The latter reveals a collapse of the ligand binding site, such that the G-quadruplexes from the two protomers directly stack on each other. The dimer must sample open and closed conformations in solution, and crystallization has likely selected the latter. In contrast to the locally asymmetric Corn-DFHO complex, the complexes of the dimeric aptamer with ThT and TO, as well as the unliganded aptamer are strictly symmetric. Thus, binding to the asymmetric ligand DFHO induces local symmetry breaking. Because ThT and TO bind DNA and RNA with limited selectivity and therefore produce high background, their complexes with Corn are not suitable for in vivo imaging. However, these complexes are 3 to 5 times brighter than the cognate Corn-DFHO complex, and thus would be useful for applications where brightness rather than selectivity is required.</p><!><p>At the same concentration of Corn aptamer (0.5 μM), and in the presence of excess fluorophore, the RNA-ThT complex fluoresces over three times brighter than the cognate complex between the RNA and DFHO (Figure 1B). Brightness is a function of extinction coefficient (7,250 M−1 cm−1 and 14,760 M−1 cm−1 at their absorption maxima, for DFHO and ThT, respectively) and quantum yield. To calculate the latter, we first established by CD spectroscopy that ThT does not alter the secondary structure of Corn, even at concentrations over 10 times above the apparent Kd (Figure 2A), and that its dimeric state is not altered upon binding ThT, by analytical ultracentrifugation (AUC, Figure 2B). Isothermal titration calorimetry (ITC) analysis yields saturable thermograms that are best fitted by a random two-site binding model with dissociation constants of 2 μM and 11 μM (Figure 2C). Assuming a 2:2 stoichiometry between the Corn and ThT [also consistent with previous Job plot analysis (Warner et al., 2017), and our co-crystal structure, see below], this indicates a quantum yield (Φ) for ThT bound to the RNA of 0.41, which is considerably higher than that reported previously (Song et al., 2017) for DFHO bound to Corn (Φ = 0.25).</p><p>The unexpectedly high quantum yield of the Corn-bound ThT led us to examine whether this RNA aptamer can turn on the fluorescence of thiazole orange, another benzothiazole derivative for which a preferential binding to DNA triplexes and quadruplexes has been reported (Lubitz et al., 2010). Upon incubation with the aptamer, TO fluoresces brightly (Figure 1B). To calculate its quantum yield, we established by titration an apparent Kd of 1.4 μM and an RNA:TO stoichiometry of 2:1 (Figure S1A,B; the poor solubility of TO in aqueous buffers with K+ precluded ITC analysis). Furthermore, AUC experiments confirmed that this fluorophore does not alter the dimeric state of the Corn aptamer RNA, even at high concentrations (Figure S1C). Assuming 2:1 stoichiometry between the Corn RNA aptamer and TO, the quantum yield for the Corn-bound thiazole orange is 0.53, even higher than that of ThT. Consistent with the high quantum yields of the Corn-ThT and Corn-TO complexes, their fluorescence lifetimes are comparable to or longer than, respectively, that of the Corn-DFHO complex (Figure 2D and Figure S1D; Warner et al., 2017). These experiments demonstrate that Corn can strongly turn on the fluorescence of two chemically related non-specific fluorophores, producing quantum yields over 2-fold higher than what it achieves with the RFP-derived fluorophore DFHO against which it was selected in vitro.</p><!><p>The Corn-ThT complex yielded rhombohedral co-crystals with one Corn protomer in the crystallographic asymmetric unit (ASU) (Experimental Section, Table 1). Structure determination revealed that a crystallographic 2-fold axis bisects the ligand binding pocket of the crystallographic Corn dimer (Figure 3A,B), and residual electron density (after building and refining the RNA) suggested that ThT occupies this pocket. The electron density features were consistent with the two bound ThT molecules arranged in an antiparallel ("head-to-tail") orientation in the same plane. At the current resolution limit (2.9 μ), the position of the methyl groups of the benzothiazole of the bound ThT is ambiguous. On steric grounds, we modeled the two heterocycles with the sulfur atoms facing each other, yielding a Rfree factor of 27.1 %. Reversing the orientation of the benzothiazole rings, such that the methyl groups face each other, did not result in an improved crystallographic residual (Rfree = 27.8 %). To estimate the crystallographic occupancy of the bound ThT, we performed parallel refinements in which the occupancy of each ThT was fixed at 0.25, 0.5 and 1.0, and compared the refined B-factors of the ligand atoms with those of the surrounding RNA. This analysis was consistent with an occupancy of 0.5 for each bound ThT. Therefore, in the co-crystal structure, there is one molecule of ThT per RNA protomer (two head-to tail molecules of ThT at an occupancy of 0.5 each), and because of the crystallographic 2-fold axis, the interprotomer interface is occupied by a total of two molecules of ThT. Moreover, because the crystallographic 2-fold axis does not coincide with the non-crystallographic 2-fold rotation relating the two bound ThT molecules, this results in two distinct alternate poses of the bound fluorophore molecules (Figure 3C,D). Since the two bound ThT molecules are in van der Waals contact with each other, sequential binding would result in the first and second ThT molecules encountering different structural environments, consistent with the ITC results. Consistent with the importance of having a G-quadruplex on each side of the bound ThT to achieve high fluorescence intensity, control experiments in which ThT fluorescence was induced by binding to Baby Spinach, in which the fluorophore binding site is flanked by one G-quadruplex and a base triple, resulted in reduced fluorescence intensity (Figure S2A).</p><p>When considering just the structure of a protomer, the structure of the Corn RNA in complex with ThT is very similar to that in its cognate complex with DFHO (Warner et al., 2017). The RNA is a stem-loop with the loop folded back on itself, producing a quadruplex in this region. The quadruplex comprises two canonical G-quartets adjacent to the interprotomer interface (T1 and T2), and two non-canonical quartets comprised of doubled non-canonical A•U pairs and doubled G•C pairs (T3 and T4, respectively). A density feature consistent with a bound K+ ion is present between T1 and T2. Although the K+ and water molecules that coordinate T3 in the Corn-DFHO co-crystal structure are not resolved in the current Corn-ThT complex electron density, the 16 residues of the quadruplex (including T3) superimpose closely between the two structures (r.m.s.d. 0.38 Å), implying that the second K+ ion and inter-base waters are also present in the ThT complex.</p><p>The cognate DFHO and non-cognate ThT complexes of the Corn aptamer differ substantially in their interprotomer dimerization interface. As described previously, binding of the asymmetric DFHO in a planar conformation (which endows it with a plane of mirror symmetry) by the chiral dimer of Corn RNA necessarily results in a quasisymmetric dimer interface, in which the three interfacial adenosines, A11, A14 and A24 from the two protomers adopt distinctly different conformations (Warner et al., 2017). Thus, for instance, only one of the two A14 nucleobases contacts the oxime function of DFHO, and one of the two A11 hydrogen bonds to an imidazolinone nitrogen while the other is extruded from the binding pocket. In contrast, the crystallographic 2-fold symmetry relating the two protomers of the dimeric RNA in the ThT complex arises from the three interfacial adenosines exhibiting precisely the same conformations in the two RNA chains. In this complex, A11, A14 and A24 make no contacts with the bound fluorophores and are arranged so that the nucleobase of A11 of one protomer stacks on that of A24 from the other, and the two pairs of stacked adenosines are situated on the same side of dimerization interface. The riboses and nucleobases of A14 of the two protomers lack interactions with adjacent residues and they are extruded from dimerization interface (Figure 3A,B). This produces an opening that may allow ThT ingress and egress from its binding site.</p><!><p>The fluorophore-free Corn aptamer dimer also yielded rhombohedral crystals with one RNA protomer per asymmetric unit (Experimental Section, Table 1). Except at the dimerization interface, the structure of the unliganded Corn RNA superimposes closely on those of the Corn-DFHO and Corn-ThT complexes (r.m.s.d. for all non-hydrogen RNA atoms omitting the three interfacial adenines of 1.63 ± 0.14 Å and 1.33 Å, respectively), and all the pairing interactions in the G-quadruplexes are conserved with the structures of the DFHO and ThT complexes. In the unliganded structure, electron density for the axial K+ ions of the quadruplex is weak, but precise conservation of the location of all nucleobases suggests that the ions and waters are bound in positions equivalent to those of the DFHO complex structure.</p><p>The most notable difference between the structures of fluorophore-bound and unbound aptamers becomes apparent when the dimerization interface of Corn dimers is compared. In the crystal structure of the unliganded Corn, the fluorophore binding site has disappeared, as the T1 G-quartets of the symmetry-related Corn protomers stack directly on each other (Figure 4A). In this rearranged structure, the nucleobases of the interfacial adenosines adopt conformations similar to those in the ThT complex, but distinctly different from those in the DFHO complex (Figure 4B,C,D). The rotational orientation of the two protomers along the 4-fold axis of the G-quadruplexes is similar in the fluorophore-free and ThT complexes, but different from that of the DFHO complex. Thus, in the unliganded and ThT-bound structures, G12 of one protomer stacks under G22 of the other protomer, and vice versa. In the DFHO complex, G12 of one protomer stacks under G15 of the other protomer, but the second does not stack on the opposite quadruplex. Overall, relative to the unliganded complex, the second protomer of the DFHO-bound complex has undergone a 60° rotation along its quadruplex axis, and this axis has also shifted by 3 Å (Figure 4D).</p><!><p>Since the structures of unliganded and ThT-bound Corn dimers determined in this work exhibit crystallographic (strict) symmetry, whereas the previously reported structure of Corn bound to its cognate, asymmetric ligand DFHO is quasisymmetric, we examined whether the asymmetry of the latter is a consequence of the asymmetry of the ligand by solving the structure of the RNA bound to a second non-specific asymmetric ligand, TO (3). Unexpectedly, the rhombohedral crystals of this complex also have a single RNA chain in the ASU. Structure determination (Methods, Table 1) revealed, consistent with the stoichiometry observed in solution, a single molecule of TO bound at the dimer interface (Figure 5). Because the bound TO is traversed by a crystallographic 2-fold axis, the ligand exists in two distinct poses in the interface (Figure 5B,C). Each asymmetric unit contains a single molecule of TO at an occupancy of 0.5 (adding to a full molecule of TO in the binding site of the crystallographic dimer).</p><p>The overall structure of the TO complex of Corn superimposes closely on the ThT-bound RNA complex (r.m.s.d. = 0.85 Å for all non-hydrogen RNA atom pairs). The similarity extends to the relative orientation of the two protomers, which is indistinguishable from that of the ThT complex. Notably, the arrangement of the interfacial adenines A11, A14 and A24 is also very similar between the two structures, despite the fact that the single TO fluorophore, which is bound in a near planar conformation, leaves a portion of the ligand binding site unoccupied (Figure 5B,C). The modest geometric fit between TO (monomer) and ThT (head-to-tail dimer) and the fluorophore binding pocket of Corn, compared to that of DFHO is also reflected in the shape complementarity statistic (Lawrence and Colman, 1993), which is 0.72, 0.74 and 0.83 for the TO, ThT and DFHO co-crystal structures, respectively.</p><!><p>Previously, point mutants of the interfacial adenines of Corn were evaluated for their ability to turn on DFHO fluorescence (Warner et al., 2017). In that study, it was found that all point mutants led to diminished fluorescence turn-on (ranging from none to 25% of wild-type). The least affected mutant was A11U, which preserved 25% of the brightness of the parental sequence, while A14U exhibited no detectable fluorescence. To complement that study, and in light of the differences in the arrangement of the interfacial adenines between the DFHO- and ThT-bound Corn structures, we tested the point mutants for their ability to turn on the fluorescence of ThT (Figure 6A). We find that, consistent with the lack of interaction of the interfacial mutants with the non-cognate fluorophore (Figure 3C,D) the point mutants substantially retain their ability to turn on the fluorescence of ThT (Figure 6A).</p><p>The A14G and A14U mutants exhibit the largest decrease in enhancement of ThT fluorescence (Figure 6A). Comparison of the CD spectra of wild-type and the two transversion point mutations of residue 14 show that the presence of a pyrimidine at this position does not alter the overall secondary structure of the RNA. Even the (A11U, A14U, A24U) triple mutant exhibits a CD spectrum that differs little from that of wild-type (Figure 6C). Analysis of the oligomerization state of the point mutants over the concentration range 280 nM to 100 μM (monomer basis) by analytical ultracentrifugation, and at 19 μM by native polyacrylamide gel electrophoresis (Figure 6D,E; Figure S3) shows that while the A11G, A11U, A24C, A24G and A24U mutants remain predominantly dimers over this concentration range, A11C exists as a mixture of monomers and dimers, and A14U is predominantly monomeric.</p><p>We further analyzed A14U, because it is predominantly monomeric and previously found to be drastically impaired in its ability to activate DFHO fluorescence (Figure 6B) (Warner et al., 2017). We determined its crystal structure in complex with ThT (Methods and Table 1), and found that its structure is overall identical to that of the wild-type Corn-ThT complex (r.m.s.d. = 0.51 Å for all non-hydrogen RNA atom pairs, excluding residue 14), and also binds to two molecules of ThT at a crystallographic dimer interface (Figure S4). Electron density for A14 is best defined in the Corn-TO co-crystal structure. Comparison of that structure with the A14U mutant co-crystal structure highlights the loss in the point mutant of a potentially stabilizing intra-protomer interaction between the sugar edge of A14 and the Watson-Crick face of G8 (Figure 6F,G). This interaction may stabilize the TO and ThT complexes of the wild-type Corn aptamer, and thus facilitate fluorescence turn-on. Analytical ultracentrifugation of Corn A14U at 100 μM concentration (at which crystallization was carried out) shows a heterogeneous mixture of monomer, dimer, and trimer indicating that the crystallographic structure represents only one of several species coexisting in the sample. If the unliganded Corn dimer transiently samples a conformation stabilized by the interaction between A14 and G8 seen in the wild-type co-crystal structures, the mutation A14U may decrease the dimer population.</p><!><p>The co-crystal structures of the Corn aptamer bound to ThT and TO provide the first experimental description of the modes of nucleic acid binding of these G-quadruplex preferent fluorophores. Both ThT and TO bind in near-planar conformations that maximize their fluorescence, and stack extensively with the exposed guanine bases of the G-quartets. Our structures show that, perpendicular to the planes of their heterocycles, the fluorophores make no hydrogen bonding interactions with the surrounding RNA, and only minimal van der Waals contacts (Figures 3,5). This is consistent with the limited hydrogen bonding potential of the fluorophores, and their reported low sequence specificity. The co-crystal structures suggest that preferential fluorescence turn-on upon binding to G-quadruplexes over duplexes or triplexes by these benzothiazole derivatives reflects both, the larger solvent-exposed non-polar area at the unstacked end of a G-quadruplex, as well as the increased planarity of G-quartets compared to base pairs or base triples, which often exhibit propeller twisting or buckling. The planar, RNA backbone-independent mode of RNA Corn binding observed for ThT and TO contrasts with that reported previously for a variety of G-quadruplex-targeted small molecules as well as the G-quadruplex-specific protein DHX36 (Chen et al., 2018; Haider et al., 2011). Those ligands interact with both, the exposed guanine nucleobases and the phosphodiester backbone. Interaction with the backbone endows some of those small molecules and the helicase protein with a preference for G-quadruplexes with a particular connectivity (e.g., parallel vs. antiparallel). The backbone-independent binding of ThT and TO suggests that the variation in their fluorescence enhancement observed for different G-quadruplexes (Gabelica et al., 2013; Li et al., 2016; Mohanty et al., 2013; Renaud de la Faverie et al., 2014; Warner et al., 2017; Xu et al., 2016) reflects instead the efficiency of DNA or RNA folding, the planarity of the bound fluorophores in the photoexcited state, and the stoichiometry of binding.</p><p>The previously determined Corn-DFHO co-crystal structure revealed that the ligand-binding site of the RNA is locally quasisymmetric (Jones and Ferré-D'Amaré, 2015) with the interfacial adenosines of each protomer participating in distinctly different RNA-fluorophore and RNA-RNA interactions (Warner et al., 2017). In contrast, the Corn-ThT and Corn-TO co-crystal structures show strictly symmetric Corn dimers, suggesting that the symmetry of the RNA binding site is broken only upon binding to DFHO. Consistent with induction of asymmetry by the cognate fluorophore DFHO, the crystal structure of the ligand-free dimeric Corn RNA is also strictly symmetric (Figure 4). In this crystal structure, the ligand binding site has collapsed, the G-quadruplexes from the two protomers stacking directly against each other. Since biochemical analyses show that Corn RNA dimers are pre-formed, it is likely that crystallization has captured a closed conformation, and that open conformations that are conducive to binding, either symmetrically to ThT and TO, or asymmetrically to DFHO, exist in solution. In this regard, it is noteworthy that the distribution of unbound Corn RNA between monomers and dimers is sensitive to mutation of the interfacial adenosines. The co-crystal structure of the A14U mutant in complex with ThT shows that the mutation disrupts an intramolecular RNA interaction that may stabilize the wild-type RNA. Thus, it is possible that A11, A14 and A24, in addition to playing a role in RNA dimerization and ligand binding, also contribute to the stability of the aptamer and, indirectly, to its oligomerization. It has been noted that mutants of Corn that are obligate heterodimers could form the basis of RNA analogues of split GFP (Warner et al., 2017). Our experiments suggest that mutation of the interfacial adenosines, as well as distal residues with which they may interact in the fluorophore-free or bound states may modulate the tendency of the RNA to homodimerize.</p><p>Although the structural basis of preferential binding and fluorescence activation of ThT by protein amyloids remains unknown, several crystal structures of ThT bound to globular proteins have been reported (Figure S5). A dimeric β2-microglobulin, proposed to be an early aggregate precursor to amyloid, was found to bind either one or four molecules of ThT at an interface between two β-barrel domains (Halabelian et al., 2015). The mixed αβ proteins acetylcholynesterase and butyrylcholinesterase were found to bind either one or two molecules of ThT in a cavity formed at the juncture of several α-helices (Harel et al., 2008; Rosenberry et al., 2017). In all these cases, the ThT binding sites are lined by aromatic amino acid side chains. In contrast to the mode of ThT binding to G-quadruplexes that we have uncovered, these protein binding sites do not accommodate more than one coplanar ThT molecule. Indeed, when multiple ThT molecules are bound, they stack on each other (Halabelian et al., 2015; Harel et al., 2008). Moreover, the protein-bound ThT molecules show variable degrees of rotation between their benzothiazole and aniline rings, unlike the near-planar conformation observed in Corn bound ThT.</p><p>Among the fluorescence turn-on aptamers structurally characterized to date, Corn is unique in that the active RNA species is a dimer. Unlike Spinach and Mango, whose fluorophore binding sites constrain their respective cognate small molecule by sandwiching between a G-quadruplex and either a base triple or unpaired nucleotide 'flaps', respectively, the Corn dimer binding site is flanked on either side by a G-quartet, one from each protomer (Huang et al., 2014; Trachman et ThT. al., 2018; Trachman et al., 2017; Warner et al., 2014; Warner et al., 2017). Comparison of the fluorescence turn-on of ThT by Spinach and Corn (Figure S2) shows that the binding site flanked by two G-quadruplexes is better able to constrain the photoexcited fluorophore. Corn RNA was selected to bind DFHO and turn on its fluorescence. Despite its limited brightness, DFHO is attractive for cellular studies because its non-specific turn-on by RNAs other than Corn is minimal. In contrast, ThT and TO, which yield much brighter fluorescence when bound to Corn also bind and are turned on by a variety of nucleic acids (as well as proteins; Figure S6). While this limits the applications of Corn-ThT and Corn-TO in cellular studies, the high quantum yields of these complexes and their intense fluorescence makes them useful in contexts where selective binding is not required. The Corn-ThT co-crystal structure also shows that a G-quartet has sufficient non-polar area to accommodate two molecules of ThT, and can thus form the basis for the design of new, larger fluorophores that better occupy the non-polar exposed face of G-quadruplex nucleic acids.</p><!><p>DFHO (1) RNA 1 (Key Resources Table) was prepared as previously described (Song et al., 2017) ThT (2) (Key Resources Table) and TO (3) (Key Resources Table) were dissolved freshly for each experiment in 100 mM KCl, 10 mM MgCl2, 50 mM Tris-HCl pH 7.5, or where indicated, in 100 mM KCl, 10 mM MgCl2, 50 mM Hepes-KOH pH 7.5, and filtered (0.22 μm cutoff). Concentrations were calculated from the dry weight of the compounds.</p><!><p>The 36 nt Corn construct previously used for crystallization (Warner et al., 2017) (RNA 1, Key Resources Table), point mutants (A11C; A11G; A11U; A14C; A14G; A14U; A24C; A24G; A24U- respectively: RNA 2, RNA 3, RNA 4, RNA 5, RNA 6, RNA 7, RNA 8, RNA 9, RNA 10, Key Resources Table), and the triple mutant (A11U, A14U, A24U- RNA 11, Key Resources Table) were prepared by in vitro transcription. DNA templates generated by PCR were transcribed in vitro by T7 RNA polymerase as described (Xiao et al., 2008). After 4h, 10 mM CaCl2 and RNase-free DNase RQ1 (Promega) were added and the reaction incubated for 30 min. RNAs were purified by electrophoresis (15% 29:1 acrylamide:polyacrylamide, 1 × TBE, 8 M urea gels), electroeluted, and washed with 1 M LiCl and water and finally exchanged into 100 mM KCl, 50 mM Tris-HCl pH 7.5 using 3 kDa cutoff centrifugal concentrators (EMD Millipore). For refolding, RNAs were heated for 2 min at 95 °C, placed 2 min on ice, then incubated at 65 °C for 5 min after addition of MgCl2 to 10 mM, and cooled from 65 °C to 25 °C in 15 min. Refolded RNAs were stored at concentration 50 μM (monomer basis) at 4 °C and diluted or concentrated by ultrafiltration as appropriate for different experiments. RNAs were quantitated assuming an extinction coefficient of 354,600 M−1 cm−1 at 260 nm.</p><!><p>Fluorescence spectra were recorded with an Easy Life-LS PTI fluorimeter at 20°C. Refolded RNA (0.5 μM, monomer basis) in 100 mM KCl, 10 mM MgCl2, 50 mM Tris-HCl pH 7.5, were incubated with ThT at concentrations ranging from 0.25 μM to 1 mM for 75 min, or for different times (0 – 96 h) at a fixed ThT concentration of 2.5 μM, at 21 °C. Excitation was at 450 nm. For competition of wild type Corn (0.5 μM) for DFHO, ThT and TO (each at 2.5 μM) fluorophores were added two at a time to the folded RNA and incubated for 30 min at 21 ° C. Excitation was at 468 nm, 450 nm, and 510 nm for DFHO, ThT, and TO, respectively. For evaluating the effect of molecular crowding and viscosity, Corn (0.5 μM) was mixed with PEG400 (0 – 15 %, v/v) or glycerol (0 – 30%, v/v) and incubated 10 min with fluorophores (2.5 μM) before fluorescence measurements. Corn affinity for TO was determined by varying the RNA concentration between 125 nM and 8.0 μM while keeping the TO concentration fixed at 2.5 μM. Excitation was at 510 nm and emission was monitored over 515-550 nm. For Corn-TO stoichiometry determination using the Job (Job, 1928) plot, the RNA and the fluorophore were incubated together in 50 mM Hepes-KOH, 100 mM KCl, 10 mM MgCl2, pH 7.5, at 20°C. The total concentration of the two was fixed at 30 μM, while each was varied in 2.5 μM steps over the 0-30 μM range. Excitation was at 510 nm and emission was monitored over 515 – 550 nm. For evaluating specificity of Corn and ThT interaction in presence of other biological macromolecules, ThT was incubated with different amounts of recombinant bovine serum albumin (0.5-50 μM), a 24 nt single-stranded DNA oligonucleotide containing bacteriophage T7 RNA polymerase promoter (0.5-50 μM), or recombinant tRNALys3 (0.5-10 μM) without any Corn or in presence of 0.5 μM Corn; fluorescence was measured after 30 min incubation at 21 ° C.</p><p>Fluorescence life time measurements were performed at 21 ° C; integration time was 1 s nm−1 and each measurement was averaged seven times. A 10-mm path length quartz cuvette was used. ThT-Corn lifetime measurement was performed on EasyLife-LS PTI system equipped with an EL445 diode (Horiba), 445/20-25 and 494/34-25 filter sets (AVR Optics); 7 μM Corn was incubated 10 min with 5 μM ThT. TO-Corn lifetime was measured on same instrument equipped with EL510 diode and 535/6 filter; 30 μM Corn was incubated 10 min with 20 μM TO. The instrument response function (IRF) was determined using Ludox (Sigma). The data shown are average of three independent experiments. Data were fitted to one, two and three exponentials using software provided by the manufacturer.</p><!><p>To determine quantum yields of Corn complexes, integrals of their emission spectra were compared with that of the reference fluorophore rhodamine 123 (Key Resources Table) which has a quantum yield of 0.9 (Kubin and Fletcher, 1982). Samples were freshly prepared in 40 mM HEPES-KOH, 100 mM KCl, 5 mM MgCl2. 100 μM Corn was mixed with 10 μM fluorophore (ThT or TO); rhodamine concentration was 10 μM.</p><!><p>Measurements were performed using a JASCO J715 spectrometer connected to a Peltier temperature controller. Spectra were acquired over the wavelength range 200-340 nm using a 1 mm path length quartz cuvette. The scanning speed was 100 nm min−1 and response time was 1 s. Fluorophores, ThT at 5-50 μM, DFHO at 10 μM, were added to prefolded RNA (10 μM) in 150 mM KCl, 10 mM MgCl2, 1 mM Tris-HCl pH 7.5, or in 10 mM KCl, 1 mM MgCl2, 1 mM Tris-HCl pH 7.5. Thermal unfolding was performed between 25 – 95 °C, at 1 °C min−1, and signal was monitored at 269 nm. Heated samples were then refolded by cooling at 1 °C min−1 back to 25 °C.</p><!><p>RNAs were in 100 mM KCl, 10 mM MgCl2, 50 mM Tris-HCl pH 7.5 (AUC buffer) at 2.8 μM (OD ~1.0), 1.4 μM (OD ~ 0.5) and 0.28 μM (OD ~ 0.1). Absorbance was measured at 260 nm at 20 °C; run speeds were 60,000 RPM. For the Corn complexes with DFHO, ThT and TO complexes, RNA at the same concentrations was mixed with either 5 μM DFHO or 30 μM ThT and TO, and absorbance was measured at 260 for DFHO and TO, or 280 nm for ThT. The reference cell contained buffer with no fluorophore. Data were analyzed using SEDFIT (Schuck, 2000) (Key Resources Table), assuming a partial specific volume of 0.53 cm3 g−1 and hydration value of 0.59 (Ramesh et al., 2011).</p><!><p>Experiments were performed in a Microcal 200 calorimeter (GE Life Sciences) with samples in 100 mM KCl, 10 mM MgCl2, 50 mM HEPES-KOH pH 7.5. ThT (1.5 mM) was injected in 25 steps (first injection 0.4 μl followed by 24 1.5 μl injections), with 3 min interval between steps. The cell contained 95 μM refolded Corn aptamer (monomer basis) (RNA 1, Key Resources Table). Thermograms were integrated using NITPIC (Keller et al., 2012) (Key Resources Table) and the resulting titration curves were fitted globally using SEDPHAT (Zhao et al., 2015) (Key Resources Table) to estimate stoichiometry and affinity. Experiments were in triplicate. Control experiments consisted of the same number of injections of 1.5 mM ThT in 100 mM KCl, 10 mM MgCl2, 50 mM HEPES-KOH pH 7.5 into the same buffer. ITC could not be employed for analysis of TO - Corn interaction because the poor solubility of TO.</p><!><p>Fluorescence of Corn (in 0.125 - 8.0 mM) in the presence of TO (2.5 mM) was monitored at six different temperatures: 10, 15, 20, 25, 30, and 37 °C, and Kd'S were derived for each temperature. The logarithm of the Kd'S were plotted against reciprocal absolute temperature. ΔH and ΔS were determined from linear least-squares fit. The logarithms of Kd'S at 10 °C, 15 °C, 20 °C, 25 °C, 30 °C and 37 °C from fluorescence titrations (0.73 ± 0.18 μM, 1.32± 0.13 μM, 1.36 ± 0.01 μM, 0.71±0.07 μM, 0.6 ± 0.01 μM, and 0.61 ± 0.01 μM, means of three replicates ± s.d., respectively), were plotted against the reciprocal absolute temperature. Scatter in the data, possibly a result of severely limited solubility of TO (that precluded ITC analysis) led to poor regression, indicative of an unreliable fit (r = 0.31). The derived thermodynamic parameters are: ΔG = 0.07 kcal mol−1; ΔH = −3.9 kcal mol−1, ΔS =−13.6 cal mol−1K−1.</p><!><p>Fluorophore-free Corn (RNA 1, Key Resources Table) was crystallized by vapor diffusion. Hanging drops prepared by mixing 200 nl of refolded Corn (100 μM, monomer basis) and 100 nl of a reservoir solution comprised of 20% (w/v) PEG 4000, 0.2 M ammonium acetate pH 6.7, 0.1 M sodium citrate pH 5.6, were equilibrated against 100 μl of reservoir at 21 °C. Rectangular plate-shaped crystals grew over two days to maximum dimensions of 100 × 50 × 20 μm3. 2 μl of 1 mM iridium (III) hexamine chloride dissolved in reservoir solution supplemented with 20% (v/v) PEG 400 were added to the drop. After 45 min, crystals were transferred to reservoir solution supplemented with 20% (v/v) PEG 400, immediately mounted in nylon loops and flash-frozen by plunging into liquid nitrogen. Co-crystals of Corn-ThT, Corn(A14U)-ThT, and Corn-TO were grown in the same manner, using reservoir solutions comprised of 20% (w/v) PEG 4000, 5% (v/v) PEG 400, 0.2 M ammonium acetate pH 6.7, 0.1 M sodium citrate pH 5.6, and 0.5 mM ThT, or 20% (w/v) PEG 4000, 5% (v/v) PEG 400, 10% (v/v) glycerol, 0.2 M ammonium acetate pH 6.7, 0.1 M sodium citrate pH 5.6, and 0.5 mM TO. ThT and TO co-crystals were grown at 15 °C and 21 °C, respectively. These crystals, which grew to similar dimensions as those of fluorophore-free Corn, were briefly soaked in the reservoir solution used for fluorophore-free Corn crystallization supplemented with 20% (v/v) PEG 400, mounted in nylon loops and flash-frozen by plunging into liquid nitrogen. Diffraction data were collected at 100 K in rotation mode at beamlines 5.0.1 and 5.0.2 of the Advanced Light Source, Lawrence Berkeley National Laboratory (ALS) using 1.0 Å or 1.1 Å X-radiation and reduced with HKL2000 (Otwinowski and Minor, 1997) (fluorophore-free Corn), XDS (Kabsch, 2010) (Corn- ThT), or DIALS (Winter et al., 2018) (Corn(A14U)-ThT and Corn-TO) (Key Resources Table).</p><!><p>The Corn-ThT co-crystal structure was determined by molecular replacement (McCoy et al., 2007) using chain A of the Corn-DFHO crystal structure (PDB ID:5BJP) as the search model. The top solution had TFZ and LLG scores of 15.4 and 309, respectively. Iterative rounds of simulated annealing, energy minimization and individual isotropic B-factor refinement (Adams et al., 2010) interspersed with manual model building (Emsley and Cowtan, 2004), produced a model with Rfree ~ 0.27. At this stage, two molecules of ThT, each with an occupancy of 0.5 were added to the model, RNA geometry was optimized (Chou et al., 2013), and further refinement carried out to yield the current model. The structure of fluorophore-free Corn was determined by molecular replacement using a search model comprised of the four tetrads and residue U19 (a total of 17 nt) of the Corn-ThT structure. The best solution had TFZ and LLG scores of 8.3 and 101, respectively. The structure of the Corn(A14U)-ThT complex was determined by molecular replacement using the Corn-ThT structure (chain A) after omitting A14 and ThT. The top solution had TFZ and LLG scores of 17.4 and 501, respectively. The structure of the Corn-TO complex was determined by molecular replacement using the Corn-ThT structure (chain A) from which ThT was omitted. The top solution has TFZ and LLG scores of 17.7 and 570, respectively. The fluorophore-free Corn, Corn(A14U)-ThT, and Corn-TO structures were refined as the Corn-ThT structure. Metal ions were identified based on electron density feature shape, B-factor analysis, and coordination geometry. Structural figures were generated with PyMol (DeLano, 2002) (Key Resources Table).</p><!><p>Measurements were performed at 20 °C on an EasyLife- LS (Photon Technology International) system equipped with an EL445 diode (Horiba), 445/20-25 and 494/34-25 filters (AVR Optics) for ThT, or an EL510 diode (Horiba) and 535/6 filters for TO. Integration time was 1 s per point and each measurement was averaged 7 times. Corn (7 μM) (RNA 1, Key Resources Table) was refolded and then incubated with 5 μM fluorophore for 1 h at room temperature. The instrument response function (IRF) was determined using Ludox (Sigma). The data shown are the average of three independent experiments. Data were fitted to one, two and three exponentials using Origin 2017 (OriginLab).</p><!><p>Native running buffer was comprised of 16.5 mM Tris, 33 mM HEPES, 0.05 mM EDTA, 20 mM KCl, 5 mM MgCl2. Samples (RNA 1-11, Key Resources Table) were prepared mixing 15 μL of 25 μM RNA and 5 μL of native loading buffer (50% glycerol, 5 × native running buffer) and loaded on a 15% 19:1 acrylamide:bisacrylamide gel (16 cm wide, 20 cm long, 0.05 cm thick) for 3 hr at 20 W, at 4 °C; native running buffer was exchanged with fresh one every 1.5 hour. For analysis of samples that contained ThT, running buffer was supplemented with 250 μM ThT.</p><!><p>DFHO (1), ThT (2), TO (3) (Key Resources Table) were dissolved freshly for each experiment in 100 mM KCl, 10 mM MgCl2, 50 mM Tris-HCl pH 7.5, or where indicated, in 100 mM KCl, 10 mM MgCl2, 50 mM Hepes-KOH pH 7.5, and filtered (0.22 μm cutoff). Concentrations were calculated from the dry weight of the compounds. Corn RNAs were quantitated assuming an extinction coefficient of 354,600 M−1 cm−1 at 260 nm. Recombinant tRNALys3 produced by T7 polymerase in vitro transcription was quantitated assuming an extinction coefficient 604,000 M−1 cm−1 at 260 nm. Known amount of synthetic T7 RNA polymerase promoter DNA (IDT) was dissolved in 100 mM KCl, 10 mM MgCl2, 50 mM Tris-HCl pH 7.5 to give 100 μM stock solution. Known amount of BSA was diluted in 100 mM KCl, 10 mM MgCl2, 50 mM Tris-HCl pH 7.5 to give 100 μM stock solution assuming an extinction coefficient of 43,824 M−1 cm−1at 280 nm. All fluorescence-based experiments were performed in triplicate of which reported values are the mean with standard deviation; triplicate measurements for each experiment were performed during the same session without switching off the xenon lamp of the fluorimeter.</p><!><p>The atomic coordinates od Corn (PDB ID 6E80), Corn-ThT (PDB ID 6E81), Corn(A14U)-ThT (PDB ID 6E82), Corn-TO (6E84) are deposited at Worldwide Protein Data Bank.</p><!><p>Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact: Adrian R. Ferré-D'Amaré</p><!><p>No experimental models and subjects were used in this study.</p>

PubMed Author Manuscript

Complex Loop Dynamics Underpin Activity, Specificity and Evolvability in the (βα)8 Barrel Enzymes of Histidine and Tryptophan Biosynthesis

Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5'-monophosphate decarboxylase, where the rapid (μs timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (βα)8-barrels of tryptophan and histidine biosynthesis, such as the specialist isomerase enzymes HisA and TrpF, and the bifunctional isomerase PriA, are decorated by multiple long loops that undergo conformational transitions on the ms (or slower) timescale. Studying the interdependent motions of multiple slow loops, and their role in catalysis, poses a significant computational challenge. This work combines conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to provide rich detail of the conformational behavior of the catalytic loops in HisA, PriA and TrpF, and the role of their plasticity in facilitating bifunctionality in PriA and evolved HisA variants. In addition, we demonstrate that, similar to other enzymes activated by ligand-gated conformational changes, loops 3 and 4 of HisA and PriA act as gripper loops, facilitating the isomerization of the large bulky substrate ProFAR, albeit now on much slower timescales. This hints at convergent evolution on these different (βα)8-barrel scaffolds.Finally, our work highlights the potential of engineering loop dynamics as a powerful tool to artificially manipulate the diverse catalytic repertoire of TIM-barrel proteins.

complex_loop_dynamics_underpin_activity,_specificity_and_evolvability_in_the_(βα)8_barrel_enzymes_of

Introduction<!>Methodology<!>System Preparation for Conventional and Enhanced Sampling Molecular Dynamics<!>Conventional Molecular Dynamics Simulations<!>Enhanced Sampling Molecular Dynamics Simulations<!>Empirical Valence Bond Simulations<!>Analysis of Conventional and Enhanced Sampling Molecular Dynamics Simulations<!>Active Site Plasticity and Substrate Binding in the Different Enzymes<!>Conformational Dynamics of Key Catalytic Loops of HisA, PriA, and TrpF<!>Conformational Dynamics of Loop 1 in HisA and PriA and Its Impact on Selectivity<!>Empirical Valence Bond Simulations of the Enzyme-Catalyzed Ribose Ring Opening Step in the Isomerization of Substrates ProFAR and PRA<!>Overview and Conclusions<!>Supporting Information

<p>Enzymes are dynamic systems able to explore many different conformations, and these dynamical properties are clearly connected to their biological function. Examples of this include allosteric regulation and product release, 1 as well as the role of conformational selection in enzyme catalysis, 2-7 promiscuity 4 and evolution. [8][9][10][11][12][13][14] In addition, such conformational dynamics can, in principle, be engineered in a targeted fashion to allow enzymes to acquire new catalytic functions and/or physiochemical properties. 8,9,[14][15][16][17] Understanding how enzymes manipulate and modulate conformational dynamics during both natural and directed evolution is an important step in this direction. In particular, understanding the dynamical behavior of decorating loops that cover enzyme active sites is important as such loops can regulate substrate selectivity, the evolution of new activities, and potentially also turnover rates. 3,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] As such, targeting the dynamics of such active site loops is attractive from an engineering perspective, 16,33 and therefore there is substantial interest in understanding loop dynamics and its impact on selectivity and catalysis.</p><p>In this context, there have been extensive studies of a wide-range of enzymes, such as triosephosphate isomerase (TPI), 34,35 orotidine 5'-monophosphate decarboxylase, (OMPDC) 36 glycerol 3-phosphate dehydrogenase (GPDH), 37,38 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 39,40 and β-phosphoglucomutase, 41 which have been demonstrated to be activated by ligand-gated conformational changes. Specifically, interactions between a key "gripper" loop decorating the active site and the non-reactive phosphodianion groups of the substrates of these enzymes trigger substantial conformational changes in the gripper loop, facilitating energetically unfavorable transitions from catalytically inactive open to catalytically active closed conformations, and these conformational transitions are central to the catalytic activities and high proficiencies of these enzymes. 22,31 It is noteworthy that several of the aforementioned enzymes have TIM-barrel folds. This fold comprises eight repeated (βα)-units, and most if not all TIM-barrel proteins possess decorating loops, 18,42 the conformational diversity of which likely plays an important role in regulating specificity and function. 25,28 These flexible loops can vary in length, 18 but are typically used to bind substrate, and to sequester the active site from solvent by closing over the active site, and it has been suggested that the active site geometries of these enzymes are shaped by the residues of these loops. 43 However, these well-characterized examples of proteins activated by ligand-gated conformational changes all focus on the roles and importance of single loops, such as gripper loop 6 in TPI. Studying the ligand-gated motion of a single loop can already pose substantial challenges; 44 systems with multiple active site loops undergoing substantial conformational changes are even more complex, and therefore unsurprisingly understudied in the literature.</p><p>We have sought to address this gap in knowledge by studying active site loop dynamics in the (βα)8-barrels of tryptophan and histidine biosynthesis. The isomerase enzymes HisA, TrpF and PriA are model systems for the evolution of specificity and activity. 28,[45][46][47][48] As shown in Figure 1, HisA catalyzes isomerization of the aminoaldose N'-[(5'-phosphoribosyl)-formimino]-5aminoimidazole-4-carboxamide-ribonucleotide (ProFAR) into the aminoketose Nʹ-[(5ʹphosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR). TrpF catalyses the same Amadori rearrangement on N-(5ʹ-phosphoribosyl)anthranilate (PRA), producing 1-(2-carboxy-phenylamino)-1'-deoxyribulose-5'-phosphate (CdRP). This rearrangement proceeds via a Schiff acid-base mechanism, that utilizes aspartate (and in the case of TrpF) cysteine residues as acid-base pairs. 49 Interestingly, many actinobacteria lack the trpF gene, instead possessing a gene for a bifunctional isomerase, PriA. 50 The PriA from Mycobacterium tuberculosis (MtPriA) has been particularly well characterized, and has kcat/KM values of ~10 4 M -1 s -1 for HisA activity and ~10 5</p><p>-10 6 M -1 s -1 for TrpF activity. 25,51 Not only are there naturally occurring bifunctional enzymes, but promiscuous TrpF activity has been detected on both ancestral and extant specialist HisA enzymes, with kcat values ranging from 10 -4 to 10 -2 s -1 . 52 HisA has also been converted into TrpF by directed evolution, 53 and in serial passaging experiments. 46 In the latter study, laboratory evolution of the ProFAR-specific HisA (lacking TrpF activity) from Salmonella enterica over 3,000 generations yielded an extensive suite of mutations in the S. enterica HisA (SeHisA) that resulted in specialist HisA enzymes, specialist TrpF enzymes and PriA-like bifunctional enzymes. green, yellow and dark red on each structure, respectively. Loop 1 in TmTrpF is short (four residues), which is why no corresponding loop is annotated on this panel. Note that for clarity, N7D and A176D reversions were applied to the structure of SeHisA in complex with ProFAR (these reversions were also applied in our simulations, as described in the Methodology section). (D) The proposed mechanism for the Amadori rearrangement leading to the isomerization of substrates ProFAR and PRA by the different enzymes. 49 As shown in Figure 1, HisA and PriA are decorated by three long catalytic loops, loops 1, 5, and 6 (or two analogous loops in the case of TrpF, which has lost most of loop 1). 25,28 Of these loops, loop 5 carries key residues that are important for substrate binding, loop 6 carries the catalytically important aspartic acid side chain, and a number of mutations important for interaction with substrate PRA have been observed at position 15 of loop 1. 28,45,54 The mutants generated by Näsvall et al. 46 were the subject of detailed structural and biochemical analyses. 28 As with PriA, 25 this analysis of the evolved bifunctional SeHisA variants indicated that the bifunctionality is driven by competition between not just the substrates ProFAR and PRA, but also between structurally distinct conformations of loops 1 and 5, in particular 28 (Figure S1).</p><p>Furthermore, although the isomerization of both ProFAR and PRA proceeds through the same Amadori rearrangement (Figure 1), ProFAR (the native substrate of HisA) is a much larger molecule, including the presence of a second phosphate group. This group forms hydrogen bonding interactions with the side chains of R83 from loop 3 and S103 from loop 4 of SeHisA, and with the corresponding side chains of R85 and T105 in MtPriA (Figure 2). Although neither of these residues are on the primary mobile loops of either enzyme (Figure 1), nevertheless, the interactions with the second phosphate group of ProFAR are very similar to analogous interactions in other enzymes activated by ligand-gated conformational changes, [34][35][36][37][38][39][40][41] suggesting that loops 3 and 4 in SeHisA and MtPriA may similarly act as "gripper loops" allowing these enzymes to attain relevant catalytically active conformations for the isomerization of the larger substrate. This effect would clearly not be present when the smaller substrate, PRA, is bound to the active site as this substrate lacks a non-reactive phosphodianion group to interact with these loops. the active site tryptophan that forms stacking interactions with the larger substrate ProFAR in SeHisA and MtPriA (W145 in both enzymes), as well as the key gripper residues that interact with the distal phosphate group of the larger substrate/product in SeHisA and MtPriA (R83 and S103 in SeHisA and R85 and T105 in MtPriA). Key hydrogen bonding interactions are also highlighted, using the distances (Å) found in the corresponding crystal structures. Note that for clarity, N7D and A176D reversions were applied in SeHisA in complex with substrate ProFAR (and this reversion was also applied in our simulations, as outlined in the Methodology section).</p><p>In the present work, therefore, we combine long-timescale conventional molecular dynamics, enhanced sampling and empirical valence bond (EVB) simulations to present a comprehensive computational study of a number of wild-type and variant forms of SeHisA, MtPriA and TrpF from Thermotoga maritima (TmTrpF). All variants studied in this work, and the corresponding structures used, are summarized in Table S1. We chose these systems because, in all three cases, there are high-quality structural data in unliganded and ligand-bound forms. For SeHisA, we chose 8 to study the unliganded and substrate (ProFAR) bound forms, 54 as well as key SeHisA variants from ref. 28 that were selected based on their specificity patterns (specialists vs. generalists, Table S2). MtPriA and TmTrpF were similarly selected on the basis of high-quality structural data of each enzyme in both unliganded and product (PRFAR) or product analogue (rCdRP) bound forms respectively, as summarized in Table S1.</p><p>Prior simulation studies of TPI by both us and others have indicated that the large ligand-gated conformational change of the gripper loop 6 is correlated with smaller conformational motions in other decorating loops on the active site. 44,56 We made similar observations when studying loop motions in PTP1B. 32 However, as these simulations indicate, even studying the motion of one large dominating conformational change is computationally non-trivial, and the current systems involve the interdependent conformational rearrangements of multiple loops simultaneously. Our current simulations of HisA, PriA and TrpF (1) provide rich detail of the conformational behavior of the catalytic loops in the different systems, and (2) provide insight into the link between conformational dynamics, catalytic activity and functional evolution in the different enzymes, in particular the role of loops 1 and 5 in regulating PriA and HisA's activity and selectivity, as well as the gripper loops 3 and 4 in driving ligand-gated conformational changes in these enzymes. 28,[45][46][47][48] This, in turn, is significant, because in recent years, there has been substantial (and increasing) interest in exploiting techniques such as loop grafting and related approaches in order to engineer flexible loops in enzymes as a means of controlling their catalytic activity. 16,33 Our data provide clear evidence that this is likely to be a powerful strategy for artificially manipulating the diverse catalytic repertoire 47 of TIM-barrel proteins.</p><!><p>Methodological details are presented here in brief. Full details of all simulations and any nonconventional parameters used in our simulations are provided in the Supporting Information.</p><!><p>Simulations.</p><p>Simulations were performed on wild-type SeHisA, MtPriA and TmTrpF, as well as relevant enzyme variants, in both their unliganded forms and in complex with various ligands (substrates ProFAR and PRA and, in the case of the enhanced sampling simulations, products PRFAR and CdRP). A total of fourteen crystal structures were used to generate starting points for these simulations, and all simulations performed as well as associated structures used are summarized in Table S1. Where present, the D7N, D11N and D176A substitutions were reverted to wild-type using the Dunbrack 2010 Rotamer Library, 57 as implemented in UCSF Chimera, v. 1.14. 58 Missing regions in the catalytic loops were reconstructed using Modeller v. 9.23. 59 The catalytic aspartic acid side chain in the active site of each enzyme (D176 in HisA, D175 in PriA and D126 in TrpF)</p><p>was kept protonated in line with the mechanism shown in Figure 1. All other residues (except H50 in PriA, which was doubly protonated) were kept in their default protonation states at physiological pH determined by use of PROPKA 3.1, 60 and visual inspection. The substrates ProFAR and PRA were manually placed into the relevant active sites in the same conformation as found in the structure of the HisA wild-type enzyme in complex with ProFAR and in the case of PRA (Figure 1D), was placed by manual overlay of the reactive part of PRA with the reactive part of ProFAR, and with the carboxylate group of PRA keeping key interactions with active site residues. Partial charges for ligands ProFAR, PRA, PRFAR and CdRP, were calculated using the standard restrained electrostatic potential (RESP) protocol using Antechamber v. 17.3, 61 and based on the vacuum electrostatic potential calculated at the HF/6-31G(d) level of theory, using Gaussian 09</p><p>Rev. E.01. 62 All other simulation parameters were described using the general Amber force field 2 (GAFF2) 63 (see Tables S3 to S6). Finally, to keep the substrate stably bound in the enzyme active sites, weak distance restraints were applied to protein-substrate distances, as described in Table S7.</p><!><p>Conventional MD simulations were performed using the CUDA version of the PMEMD module of the AMBER 16 simulation package. 64 The protein, ligands and solvent were described using the ff14SB force field, 65 the General AMBER Force Field 2 (GAFF2), 63 and the TIP3P water model, 66 respectively. Following initial minimization and equilibration, each system (summarized in Table S1) was subjected to 10 x 500 ns of molecular dynamics simulations controlled by the Langevin thermostat with a collision frequency of 2 ps -1 , 67 and the Berendsen barostat with a 1 ps coupling constant. 68 This led to a cumulative 5 μs of production simulations per system, and a cumulative total of 70 μs of conventional MD simulations over all systems studied (Table S1).</p><!><p>Steered molecular dynamics simulation (sMD) were performed using GROMACS 2018.4 in order to pull products PRFAR and CdRP out of the active site of the SeHisA(dup13-15/D10G/G102A/Q24L) variant, as described in the Supporting Information. The system preparation was performed as for the conventional MD simulations, and using the same force fields and water models as the conventional MD simulations. Following initial minimization and equilibration, 10 x 50 ns production MD simulations were performed on each system. The first 5 ns of production MD were unrestrained, after which an external force with a force constant of 10 kcal mol -1 Å -2 was applied to pull the product out of the active site. This external force was then released for the last 5 ns of the MD simulation run.</p><!><p>Following our prior success in using the empirical valence bond (EVB) approach 69 to study a wide range of analogous ring opening reactions, such as lactone 70,71 and epoxide 72,73 hydrolysis, we extended this approach to study the enzyme-catalyzed opening of the ribose ring of substrates ProFAR and PRA (Figure 1), as catalyzed by wild-type and variant forms of HisA, PriA, and TrpF. Our focus for our EVB simulations was specifically on the ribose ring-opening reaction (the first step of the mechanism shown in Figure 1D), as motivated in the Results and Discussion, and described using the valence bond states shown in Figure 3. Simulations were performed on wild-type SeHisA, MtPriA and TmTrpF as well as selected variants, as described in the Supporting Information. All simulations were performed using the Q6 simulation package, 74,75 using the OPLA-AA force field. 76 All EVB parameters necessary to reproduce our work, as well as a detailed description of the computational methodology and subsequent simulation analysis can be found in the Supporting Information, with the full parameters used in our simulations updated to Zenodo (DOI: 10.5281/zenodo.5893598). Each system was simulated using 30 individual replicas, with each replica first equilibrated for 20 ns and the endpoint of that equilibration being used as the starting point for propagating an EVB trajectory. Each EVB free energy perturbation/umbrella sampling (EVB-FEP/US) 69</p><!><p>Unless stated otherwise, all analysis of all conventional and enhanced sampling molecular dynamics simulations was performed using CPPTRAJ. 77 Hydrogen bonds were defined as formed if the donor−acceptor distance was ≤3.0 Å and the donor-hydrogen-acceptor angle was within 180 ± 45°. Principal component analysis (PCA) was performed by first RMS fitting to a whole protein Ca carbon atoms and then performing PCA on the Cα carbon atoms of loops 1, 5 and 6, as well as loop 1 for HisA loop 1 elongated systems analysis. Other analyses were performed as described in the Supporting Information.</p><p>13</p><!><p>The TIM-barrel structures of SeHisA and MtPriA are similar, with an RMSD of 1.09 Å between them (comparing the Ca atoms in PDB IDs 3ZS4 55 and 5A5W 54,55 ). TmTrpF, in contrast, is a smaller enzyme with 40 fewer residues in the sequence than MtPriA and SeHisA. We performed 10 x 500 ns conventional molecular dynamics simulations of unliganded HisA, TrpF and PriA, and calculated the average and standard deviations of the active site volumes of each enzyme using the MDpocket 78 tool, which is provided as part of the fpocket 79 suite of pocket detection programs, as described in the Supporting Information. The resulting calculated volumes are shown in Table S8. We obtained average volumes of 745.4 ± 129.6 Å 3 , 1033.8 ± 217.0 Å 3 and 1173.5 ± 158.0 Å 3 , for the active sites of TrpF, PriA and HisA, respectively during our simulations. From this, it can be seen that the TrpF active site is more compact than that of PriA and HisA, which have successively larger active site volumes, with more "flexible" pockets than TrpF (using the standard deviation on the volume as a proxy for this flexibility). For comparison, the substrates ProFAR and PRA have volumes of 829 and 559 Å 3 , respectively, calculated using Alexander Balaeff's Mol_Volume program Version 1.0, with default radii of 1.7Å and a probe sphere of 0.5Å. This confirms the structural data indicating that the active site pocket of TrpF is too compact to accommodate the much larger substrate, ProFAR, leading to the selectivity of this enzyme towards PRA. 80 Furthermore, the PriA active site is the most flexible of the three, in line with structural data 25 that indicates that PriA is capable of significantly rearranging its active site (in particular loop 5 conformation) when accommodating the different substrates ProFAR and PriA (Figure S1).</p><p>Structures and mutagenesis experiments have identified two key active site side chains in HisA and PriA, which are important for binding of the substrate ProFAR. 25,28,54,81 These are W145, which forms a stabilizing stacking interaction with the substrate, and R83 (R85), which interacts with the second phosphodianion group of the substrate (Figure 2). To further explore the conformational diversity of these key tryptophan and arginine residues in HisA and PriA, respectively, we examined the joint dihedral angle distribution of the side chains of these residues in simulations of unliganded HisA and PriA, as well as HisA and PriA in complex with both From these data it can be seen that both the tryptophan and arginine side chains are highly conformationally flexible in the unliganded enzymes. However, while the binding of the substrate ProFAR to HisA restricts the conformational space of the arginine side chain on the "gripper" loop 3 to a catalytically competent position that helps stabilize the bound substrate, in PriA, the R143 side chain is only 4.8Å from the "gripper" residue R85 (distance between the two side chain carbon atoms, based on PDB ID: 3ZS4 55 ). This in turn creates electrostatic repulsion between the two arginine side chains, thus destabilizing loop 5 as well as the interaction between the substrate ProFAR and the R85 side chain (Figure S3). In contrast, when PRA, that lacks the second phosphodianion group, is bound to the PriA active site, the R83/R85 side chains increase their conformational flexibility again, sampling more or less the same conformational space as in the case of the unliganded enzyme (Figure S2). Therefore, the interaction of these residues with the larger substrate ProFAR is likely playing an important role in the ability of these enzymes to bind and isomerize this compound.</p><p>In the case of W145, this side chain slightly increases its conformation flexibility when the smaller substrate PRA is bound to HisA (note that PRA was placed manually in the active site by overlay with substrate ProFAR, as described in the Supporting Information). However, in the case of PriA, both W145 the and R143 side chains are conformationally restricted to a catalytically competent position due to a rearrangement of loop 5 upon PRA binding, that swaps the position between these two residues compared to when ProFAR is bound to PriA, preventing the electrostatic repulsion between the R85 and R143 side chains that is observed when ProFAR (PRFAR) is bound to the active site (Figure S1). 25 As noted previously, the R83/R85 side chain is one of a number of key residues on the "gripper" loop that interact with the distal phosphodianion group of the larger substrate ProFAR, contributing to the stabilization of the substrate in HisA active site. Hence, in PriA, the loop 5 rearrangement required for ProFAR substrate binding 25 prevents R85 from gripping the second phosphodianion group and thus showing clear preference for the isomerization of PRA substrate.</p><p>We note the similarity of these gripper interactions to corresponding interactions in enzymes such as TIM, OMPDC and GPDH, [34][35][36][37][38] where interactions with the non-reactive phosphodianion group of the substrate drives a ligand-gated conformational change. This in turn stabilizes otherwise energetically unfavorable but catalytically important closed conformations of key catalytic loops over the respective active sites of these enzymes. In the case of the current systems, the interaction between the HisA gripper residues and the remote phosphodianion group of ProFAR appears to be similarly important for maintaining the closed conformation of loop 5, and when this interaction is lost, as in PriA, we see corresponding opening of loop 5 (Figure S3). This supports the likelihood that HisA and PriA are also activated by ligand-gated conformational changes, albeit with more complex loop dynamics (due to the involvement of not one but three highly mobile and long catalytic loops) than in other previously characterized systems.</p><!><p>To further explore the impact of the binding of the two substrates on loop dynamics, we extended our simulations to also included simulations of TrpF in both its unliganded form, and in complex with PRA (Table S1). We then performed Principal Component Analysis (PCA) to characterize the motion of the key catalytic loops during our simulations in each of the individual systems, similarly to prior analysis we have performed on triosephosphate isomerase, 44 except in our prior work substantial conformational changes take place in only one and not two (TmTrpF) or three (SeHisA; MtPriA) distinct loops. The PCA analysis was performed on the mass-weighted Cartesian coordinates of each enzyme compared to the coordinates of the corresponding closed state, allowing us to explore the variation of the conformations of these loops in coordinate space. We subsequently projected the free energies for each enzyme along the most dominant motions, PC1 and PC2, from simulations of each of HisA, PriA and TrpF in their unliganded forms as well as in complex with substrates ProFAR and PRA, respectively (here, the smaller substrate PRA was artificially placed in the HisA active site by manual overlay with the reactive part of ProFAR, as outlined in the Supporting Information). This allowed us to compare the free energy surfaces defined by these two principal components both between the different enzymes, and the effect of ligand binding on these surfaces. The resulting data are shown in Figure 6. Note that, as shown in Figure 5, these projected free energy surfaces show the combined motion of all key catalytic loops along each principal component. In the unliganded forms of all three enzymes, the catalytic loops can explore a range of "wideopen" conformations and are overall highly conformationally diverse (Figure 5). This observation is consistent with our prior simulation studies on both triosephosphate isomerase, 44 and the protein tyrosine phosphatases PTP1B and YopH, 32 as well as chimeric forms of these enzymes. 83 However, and consistently with structural data, the binding of ProFAR to HisA fully restricts the conformational sampling of all three loops (Figure 6B). In the case of PriA, the binding of both ProFAR and PRA also stabilizes the closed conformation of the three active site loops, but still allows for some conformational flexibility in these loops (Figure 6, based on both the topologies of the projected free energy surfaces, and the corresponding energies). In sharp contrast, in the liganded form of TrpF, our MD simulations show that PRA is not stable in the active site due to the flexibility of loop 6, which explores transitions towards open conformations, similarly to the unliganded system (Figures 5E, F and 6G, H). In the crystal structure of the unliganded enzyme (PDB ID: 1NSJ 55,82 ), this loop is present in a closed position but with missing density, whereas in our simulations of both the liganded and unliganded form of the enzyme, loop 6 samples open conformations, suggesting that it is not the correct closed state to stabilize the substrate PRA in the active site. We note that the structure used for these simulations (PDB ID 1LBM 49,55 ) was solved in complex with the product analog rCdRP. Our simulations suggest that the loop conformations observed in this structure are a conformational state on the trajectory to product release, rather than an ideal conformational state for stabilizing the Michaelis complex.</p><p>In addition, in HisA and PriA, we observe the formation of a stacking interaction between the substrate ProFAR and the side chain of W145 in our conventional MD simulations (Figure S4), with an average distance of 3.9 ± 0.3 Å and an angle g = 11.7 ± 5.5° between the center of mass of the imidazole ring of ProFAR and the indole ring of the W145 side chain during our simulations.</p><p>Our results confirm the role for W145 that was proposed previously, based on experimentally determined structures. 54 This is furthermore consistent with prior structural analysis that indicates that HisA activity is abolished in SeHisA(dup13-15), because the extended conformation of loop 1 blocks this side chain from interacting with ProFAR. 28 In contrast, in the case of PriA, and again in agreement with prior structural analysis, 25 we observe two possible conformations of loop 5, depending on which substrate is bound to the active site. That is, when ProFAR is bound to the active site, we sample a conformation similar to that observed in wild-type HisA (Figure S1A) with a similar stacking interaction between ProFAR and W145 (Figure S4), however, the loop 5 rearrangement required to optimize the stacking position of W145 with ProFAR creates transient electrostatic repulsion between loop 5 and the rest of the enzyme, making this loop more conformationally dynamic, which we observe in our analysis in the form of an increased standard deviation in the distance and angle of the corresponding stacking interaction (d = 4.1 ± 0.6 Å, g = 19.2 ± 13.9°) (Figures S3 and S4). The greater plasticity of this interaction, in turn, decreases substrate stability in the active site (the ProFAR RMSF increases from 18.4 Å in our simulations of wild-type HisA to 22.2 Å in our simulations of wild-type PriA), and thus the corresponding ProFAR isomerization activity of PriA.</p><p>For comparison, in our simulations of PriA in complex with substrate PRA, we sample an active site conformation in which the R143 side chain (loop 5 residue) forms salt bridges with the side chains of with D130 and D175, and the arginine acts as a "shield" dampening the electrostatic repulsion between the D130 side chain and the anthranilate carboxylate group of PRA. This interaction also stabilizes the catalytic aspartic (D175), placing it in an optimal position for catalysis (Figure S5 and Table S9), as shown in previous studies 25 . This PriA conformation is similar to the "TrpF-active" conformation observed in the SeHisA(dup13-15/D10G/Q24L/G102A) crystal structure 28 (PDB ID: 5AB3 28,55 , Figure S6, with manual placement of PRA in the active site), where the arginine is close to residue D129. While we observe this conformation in our PriA simulations, we do not observe the formation of a corresponding interaction in our simulations of wild-type HisA in complex with PRA, the negative charge repulsion between PRA and the D129 side chain destabilizes the position of the substrate in the active site (Figure S7), as well as the stability of the loop 6 carrying the key catalytic aspartic acid side chain (Figure S7). We do, however, observe a similar interaction with the R169 side chain in the SeHisA(L169R) variant, with interactions with D129 and, in this case, a salt bridge interaction with the anthranilate carboxylate group of PRA (Figure S7 and Table S9), consistent with experimental work that demonstrated that the introduction of the L169R substitution in HisA induces TrpF activity (Table S2). 46 Finally, in the case of TrpF in complex with PRA, we observe a salt-bridge interaction between E184 and the side chain of R36 on loop 3 (Figure S6 and Table S9), and we can see that as for HisA and PriA, that R36 is again acting as a "shield" avoiding possible negative repulsion interactions between the substrate and the negatively charged side chain. However, we do not observe clear interactions between the R36 side chain and the substrate PRA (Figure S6 and Table S9, with the fraction of simulation time in which this interaction is observed being <0.1).</p><p>Overall, we observed that this arginine plays an essential role in the introduction of TrpF activity, by shielding electrostatic repulsion between negatively charged side chains and the anthranilate carboxylate group of PRA. This is in agreement with experiments where introducing an arginine or removing the negative residue introduce TrpF activity in HisA systems. 46</p><!><p>While loops 5 and 6 of HisA and PriA have been clearly identified as being important for binding and catalysis (loop 6 carries the catalytic aspartic acid side chain, Figure 1), 25,28,54 the precise catalytic role of loop 1 remains unclear, although extending the conformation of loop 1 through duplication of residues 13-15 (HisA(dup13-15)) plays an important role in the acquisition of bifunctionality 28,84 in a real-time evolution experiment on HisA, 46 and substitutions at position 15 on this loop appear to be important for facilitating the TrpF activity of this enzyme. 28,54 Therefore, we also performed simulations of the SeHisA(dup13-15/D10G) variant, as described in the Methodology section. SeHisA(dup13-15/D10G) is a bifunctional enzyme that can catalyze the isomerization of both ProFAR and PRA with modest catalytic efficiencies, 28 and the corresponding crystal structure (PDB ID: 5AC7 28,55 ) shows the enzyme in a 'PRA-active' conformation with loops 1 and 6 in a closed state, and loop 5 in an open state.</p><p>When initiating simulations of the unliganded SeHisA(dup13-15/D10G) variant starting from these loop 1 closed conformations, we did not observe any opening of loop 1. This is in contrast to our simulations of the wild-type enzyme, where we sampled open conformations of this loop when we started from the unliganded closed conformation observed in PDB ID: 5A5W, 54 removing the ProFAR substrate. This provides evidence, in addition to the discussion in ref. 28 , that the elongation of loop 1 heavily stabilizes this PRA-active conformation. This is further supported by examining the root mean square fluctuations of all Cα atoms in our simulations of these two enzymes (Figure 7), where we observe that loop 1 is more flexible than either of loops 5 or 6 in simulations of the wild-type enzyme, but has reduced flexibility in simulations of the SeHisA(dup13-15/D10G) variant. S2).</p><p>Interestingly, while the elongation of loop 1 through the duplication of residues 13-15 (VVR) appears to be essential for the change of specificity towards the isomerization of PRA (facilitated by the presence of a new stabilizing arginine side chain close to the active sites, Figure S1, 28 simply the loop duplication by itself is not enough to induce bifunctionality. That is, while the duplication elongates loop 1, it also rigidifies it, such that SeHisA(dup13-15) does show some ability to isomerize PRA (k cat > 0.15 s -1 ), but at the expense of losing all ability to isomerize the larger substrate ProFAR (no detectable activity). 28 Therefore, the duplication by itself simply leads to a switch in activity from a modestly efficient isomerase towards ProFAR (k cat 7.8 s -1 ) towards a less efficient isomerase with activity towards PRA. Critical to the bifunctionality is the inclusion of an additional substitution, present in both variants studied above, namely D10G. This substitution increases the flexibility of the elongated loop 1 (Figure 8), allowing for the loop to take on wide-open configurations which in turn facilitate the entry and binding of ProFAR to the active site (Figure 8B, wide-open conformation). Our conventional MD simulations (10 x 500 ns per system) are overall short, taking in particular into account the slow turnover numbers of these enzymes (that suggest loop motions on the ms to s timescale). 28 However, our observations that the D10G substitution leads to increased flexibility of loop 1 are in good agreement with prior NMR relaxation dispersion experiments. 28 These detected μs to ms motions at 14 backbone 15 N positions in the SeHisA(dup13-15/D10G) variant, compared to only three positions for the SeHisA(dup13-15) variant, and with two resonances that are unique to SeHisA(dup13-15/D10G). As a result, adding this substitution is sufficient to convert SeHisA(dup13-15/D10G) back to a bifunctional enzyme, through exploitation of conformational dynamics, with k cat of 0.09 s -1 for the isomerization of PRA, and 0.05 s -1 for the isomerization of ProFAR (Table S2). 28 To explore this further, we therefore examined simulations of four loop elongated variants, specifically: dup13-15 (PDB ID: 5G2I 28 ), dup13-15/D10G (PDB ID: 5AC7, 28,55 ), dup13-15/D10G/G102A (PDB ID: 5AC8 28 ), and dup13-15/D10G/G102A/Q24L/V15[b]M (PDB ID:</p><p>5G1Y 28 ). The first and last of these variants are only active towards the isomerization of PRA, whereas the middle two variants are bifunctional towards both PRA and ProFAR (Table S2). In all cases, when performing conventional MD simulations starting from the closed conformation of loop 1, this loop is very stable, and remains closed over our simulation timescales (Table S1). We From analysis of our MD simulations (Table 1), we clearly see how all variants carrying the D10G substitution are able to populate all three of closed, open and wide-open conformations.</p><p>However, the relative populations of these states depends strongly on enzyme variant: already, the D10G substitution by itself appears to be sufficient to cause a conformational shift towards a closed conformation, and the SeHisA(dup13-15/D10G/Q102A/Q24L/V15[b]M) variant, which shows the highest TrpF activity of all variants studied in ref. 28 (Table S2), also shows the most significant population shift towards sampling a closed conformation of loop 1. This is consistent with structural analysis, 28 which indicated that the Q24L substitution is important because it introduces a new stabilizing interaction with V15b, as well as an even better interaction with V15[b]M, such that the Q24L interaction is just as important as the VVR duplication for the adaptive benefit of the V15[b]M substitution to be realized. Finally, the wide-open conformation is also only rarely sampled in the SeHisA(dup13-15) variant, which does not carry the D10G substitution. We hypothesize that in the case of these variants, this is due to the presence of a Gly-Gly dyad in the hinge of loop 1, which provides the loop with enough flexibility to explore these wide-open conformations. Clearly, dup (13-15), as well as the inclusion of additional substitutions, is significantly impacting the conformational space sampled by this loop, shifting towards a loopclosed conformation of loop 1 that is favorable for TrpF activity.</p><!><p>To further probe the role of loop 1 in HisA and PriA, we have complemented our conventional and enhanced sampling molecular dynamics simulations with empirical valence bond (EVB) 69 simulations of the initial ribose ring opening step in the Amadori reaction of substrates ProFAR and PRA (Figure 1), as catalyzed by wild-type and variant forms of the two enzymes (see the Methodology section for details of the simulation and parameterization procedures).</p><p>We note that the calculated activation free energies for this step are not trivial to directly compare with the experimental turnover numbers: no kinetic data exists on the rates of the individual chemical steps. Furthermore, the observed kcat values for these systems are extremely low -on the order of 1 s -1 (or lower) for both the HisA and TrpF reactions catalyzed by HisA and its variants. 28 However, E. coli TrpF has a kcat value of 30-40 s -1 , with the rate-limiting step being a spontaneous keto-enol tautomerization step that occurs off the enzyme, after the ring-opening step. 86 When also taking into account the potential involvement of loop dynamics in determining the turnover rates, as is the case in other enzymes with catalytically important conformational changes such as protein tyrosine phosphatases, 26,29,32,83 this means that the experimental turnover numbers for the isomerization of ProFAR and PRA by the enzymes of interest do not correspond to a chemical step occurring in the enzyme active site. However, as the Amadori reaction that occurs between the enzyme-catalyzed ring-opening reaction and the non-enzymatic tautomerization is likely to be fast (even the uncatalyzed reaction occurs spontaneously at 25 °C85 ), the ring-opening reaction is likely the slowest enzyme-catalyzed chemical step in the catalytic cycle. This is supported by QM/MM studies of the mechanism of the HisA-catalyzed reaction, 84 although this work does not take into account that chemistry is not rate-limiting here. However, even if the experimentally measured turnover numbers (kcat) do not directly correspond to this step, they do produce a lower limit for the rate of this step (and thus an upper limit for the corresponding activation free energy for the rate-limiting step of the enzymatic reaction). Thus, comparing the calculated activation free energies for the ring-opening in different variants, and in different conformational states of loop 1, can still provide insight into the impact of loop dynamics and key amino acid substitutions on the rate of the slowest enzyme-catalyzed step.</p><p>Taking these limitations into account, the resulting experimental and calculated activation energies are shown in Tables S10 and S11 and S10 and S11).</p><p>The experimental activation free energies (∆G ‡ exp) were derived from the kinetic data presented in refs. 25,28 . Structures were selected based on clustering analysis using the hierarchical agglomerative algorithm, as implemented in CPPTRAJ. 77 Note that the annotated catalytic distances are average values over 6000 snapshots extracted for each state from our EVB trajectories (from 30 x individual 200 ps EVB mapping windows per stationary point/system). For a full list of reacting distances across all variants, see Tables S12 and 13.</p><p>As can be seen from these data, our calculations reproduce the experimental trend in activation free energies derived from the turnover numbers relatively well for both substrates ProFAR (Table S11) and PRA (Table S12), reproducing these values to within 2.0 kcal mol -1 for either substrate.</p><p>This was unexpected, considering the experimental turnover number does not correspond to a chemical step in the enzyme, as discussed above, and indicates that the observed changes in activity nevertheless do have a chemical component.</p><p>While both wild-type PriA and HisA are capable of catalyzing isomerization of the substrate ProFAR, only PriA can catalyze the isomerization of the smaller substrate PRA. As already seen 33 from the PriA crystal structures with both products bound, loop 5 can be rearranged either to accommodate one substrate or the other, displaying two slightly different conformations of the loop (Figure S1). 25 These are a "knot-like" pro-ProFAR conformation of loop 5, with W145</p><p>pointing "in" towards the substrate ProFAR (PDB ID: 3ZS4 55 ), and a pro-PRA β-hairpin conformation of loop 5, with R143 pointing "in" towards the substrate PRA (PDB ID: 2Y85 25,55 ), extrapolating the substrate positioning from the position of the analogous product PRFAR and product analog rCdRP in the respective conformations. Here, we used PriA in its pro-ProFAR conformation as a reference state to calibrate our EVB simulations of the initial ring-opening of ProFAR. The resulting EVB parameters were then used unchanged in all relevant systems.</p><p>Based on these parameters, we obtain an activation free energy of 17.5 ± 0.6 kcal mol -1 for the analogous reaction catalyzed by wild-type HisA, which is only 1.1 kcal mol -1 higher than the experimental value (derived from kcat) of 16.4 kcal mol -1 . To further validate our PriA/HisA-ProFAR results, we performed single amino acid substitutions in each enzyme (R19A and D130A</p><p>in PriA and S202A, D129N and D10G in HisA), and performed EVB simulations on these variants.</p><p>In most of the systems we obtain activation free energies ~1 kcal mol -1 higher than the corresponding experimental values, as for wild-type HisA. However, for D130A in PriA and S202A in HisA, we underestimate the activation free energies by 1.5 -2.0 kcal mol -1 in comparison with the experimental value, suggesting that the experimental effect is due to either a change in substrate positioning or loop conformation or dynamics, which we are unable to capture in our simulations when simply starting from the wild-type crystal structure and manually truncating these residues.</p><p>In the case of the substrate PRA, we again used the reaction catalyzed by wild-type MtPriA as our EVB reference state, this time with the loop 5 in its pro-PRA conformation. We note that as 34 shown in Table S2, wild-type SeHisA does not show TrpF activity, whereas variants in which loop 1 is extended through dup13-15 do. 28 In the SeHisA(dup13-15) variant, we obtained an activation free energy of 19.9 ± 1.2 kcal mol -1 , which is within 1.3 kcal mol -1 of the experimental value (derived from kcat) of 18.6 kcal mol -1 (noting again that the rate-limiting step for the enzymecatalyzed reaction occurs off the enzyme, 86 so this value is only a proxy for the barrier for the ringopening). We then extended our EVB calculations to model PRA ring-opening as catalyzed by a set of variants of MtPriA and SeHisA(dup13-15) (Figure 9 and Table S11). In the case of the SeHisA(dup13-15) variants, all the variants yielded results within reasonable agreement (~1 kcal mol -1 ) with experimental values. We note as an aside that we also performed simulations on wildtype HisA for PRA substrate (which is not active toward this substrate) and obtained an activation free energy of 19.5 ± 0.6 kcal mol -1 , very similar to the one obtained for SeHisA(dup13- 15), suggesting that in theory the wild-type enzyme could catalyze this reaction if all loops are in the correct conformation and the substrate is optimally positioned, and that the experimental lack of activity is not due to a high barrier to the chemical reaction catalyzed by this enzyme. Tying in with this, as described in the Supporting Information, our PRA simulations are initated from an idealized position of this substrate in the active site, based on overlay with the position of the larger substrate ProFAR. However, the stability of this ProFAR conformation in the active site is facilitated by interaction with the gripper loop 3, whereas PRA lacks the distal phosphodianion group of ProFAR and is thus not able to make this interaction (Figure 3). This suggests that if PRA could be gripped properly (and thus optimally aligned), turnover could in turn happen.</p><p>In the case of MtPriA we modeled three single amino acid substitutions (R19A, D130A and R143A) and extended our EVB simulations to model the effect of these substitutions (Table S11, Figure 9), in order to specifically capture the impact of the loss of electrostatic contribution of each truncated side chain on the activation free energy. In the case of the R19A variants, we obtain excellent agreement with the experimental value. However, in the case of the D130A variant, our model significantly under-estimates the activation free energy difference compared to experiment, again suggesting that the experimental effect is rather related to a change in substrate positioning or loop dynamics, that is not captured in our EVB simulations. In the case of the R143A variant, we obtain an activation free energy 1.9 kcal mol -1 lower than the wild-type enzyme. We note that, experimentally, this substitution has been shown to significantly impair the isomerization activity of MtPriA towards PRA (kcat/KM reduced from 1.7 x 10 5 M -1 s -1 to 6.0 x 10 3 M -1 s -1 on introduction of this substitution 25 ). However, it is unclear if this is an effect on kcat, KM or both, and it is plausible that the loss of activity is due to structural effects that prohibit productive substrate binding, that are not captured in our simulations. 38 This latter issue would be similar to our observations from a recent study of an analogous system activated by a ligand-gated conformational change, glycerol-3-phosphate dehydrogenase (GPDH). 38 In this system a substantial loss of activity upon truncation of a key catalytic arginine to alanine could only be explained by structural rearrangements (predominantly blocking of the closure of a key catalytic loop), that were observed upon crystallization of this variant. In contrast, this loss of activity could not be captured simply by performing a truncation of this side chain on the wild-type enzyme and considering only electrostatic effects which only accounted for a smaller part of the observed change in activity. S11 and S12). Due to the lack of relevant crystal structures, we used structures with loop 1 in an open conformation extracted from our conventional 36 MD simulations as starting points for the EVB simulations. As can be seen from these data, for the catalyzed isomerization of PRA, when the reaction was modelled with loop 1 in an open conformation, we obtain much higher activation free energies for ring-opening than when modeling the reaction from a loop-closed conformation, due to a combination of the loss of key interactions between loop 1 and the substrate, and also extra solvent-exposure of the active site when this loop opens up. However, we observe no impact on the activation free energy when modelling the catalyzed isomerization of ProFAR, staring with loop 1 in open conformation.</p><p>Therefore, while the catalytic importance of loops 5 and 6 is well-established, 25,28,54 our EVB calculations show a clear role also for correct closure of loop 1 for PRA isomerization, with full closure of the loop into a catalytically competent conformation being essential for efficient isomerization of PRA.</p><!><p>In the present work, we use a combination of conventional and enhanced sampling molecular dynamics simulations, as well as empirical valence bond calculations, to explore the role of loop dynamics in dictating the selectivity and evolvability of the evolutionarily important model enzymes, HisA and TrpF, 28,[45][46][47][48] which selectively catalyze the isomerization of substrates ProFAR and PRA, respectively (Figure 1), as well as the bifunctional isomerase PriA, which catalyzes both reactions in bacteria such as M. tuberculosis. 87 The roles of loop dynamics and ligand-gated conformational changes in TIM-barrel proteins and proteins from other folds have been a topic of substantial research interest (e.g. refs. 18,[34][35][36][37][38][88][89][90][91][92][93][94][95][96][97] , among many others). However, what makes the current enzymes stand out from these prior studies is the importance of not one but two (TrpF) or even three (HisA and PriA) long, mobile loops (Figure 1), the specific conformations of which have been suggested to play an important role in facilitating the selectivity of PriA and evolved HisA variants. 25,28,54 Thus, these enzymes undergo more complex loop dynamics than the aforementioned systems. In addition, prior enzymes that have been characterized as being activated by ligand-gated conformational changes, such as TPI and OMPDC, are extremely proficient enzymes. [34][35][36][37][38][98][99][100] In contrast, all enzymes studied here are relatively inefficient (Table S2), 25,28,80 , with turnover numbers of ~10 s -1 or less, 28,52,101 despite catalyzing a reaction that is intrinsically very fast. 85 Related to this, while loop motions in highly proficient TIM-barrel enzymes such as TPI are relatively fast (on the μs timescale 96 ), motions of up to the ms timescale have been detected in the evolved HisA variants, 28 and thus loop motions are likely to be (at least partially) rate-limiting in these enzymes.</p><p>At the simplest level, our simulations show, in agreement with structural data, 25 that the enzymes TrpF, PriA and HisA have increasingly large (in terms of active site volume) and "breathable" active sites (Table S8), allowing for the accommodation of substrate ProFAR by HisA and PriA unlike PRA-specific TrpF, the active site of which is clearly too small to accommodate the larger substrate. 25 More importantly, HisA and PriA both possess "gripper residues" interacting with the non-reactive phosphodianion group of ProFAR (R83 and S103 in HisA, R85 and T105 in PriA, Figure 2) that are very similar to analogous interactions in other enzymes activated by ligand-gated conformational changes, such as TPI. 34,35 Of note, however, is that the HisA/PriA "gripper" residues are contributed from the less mobile loops 3 and 4, unlike the primary gripper loop, loop 6 in TPI, which undergoes a substantial conformational change upon ligand binding. 22 Other TIM-barrel proteins such as OMPDC possess analogous gripper loops to TPI loop 6, 22 showing evidence for convergent evolution on these different (βα)8-barrel scaffolds.</p><p>Our simulations show that while the gripper interaction is stable in HisA throughout the simulations, in PriA, there is electrostatic repulsion between R85 and an additional active site arginine, R143, which causes instabilities in the catalytic loops (in particular loop 5, Figure S3), as well as in the substrate positioning in the active site, such that the larger ProFAR is bound less stably in the PriA active site than in the HisA active site. This is in effect a ligand-gated effect, where interaction with the non-reactive phosphodianion (which is not present in the smaller substrate, PRA) facilitates the stability of catalytically important loop 5. Thus, the underlying principles driving loop stability are similar to those of other enzymes that are activated by ligandgated changes.</p><p>Following from this, PCA analysis on our simulations shows that the active site loops in these enzymes are not rigid, but can sample a range of wide-open conformations with transitions between them, with their conformational flexibility being stabilized by ligand binding (although less so in the bifunctional PriA than the ProFAR-specific SeHisA). In contrast, in TrpF, which binds a smaller substrate and lacks the gripper, the active site loops remain dynamic, in particular loop 6 (Figure 1), which samples a range of open conformations even with substrate PRA bound to the active site. Related to this, all HisA variants from the real-time evolution experiment 46 studied here also sample a range of open and wide-open conformations. In this context, however, the single D10G substitution on loop 1 appears to be sufficient by itself to increase the population of the closed conformation sampled during our simulations (Table 1), and the highest proportion of closed conformation is observed in simulations of the SeHisA(dup13-15/D10G/Q102A/Q24L/V15[b]M) variant, which has the highest TrpF activity 28 (Table S2).</p><p>Furthermore, pulling simulations where we pull products PRFAR and CdRP out of the SeHisA(dup13-15/D10G/G102A/Q24L) active site (the only variant from the real-time evolution experiment 28 with a PRFAR-bound crystallographic structure) show significant conformational changes in both loops 1 and 6 when pulling PRFAR out of the active site, whereas when pulling CdRP out of the active site, loop 1 is much more stable and the main requirement is for loop 6 to open. This suggests that loop 1 dynamics are more important for binding of ProFAR and subsequent release of PRFAR, than for the smaller substrate PRA and its product CdRP, whereas loop 1 dynamics appears to be critical to catalysis (Table S11). In addition, the substantial rearrangements that we observe for both compounds suggests that a slow (potentially rate-limiting) product release step is the reason for the low turnover numbers observed for an otherwise facile reaction, further providing evidence that turnover rates are being regulated by loop dynamics.</p><p>In addition, in contrast to HisA, which undergoes conformational changes of loop 1 during the real-time evolution experiment that changes its selectivity from ProFAR-specific to PRAspecific, 28,46 the bifunctional enzyme PriA is already able to rearrange its active site in its wildtype form, to accommodate the different substrates through alternation between Pro-ProFAR and Pro-PRA conformations of loop 5. 25 These conformational changes both reduce repulsion between the two active site arginine side chains that in turn destabilize loop 5 dynamics (Figure S3), as well as disrupting the stacking interaction between the W145 side chain and the substrate ProFAR (Figure S4). This rationalizes the preference of this enzyme towards PRA rather than ProFAR, despite its similarities with the HisA active site, and we note also that loss of the stacking interaction between W145 and the substrate ProFAR was also presented as one aspect of the gain of PRA-isomerization activity in the SeHisA(dup13-15) variant from the real time evolution experiment. 28,46 Finally, we performed EVB simulations of the first ring-opening step of ProFAR and (where relevant) PRA isomerization (Figure 1) by wild-type HisA, PriA and variants. As described above, the actual rate of the chemical step in these enzymes is unknown, since the rate-limiting step is likely to occur off the enzyme. 86 However, of the steps that occur in the enzyme active site, this is the step that is likely to be the slowest, and therefore it is of interest how the substitutions affect the rate of the ring-opening reaction. Here, we see that our calculated activation free energies for the ring-opening reaction trend well with the differences in activation free energies derived using the measured turnover numbers as an upper limit for this value, suggesting there is both a chemical and a dynamical component to the observed changes in activity upon substitution and/or duplication of key residues. Furthermore, in order to reproduce the relevant PriA activation free energies, it was necessary to start from different structures of loop 5, following earlier structural analysis that demonstrate the loop can exist in either a knot-like pro-PRA or beta-hairpin pro-ProFAR conformation (Figure S1), depending on what product is bound to the active site. 25 Clearly, the ease with which this rearrangement can occur will also impact the selectivity of this enzyme. Also, EVB simulations of wild-type SeHisA, MtPriA and the SeHisA(dup13-15/D10G/G102A/Q24L/V15[b]M) variant with loop 1 in an open conformation all yield substantially higher energies for PRA isomerization, whereas ProFAR isomerization (by wild-type HisA) seems to be unaffected (Tables S10 and 11). This further emphasizes the importance of the correct closure of loop 1 for isomerization of the smaller substrate PRA. This is in contrast to substrate binding, where the conformational plasticity of loop 1 appears to be far more important for facilitating correct binding of ProFAR than PRA (Figure S8).</p><p>Taken together, these observations highlight the critical role of multiple decorating loops in HisA, PriA and TrpF in facilitating catalysis. These enzymes stand out from prior systems that have been demonstrated to be activated by ligand-gated conformational changes [34][35][36][37][38][39][40][41] due to a number of factors. First, we have shown the inter-dependent motion of three long loops (or two in TrpF), none of which dominates and each of which is capable of undergoing substantial conformational changes to facilitate the turnover of different substrates. Second, unlike prior systems which show substantial rate accelerations compared to the uncatalyzed reactions with comparatively rapid loop motions, in these enzymes, the catalyzed reaction is already intrinsically fast 85 whereas loop-motion is slow and appears to be controlling the reaction rate.</p><p>It has been argued that a PriA-like gene product could have been the common evolutionary ancestor for both HisA and TrpF. 53,87,102 Ancestral sequence reconstruction has also been used to suggest that ancient HisA precursors were likely bifunctional, and that this bifunctionality persisted over at least a two-billion-year time span. 52 However, as shown in Figure 5, HisA and PriA exploit loops 1, 5 and 6 to facilitate activity, whereas TrpF lacks an analog for loop 1, and isomerizes PRA harnessing just two catalytic loops, 3 and 6. Our results suggest that an evolutionary trajectory from a PriA-like ancestor to an extant TrpF would be surprisingly complex.</p><p>Loop 1 would need to be truncated (and not extended, as when SeHisA was artificially evolved into a TrpF 28 ) and the inter-dependency of this third loop would need to be lost, raising the question of what evolutionary path would take a PriA-like precursor to TrpF, while completely abolishing this loop.</p><p>Despite the novel aspects of the systems studied here, a key similarity with prior systems is the generalizability of ligand-gated conformational changes across a wide range of systems, in particular TIM-barrel proteins, 34 which tend to possess flexible loops decorating their active sites.</p><p>The conservation of such ligand-gated conformational changes-albeit triggered in different loops-suggests that these decorating loops evolve independently of the barrel providing a starting point for the emergence and divergence of new enzyme activities. 31,47 In addition, TrpF, for example, has been shown to be highly tolerant of variations in loop 6 sequence, such that grafting sequences from related enzymes such as TrpA, HisA and PriA onto the TrpF scaffold did not abolish activity. 103 This is significant considering the high evolvability of this scaffold, 18 and the wide range of chemistry it supports, 47 which makes it very desirable as a starting point for protein engineering efforts. In addition, it could be argued that the real-time evolution experiment that bestowed PRA-isomerization activity to HisA 46 effectively performed "natural" loop-engineering by altering the conformations of key active site loops. 28 Our work suggests therefore that, more broadly, loop grafting and engineering is a powerful tool for generating novel enzymes with tailored activities and specificities, even in complex systems with multiple highly mobile and interdependent catalytic loops.</p><!><p>Full details of computational methodology, additional structural and simulation analysis.</p><p>Representative EVB input files, starting structures, parameter files, and Q6 topology files have been uploaded to Zenodo, DOI: 10.5281/zenodo.5893598.</p>

ChemRxiv

Superconcentrated electrolytes widens insertion electrochemistry to soluble layered halides

Insertion compounds provide the fundamental basis of today's commercialized Li-ion batteries. Throughout history, intense research has focus on the design of stellar electrodes mainly relying on layered oxides or sulfides, and leaving aside the corresponding halides because of solubility issues. This is no longer true. In this work, we show for the first time the feasibility to reversibly intercalate electrochemically Li + into VX3 compounds (X = Cl, Br, I) via the use of superconcentrated electrolytes, (5 M LiFSI in dimethyl carbonate), hence opening access to a novel family of LixVX3 phases. Moreover, through an electrolyte engineering approach we unambiguously prove that the positive attribute of superconcentrated electrolytes against solubility of inorganic compounds is rooted in a thermodynamic rather than a kinetic effect. The mechanism and corresponding impact of our findings enrich the fundamental understanding of superconcentrated electrolytes and constitute a crucial step in the design of novel insertion compounds with tunable properties for a wide range of applications beyond Li-ion batteries.

superconcentrated_electrolytes_widens_insertion_electrochemistry_to_soluble_layered_halides

Introduction<!>Results<!>Rationale for the decreased solubility of halides in superconcentrated electrolytes<!>Synthesis<!>Liquid electrolytes<!>Solid-state batteries<!>Materials characterization<!>X-Ray Absorption Spectroscopy<!>Solubility measurements

<p>Redox chemistry provides the fundamental basis for numerous energy-related electrochemical devices, among which Li-ion batteries (LIB) have become the premier energy storage technology for portable electronics and vehicle electrification. Throughout its history, LIB research has witnessed a frenetic race for designing new intercalation compounds, so that most of the crystallographic families with open framework have been investigated and though chances to discover new promising phases are slim. Nevertheless, some compounds sharing similar structures with those of archetypal LIBs cathode materials have never been envisioned as potential host materials. For instance, while transition metals oxides and sulfides have been widely studied, members of the vanadium tri-halide family VX3 (X = Cl, Br or I) were never investigated as battery intercalation compounds despite sharing similar structure with the iconic Li-ion cathode layered materials TiS2 and LiCoO2. Yet, recent studies highlight the interesting physical properties of theses vanadium halide phases. 1,2 For instance their magnetic and electronic structures can be finely tuned by playing with the interlayer coupling between the Van der Walls gap. 3 Such structural modifications are easily achieved through cation intercalation, 4 reinforcing our motivation for testing these materials as Li intercalation hosts.</p><p>However, the absence of reports on layered halides intercalation compounds is partially explained by their high solubility in polar solvents, as highlighted by their use as vanadium precursors in redox-flow batteries. 5 The delicate balance between materials and liquid electrolytes was already one of the main hurdles that delayed the commercialization of secondary lithium-ion batteries relying on insertion reactions. For instance, the physical and chemical integrity of carbonaceous electrodes were found altered upon lithium intercalation in polypropylene carbonate (PC) based electrolytes initially employed. 6 Such difficulty was later on addressed by tuning the lithium solvation shell with the use of ethylene carbonate (EC), suppressing the co-intercalation of PC and forming a stable passivating layer on the carbon anode, 7 or more recently by using superconcentrated electrolytes. [8][9][10] In turn, history teaches us that an electrolyte engineering approach can be used to unlock lithium intercalation in phases hitherto believed to be too unstable for the application.</p><p>Besides graphite intercalation, superconcentrated electrolytes have also shown promising functionalities to solve many fundamental drawbacks relative to the use of carbonate based electrolytes, such as incompatibility with Li-metal, instability at high voltages, flammability or dissolution of transition metals from the cathode. [11][12][13][14][15][16][17][18] Hence, the use of superconcentrated electrolytes was identified as an attractive direction to explore the reversible intercalation of Li + into halide-based compounds such as the members of the VX3 family.</p><p>Herein we demonstrate the feasibility, via the use of superconcentrated electrolytes, to unlock the reversible electrochemical intercalation of lithium in vanadium halides previously considered as transition metal salts too soluble to be used in Li batteries. As a proof of concept, we studied the electrochemical behavior of three vanadium-based halides: VCl3, VBr3 and VI3. Combining electrochemical measurements with structural (synchrotron X-ray and neutron diffraction) and solubility measurements, we successfully demonstrate that nearly one Li + per formula unit can be reversibly intercalated into these materials, hence opening the door to new intercalation chemistries going beyond our current knowledge regarding oxides, sulfides or polyanionic compounds.</p><!><p>Intercalation of lithium into vanadium halides enabled thanks to superconcentrated electrolytes While VCl3 is commercially available, VI3 and VBr3 were grown in evacuated quartz sealed tubes by reacting elemental vanadium, iodine and bromine in nearly stoichiometric conditions (see methods). Their structure was found to match those reported in the literature, where edge-shared VX6 octahedra form honeycomb layers stacked along the c direction with a AB sequence (O1 type structure 19 ) (Figure 1. a, Supplementary Figure 1, Supplementary Tables 1 and 2). These materials were tested by assembling battery half-cells to investigate their ability to electrochemical insert lithium cations (Li + ), using state-of-the-art LIB electrolyte For these three compounds (Figure 1, b), a relatively small discharge capacity is obtained and the process is clearly irreversible as no capacity is observed during the subsequent charge. Moreover, large amount of vanadium traces are observed after discharge on the negative electrode (Supplementary Figure 2), pointing to the dissolution of the cathode material and an irreversible solution process as the origin for the limited discharge capacity. Similar poor performances were encountered using ionic liquid electrolytes (Supplementary Figure 3). Hence, there is a need to develop a new strategy to overcome these dissolution issues. The first attempt consists in replacing the liquid electrolyte by a solid-state electrolyte in which no dissolution can occur. Effectively, when composite cathodes are prepared and tested in an all-solid-state battery configuration (see methods), the electrochemistry is greatly improved compared to that in LP30, thus hinting towards the electrochemical intercalation of Li + into VX3 compounds (Figure 1. c). However, in such configuration the complex interplay between ionic conductivity of the solid electrolyte, chemical compatibility between components and electrode microstructure requires a separate optimization of electrode formulation and testing conditions for each compound. This is indeed exemplified in the chemical incompatibility found between VBr3 or VI3 and argyrodite solid-state electrolyte, evidenced as a clear color change upon mixing. This observation, combined with the difficulty of assembling ASSB and characterizing active materials in such configuration, renders critical the search for an adequate liquid electrolyte in which halides would be stable.</p><p>To select the correct electrolyte, reconsidering the physico-chemical properties of classical aprotic Li-ion batteries electrolytes proves to be insightful. Commercial LP30 electrolyte is made from a mixture of carbonate solvents (EC and DMC) in which a salt (LiPF6) is dissolved. While EC is a polar component essential to ensure both ion-pairs dissociation and the formation of a stable solid electrolyte interphase (SEI) at the negative electrode, its high dielectric constant is presumably responsible for the VX3 dissolution. Hence, not only the chemical composition of the electrolyte must be modified to prevent vanadium cations and/or halides solubility but also its solvation properties. Inspired by the recent observation of the non-miscibility of lithium halides salts with lithium imide salts, 20,21 we then investigated the electrochemical behavior of vanadium halides in superconcentrated electrolytes. As shown in Figure 1 d, when LP30 is substituted with a 5 M lithium bis(fluorosulfonyl)imide (LiFSI) in DMC electrolyte (denoted LiFSI 5 M hereafter), the electrochemical behavior of the three different phases is drastically even at C-rates as low as C/40, indicating that superconcentrated electrolytes can tackle the solubility issue observed in LP30. Indeed, albeit some initial irreversible capacity being observed in the first cycle, these results suggest that almost 1 Li + per formula unit can be reversibly inserted into these hosts. Furthermore, no vanadium was found at the negative electrode at the end of discharge (Supplementary Figure 2). Overall, switching from regular diluted carbonate-based electrolytes to a superconcentrated electrolyte appears as a key to unlock the electrochemical intercalation of Li + into halide layered compounds. To grasp further insights into the behavior upon cycling of these halides, their structural evolution was monitored by operando synchrotron X-ray diffraction (SXRD). For LixVI3 (Figure 2 a), two successive biphasic processes can be distinguished upon discharge, consistent with the two plateaus previously observed (Figure 1 d). For 0 < x < 0.6, the pristine phase disappears at the expense of an intermediate phase which is then replaced by a fully lithiated phase. On charge, the process is found reversible with the discharged phase being converted back to the pristine one; similar reversible bi-phasic intercalation processes are observed for both VCl3 and VBr3 (Supplementary Figures 4 and 5). Having proved the full structural reversibility of the insertion process and the absence of vanadium dissolution, we believe that part of the observed irreversible capacity is mostly nested in minute amounts of amorphous impurities of chemically absorbed I or Br in our starting materials that were made by gas phase reactions. The structure of every intermediate and fully lithiated phases were then determined by Rietveld refinement. This analysis reveals that the O1type layered structure (𝑅3 space group) of the pristine is preserved for every intermediate phase, with the sole evolution of the lattice parameters (Supplementary Figure 6 and Supplementary Tables 3 and 4).</p><p>Unlike for the intermediate phases, the structure of the fully discharged phases is dependent on the nature of the anion. Hence, while for VI3 the fully discharged phase possesses the O1 structure, for VCl3 and VBr3 a distortion to an O3 layered structure (𝑅3 𝑚 space group) is observed (Supplementary Figures 7 and 8, Supplementary Tables 5,6 and 7), in agreement with a previous report on a VCl3 lithiated phase prepared by a solid-state route. 22 Such a subtle Li-driven structural difference depending upon the nature of the halide may simply be rooted in their size, following the ionic radii Cl < Br < I. Finally, and to no surprise for layered compounds, it was confirmed by neutron powder diffraction experiments that lithium cations sit in the interlayer of the phases (Supplementary Figure 7). To gain a deeper understanding of the kinetics of the redox process taking place during intercalation, galvanostatic intermittent titration technique (GITT) was performed. Interestingly, the quasi-equilibrium path is almost identical for the three halides. Two discharge plateaus are observed, only differing in their potentials which correlate nicely with the ligand electronegativity (the more electronegative the halide, the higher the potential, Figure 3 a,b). This result suggests that the electrochemical trace is solely related to the redox potential of the V(III)/V(II) couple. To validate this charge compensation mechanism, operando X-ray absorption spectroscopy was performed on VCl3 (Figure 3 c). During the discharge, a shift of the V K-edge position to lower energy (Figure 3 c) reveals a shift from V(III) (pristine) to V(II) (Figure 3 d). The overall evolution during the discharge can be fully described using three principal components: the pristine phase, the end of discharge phase and an intermediate phase (Supplementary Figures 9, 10 and 11), confirming the existence of an intermediate phase as previously observed during operando synchrotron XRD.</p><!><p>In summary, we directly proved that superconcentrated electrolytes can be used to explore new intercalation compounds and synthesize novel phases for chemistries previously disregarded as highly soluble in liquid electrolytes. To rationalize this effect, the solubility of VCl3 was measured at different concentrations of LiFSI as supporting salt in DMC by inductively coupled plasma mass spectrometry (ICP-MS). The results, shown in Figure 4 a, reveal an initial increase of the vanadium solubility as a function of the LiFSI concentration followed by a drastic decrease when LiFSI concentration is greater than 1 M, reaching values as low as few mM for the LiFSI 5 M superconcentrated electrolyte. Furthermore, vanadium chloride powder was mixed with 5 M LiFSI in DMC solutions for 3 days at 55°C and 85°C, before allowing the solutions to rest at room temperature and monitoring the amount of dissolved vanadium. Since the measured concentrations were extremely close to the ones obtained at room temperature (cV(RT) = 6.3 mM, cV(55°C) = 7.6 mM and cV(85°C) = 5.5 mM), any scattering of the results due to a kinetic hindering of the powder dissolution can be discarded to explain the aforementioned trend. Moreover, similar results were obtained for VI3 and VBr3 (Supplementary Figure 12). Thus, this peculiar bell shape reflects a thermodynamicallydriven phenomenon. To derive the equilibrium law that governs the solubility of vanadium at different supporting salt concentrations, we first need to understand the nature of the dissolution reaction by identifying the chemical environment of the vanadium cations dissolved in solution. Visually, every 1 M LiFSI solution saturated with VX3 exhibits a pronounced coloration, as evidenced by the presence of absorption peaks in their UV-vis spectra (Figure 4 b). Interestingly, the wavelengths of the absorption peaks are halidedependent, suggesting the formation of a vanadium-halide complex in solution. Moreover, the initial increase of the solubility with LiFSI concentration (at concentrations below 1 M) advocates for the participation of the salt anion in the formation of this complex. Such observations can be rationalized by the formation of adducts in solution in the form of [VX3-nFSIp] n-p , through a chelation or a ligand exchange mechanism, as proposed below:</p><p>where n and p are integers with 0 ⩽ n ⩽ 3 and p > 0 (n = 0 for the chelation mechanism).</p><p>Moreover, the solubility of LiCl is very low in pure DMC (1.3 mM). 23 Hence, since the VCl3 solubility in 1 M LiFSI electrolyte is around 100 times greater than this value, Clions must be generated from the VCl3 dissolution and should undergo an almost complete re-precipitation:</p><p>This second step, which can hardly be observed due to the very limited amount of LiCl precipitated (see discussion in the SI), can drastically shift the first equilibrium towards the formation of the product, as expressed by the equation below:</p><p>At the thermodynamic equilibrium, the Guldberg and Waage law of mass action gives:</p><p>Thus, considering the activity of a solute as the product of its concentration c and its activity coefficient , divided by the standard concentration (c° = 1 mol L -1 ), the solubility s of the VX3 salt can be expressed by the following equation:</p><p>Since the solubility of LiBr (sLiBr) and LiI (sLiI) are expected to be greater than the one of LiCl (e.g. sLiBr = 4 mM in DMC at 25°C, 3 times that of LiCl) 23 , one would expect K°2(Cl) > K°2(Br) > K°2(I), which is consistent with the trend observed for the solubilities of the VX3 compounds that follows VCl3 > VBr3 > VI3 (Figure 4 c). For a chelation mechanism, K°2 = 1 and the solubilities of the different vanadium halides are expected to vary less than for the ligand-exchange mechanism. Hence, our experimental observations point towards dissolution through a ligand-exchange mechanism (see SI for a deeper discussion). Neglecting a variation of the activity coefficients, hypothesis which usually holds for low salt concentrations, 24 the VX3 solubility should thus increase with the LiFSI concentration, in agreement with our experimental observations made for concentrations < 1 M (Figure 4 a). However, such explanation does not hold anymore at high ionic strength, regime in which the activity coefficients are known to largely deviate from unity. 24 First, as expressed in equation ( 5), a large decrease of 𝛾 and 𝛾 could potentially explain the lowering of the VX3 solubility.</p><p>Nevertheless, as suggested in a recent theoretical work, the activity coefficients of single ions are more likely to be greater at high salt concentrations. 24 Hence, the decreased solubility is rather explained by a large increase of 𝛾 ( )</p><p>which, owing to the interaction of the vanadium adduct with surrounding solvent molecules, would require developing a refined model to accurately account for the variation of its activity coefficient with the ionic strength. We should further emphasize that independently on the dissolution mechanism (ligand-exchange or chelation), the VX3 solubilities are expected to vary in a similar manner depending on the LiFSI concentration (Figure 4 d).</p><p>Our study provides the rationalization for the observed bell shape behavior: the initial increase of the solubility originates from the increased concentration of FSI anions, while the decrease observed in the concentrated regime arises from a large deviation from unity of the transition-metal complex activity coefficient value (Figure 4e). More importantly application-wise, the solubility of the fully discharged LiVCl3 phase shows similar bell shape (Supplementary Figure 13), albeit the solubility is found lower than for VCl3 which ensures a good resistance to dissolution for the material even upon reduction. However, we note that upon long cycling a severe capacity decay is observed and can be attributed to vanadium dissolution accumulated over time (Supplementary Figure 14). Obviously, future work ranges in better tuning the delicate electrolyte-solvent balance to improve the capacity retention both at RT and 55°C. In conclusion, these findings confirm that the fundamental framework developed in this work captures every stage of lithiation and can be transposed to the study of other intercalation compounds.</p><p>Overall, we established the use of superconcentrated electrolyte as a platform to discover new families of materials for Li + intercalation. Driven by the broad interest for their physical properties, we (reversibly) intercalated Li + into layered vanadium halides to form new layered phases, the impact of which goes beyond the sole development of intercalation electrodes for secondary batteries. Indeed, preliminary nuclear magnetic resonance (NMR) results (Supplementary Figure 15) suggest a fast exchange of Li + cations between different crystallographic sites, which does not come as a total surprise owing to the ongoing interests for halides as solid state ionic conductors. Moreover, these layered halides have been widely investigated for their promising magnetic properties, and preliminary magnetic susceptibility measurements show that Li + intercalation can be used to tune their magnetic structure. Aside from unlocking the synthesis of new layered structures, our study also lays the fundamental background to comprehend a so far ill-understood effect related to the use of superconcentrated electrolytes, i.e. the decreased solubility of active material. Indeed, we demonstrate that the low solubility of transition metal compounds in superconcentrated electrolytes originates from a shift of the solubility equilibrium, i.e. from a thermodynamic effect rather than a kinetics one, which also applies to current collectors whose stability in presence of corrosive anions (e.g. FSI, TFSI) was observed to increase in superconcentrated electrolytes. In general, our study highlights the critical need for solid-state chemists to include knowledge about the physical chemistry of liquids when developing novel intercalation compounds. Evidently, the future success of such explorative and collaborative work will rely on answering fundamental questions regarding equilibrium and ion activities in this novel class of electrolytes.</p><!><p>VBr3 and VI3 were grown in evacuated quartz sealed tubes by reacting elementary vanadium (vanadium powder, -325 mesh 99.5% -Alfa Aesar), iodine (99+%, Alfa Aesar) and purified bromine (Sigma-Aldrich) with a slight excess of halide. Because bromine is a liquid, the tube was place in a liquid nitrogen bath to freeze bromine before being evacuated and quickly flame-sealed. The tubes were placed in a tubular oven, the extremity containing the reactants being heated at 400°C for VBr3 and 450°C for VI3 while the other extremity was placed at almost room temperature, as reported elsewhere. 1,2 After a 72h synthesis, large crystals were collected at the cold extremity of the tube, placed in a Schlenk tube and further heated at 200°C for few hours under vacuum (10 -2 mbar) to eliminate the excess bromine or iodine traces. VCl3 (97%, Sigma-Aldrich)</p><p>was used as received. Lithiated phases were obtained by adding the VX3 phases in a 3 times excess of nbutyllithium solution (2.5 M in hexane, Sigma Aldrich) and stirring the suspension for at least 1 hour. The solution was further centrifuged and the collected powder was washed 3 times with hexane before drying under vacuum in the glovebox antechamber.</p><p>The Li3PS4 solid electrolyte was obtained in our laboratory via a THF-mediated route proposed by Liu et al. 25 and with a room temperature ionic conductivity of RT= 0.21 mS/cm. On the other hand, commercial</p><p>Li6PS5Cl electrolyte was used (NEI), having an ionic conductivity of RT= 3.8 mS/cm.</p><!><p>All the electrochemical experiments were carried out in an Ar-filled glovebox. All the materials tested were mixed with conductive carbon super-P in an active-material/carbon ratio of 7/3. For all the experiments using a liquid electrolyte (LP30 1 M LiPF6 dissolved in 1:1 v/v ethylene carbonate/dimethyl carbonate, Solvionic or LiFSI 5 M in dimethyl carbonate, Solvionic) the as prepared composite were tested in a coin-cell configuration versus a Li metal negative electrode separated by one Whatman glass fiber separator, soaked with ~ 100 L of electrolyte. The mass of composite loaded in the coin cells was comprised between 3 and 5 mg. The cells were cycled on VMP or MPG potentiostats from BioLogic at room temperature, except for the GITT for which the cells we placed in a 25°C oven.</p><!><p>Both anode and cathode composites were prepared by hand grinding the components in the proportions mentioned below with mortar and pestle within an Ar-filled glovebox. The electrochemical testing of the VX3 compounds (X = Cl, Br, I) in all-solid state configuration was conducted in a three-pieced homemade cell consisting in two stainless steel pistons which are inserted into a PEI body (Supplementary Figure 16).</p><p>The cell is closed by means of six axial screws which also provide the pressure required for correct operation.</p><p>Additionally, a ferrule-cone pair is also integrated in each piston making the setup airtight.</p><p>For the battery assembling 35 mg of Li6PS5Cl electrolyte were firstly loaded into the cell and cold pressed at 200 kg/cm 2 for 1 minute. Next, 10 to 12 mg/cm 2 of catholyte (VX3:Li3PS4:VGCF, 65:30:5 wt.%) were evenly spread onto one side of the compressed solid electrolyte and a pressure of 1000 kg/cm 2 was applied during 1 minute. Lastly, 35 mg of anode composite (Li0.8In: Li6PS5Cl, 60:40 wt.%) were added onto the opposite side and the whole stack was densified at 4000 kg/cm 2 for 15 Min. The fully assembled cell was later closed with a torque key applying 2.3 Nm torque in each screw, which yields an internal pressure of ~1000kg/cm 2 .</p><p>Galvanostatic cycling of the cells was carried out at room temperature, in the voltage range at C/10 using the VMP3 electrochemical workstation by Bio-Logic Science Instruments SAS.</p><!><p>Ex Situ synchrotron X-ray diffraction (SXRD) patterns were collected on the BL04-MSPD beamline of the ALBA synchrotron (Barcelona area, Spain) at wavelength λ = 0.41378 Å using the Position Sensitive Detector MYTHEN. Powder samples were filled in 0.6 mm diameter borosilicate capillaries inside an Ar-atmosphere glovebox and subsequently flame-sealed. Operando measurements were carried out in transmission mode using dedicated coin cells 26 assembled under argon filled glovebox and mounted on an ALBA designed 4 samples changer. Constant wavelength (λ = 1.622 Å) neutron powder diffraction (NPD) data were collected for LiVX3 (X= Cl, Br, I) at room temperature using the ECHIDNA high angular resolution powder diffractometer installed at the OPAL research reactor (Lucas Heights, Australia). 27 To prevent reaction of the samples with ambient atmosphere, the samples were loaded into 9 mm diameter cylindrical vanadium cans in Ar-filled glove box and sealed with In seals. All diffraction patterns were refined using the FullProf program.</p><p>The scanning electron microscopy images were measured on a FEI Magellan scanning electron microscope equipped with an Energy dispersive X-ray spectroscopy (EDX) Oxford Instrument detector. EDX measurements were carried out using an acceleration voltage of 10 kV. The Li metal anode samples were collected from cycled cells, washed with dimethyl carbonate and sealed in an air tight container. The transfer from the container to the microscope vacuum chamber were realized rapidly (~ 10 s) to minimize air exposure.</p><!><p>Synchrotron X-ray absorption spectroscopy was performed at the vanadium K-edge at the ROCK beamline of the SOLEIL synchrotron facility (Saint-Aubin, France). 28 The Si(111) quick-XAS monochromator with an oscillation frequency of 2 Hz was employed to select the incident photons energy. The spectra were collected in transmission using three gas ionization chambers in series as detectors. A vanadium metal foil was placed between the second and the third ionization chambers to ensure the energy calibration. An average of 900 scans per spectrum (corresponding to 15 minutes of acquisition time for one merged XAS spectrum) was recorded to ensure the reproducibility and to increase the signal-to-noise ratio. The VCl2 and VCl3 reference samples were prepared by mixing uniformly the active material with carbon, then pressed into pellets of 10 mm in diameter. For the operando measurements, a self-standing electrode was prepared by mixing VCl3 active material with carbon black and polytetrafluoroethylene (PTFE) in the ratio of 20:70:10 (by wt.%). The electrode was placed in the in situ electrochemical cell 29 and cycled against metallic Li using a Celgard membrane as separator and ~100 L of 5 M LiFSI in dimethyl carbonate (DMC) as electrolyte. Then, the in situ cell was discharged from the OCV (3.025 V) to 2.5 V vs Li + /Li at a C-rate of C/20 (corresponding to 1 mole of Li + inserted in 20 hours), and a XAS spectrum was recorded every 15 minutes. The details for the principal component analysis are given in the SI.</p><!><p>To ensure that the concentrations of vanadium measured in the electrolytes match the thermodynamic limit of solubility for VX3 materials, a large excess of powder (~100-500 mg, depending on the material) was added in 1 mL of organic electrolyte and stirred for 3 days at room temperature in a Ar-filled glovebox. The as prepared solutions were centrifuged (6 000 rpm, 1 hour), and the supernatant was collected and further filtered on 0.2 m pore size PTFE syringe filter in an Ar-filled glovebox. Outside of the glovebox, the as prepared solutions were diluted 1 000 or 10 000 times in a 2w% HNO3 (prepared from HNO3 99.999% metal basis Alfa Aesar and Mili-Q ultrapure water) to reach final vanadium concentrations below 1 ppm. The vanadium concentration in the as prepared aqueous solutions were measured by ICP-MS (Nexion 2000 Perkin Elmer) using a calibration curve obtained by diluting a vanadium standard solution for ICP-MS (TraceCERT, 1 mg/L V in nitric acid, Sigma Aldrich).</p>

ChemRxiv

Magnetic Electrochemical Immunoassays with Quantum Dot Labels for Detection of Phosphorylated Acetylcholinesterase in Plasma

A new magnetic electrochemical immunoassay has been developed as a tool for biomonitoring exposures to organophosphate (OP) compounds, e. g. insecticides and chemical nerve agents through directly detecting OP phosphorylated acetylcholinesterase (OP-AChE). This immunoassay uniquely incorporates highly-efficient magnetic separation with ultra-sensitive square wave voltammetry (SWV) analysis using quantum dots (QDs) as labels. A pair of antibodies were utilized to achieve the specific recognition of OP-AChE that was prepared using paraoxon as an OP model agent. Anti-phosphoserine polyclonal antibodies (Ab1) were anchored on amorphous magnetic particles (MPs) preferably chosen to capture OP-AChE from the sample matrixes through binding their phosphoserine moieties that were exposed through unfolding the protein adducts, which was validated by electrochemical examinations and enzyme-linked immunosorbent assays. Furthermore, anti-human AChE monoclonal antibodies (Ab2) were labeled with cadmium-source QDs to selectively recognize the captured OP-AChE, as characterized by transmission electron microscopy (TEM). The subsequent electrochemical SWV analysis of the cadmium component released by acid from the coupled QDs was conducted on disposable screen-printed electrodes (SPEs). Experimental results indicated that the SWV-based immunoassays could yield a linear response over a broad concentration range of 0.3 \xe2\x80\x93 300 ng/mL OP-AChE in human plasma with a detection limit of 0.15 ng/mL. Such a novel electrochemical immunoassay holds great promises as a simple, selective, sensitive, and field analysis tool for the effective bio-monitoring and diagnosis of potential exposures to nerve agents and pesticides.

magnetic_electrochemical_immunoassays_with_quantum_dot_labels_for_detection_of_phosphorylated_acetyl

Introduction<!>Reagents and materials<!>Instruments<!>Preparation of MP-Ab1 conjugates<!>Preparation of QD-Ab2 labels<!>Preparation of OP-AChE<!>ELISA for antibody immuno-affinities to OP-AChE<!>Magnetic electrochemical SWV immunoassays<!>Principle of magnetic electrochemical immunoassay using QD labels<!>Evaluation of antibody immuno-affinities to OP-AChE<!>Minimization of nonspecific adsorption of the immunoassay<!>Optimization of major parameters for electrochemical immunoassays<!>Analytical characteristics of SWV immunoassays<!>Conclusions

<p>Neurotoxic organophosphates (OPs) have been widely used as pesticides in agricultural industry and as chemical warfare agents.1, 2 As a result, OP contaminations have been widespread in air, water, soil, and food, such that there is a potential for human exposures. Therefore, public concern about the development of effective detection devices for effective monitoring OPs and evaluation of human health risk of OP exposure has grown steadily in recent years.3–5 Moreover, after the Tokyo subway attack in 1995,6 the needs for feasible OP detection methods have become increasingly urgent for purpose of early warning of potential terrorist attack and diagnostic mitigation of the effects from alleged nerve-agent exposures.2, 3, 5–7 </p><p>In recent decades, numerous analysis methods have been developed for the assessment of OP exposures, and the relevance of end points to human health is of utmost concern.3, 8–10 In this regard, biomonitoring of OP exposures is recognized to be one of the best approaches.8 The internal dosages of OP agents or their metabolites are quantitatively and / or qualitatively measured on the basis of our knowledge of the metabolic fate of the toxicants, thus providing the accurate evaluation of the health risk of integrated OP exposure. Unfortunately, the majority of current biomonitoring protocols for OP exposures, 3, 8, 11, 12 may still suffer from some intrinsic disadvantages of either low detection specificity and sensitivity (i.e., Ellman colorimetric assays13, 14 ), or expensive analysis settings entailing well-trained personnel and inconvenience for field applications (i.e., gas or liquid chromatography coupled with mass spectrometry (GC- or LC-MS) 4, 15–17). Hence, simple, sensitive, selective and field-deployable tools are still highly desired for biomonitoring and diagnostic evaluation of OP exposures at present, especially for the enhancement of our response to a sudden emergency and the improvement of our ability to medically counteract the effects.</p><p>It is generally recognized that selection of suitable biomarkers for OP detections is of central importance for developing a biomonitoring strategy.8, 12 Biomarkers that are currently used include: free OPs in blood, or their metabolites in urine, and cholinesterase (ChE) inhibition in blood.3, 8, 9, 12 OPs can stoichiometrically bind with ChE and inhibit the enzyme activity, at the same time, they are metabolized by organophosphorus hydrolase to form inactive phosphonic acids that are then renally excreted.8 The high reactivity of OPs with these enzymes suggests that the levels of free OPs will be inherently low (typically in the range of nanogram per liter or parts per trillion in blood 12), such that ultra-sensitive detection methodologies are thereby required; yet, the formidable false positive signals might be difficult to avoid. Moreover, while OP metabolite level in urine is also considered a sensitive indicator of OP exposure,8, 18 the fact that not all toxicant specific metabolites are derived solely from OPs is a real concern.12 Also, ChE inhibition as a biomarker of OP exposure effect has historically been an important strategy, but the inhibition-based quantification can also be problematic.12, 19 For example, for a quantitative assessment a baseline enzyme level is required to accommodate the individual fluctuations in enzyme levels. All of these factors thereby make the blood ChE measurements less viable for assessing some OP exposures.12 Therefore, exploring selective, sensitive and reliable alternative biomarkers is an important consideration for biomonitoring of OP exposures.</p><p>Electrochemical immunoassays with high selectivity and sensitivity have evolved rapidly over the past decades.20–23 Their detection sensitivity may be enhanced by using various nano-scale materials newly emerged, i.e., quantum dots,24 for electrochemical signal amplifications.23, 25 Such a versatile analysis tool can possess some advantages over the present standard methods for assessment of OP exposures that are based on GC-MS and LC-MS.20, 21, 26 More importantly, their simple operation and miniaturized analysis instruments can meet the requirements of decentralized point–of-care tests or field detections.21, 22, 27 Moreover, according to the biochemical mechanism widely accepted for ChE phosphorylation,28, 29 the inhibition event may produce very stable enzyme complexes with structurally precise phosphoserine esters,30 suggesting that these products may serve as selective indicators directly correlated to the severity of OP exposures. However, a challenge may lie in the current unavailability of recognition elements or appropriate receptors, i.e., antibodies, for specifically targeting phosphorylated ChE. Although some specific antibodies against OP agents, i.e., paraoxon, have been recently developed and commercially available for immunoassays,31–33 they might be unable to selectively recognize modified or aged OP moieties of OP-AChE., which is addressed in this study.</p><p>In this work, we present preliminary studies to establish a novel electrochemical immunoassay of phosphorylated AChE as a biomarker for biomonitoring and diagnosis of OP exposures. OP-AChE, which was prepared by incubating human AChE with paraoxon, was used as the model targets. In order to circumvent the current limitations of OP-AChE recognition, two kinds of antibodies, anti-phosphoserine polyclonal antibodies (termed as Ab1) and anti-human AChE monoclonal antibodies (termed as Ab2), were employed to facilitate the specific recognitions of OP-AChE. Amorphous magnetic particles (MPs) with large surface-to-volume ratio were chosen to load Ab1 to capture the OP-AChE from the sample matrixes through binding the phosphoserine moieties, which were disclosed through reductively unfolding the protein adducts via dithiothreitol (DDT), followed by the second recognition of Ab2 labeled with QDs serving as the signal-amplifying tags. The sandwich immunoreaction events were subsequently quantified by square wave voltammetric (SWV) analysis. Main parameters governing the SWV responses were optimized including the MP-Ab1 conjugate dosage, QD-Ab2 label concentration, and reaction time. Moreover, a blocking agent consisting of 3 % BSA and 1 % PEG was introduced for effective minimization of nonspecific adsorptions in the immunoassays. The magnetic-aided immunoassay was overall proofed with simple, selective and sensitive analysis features by spiking human plasma with known OP-AChE concentrations.</p><!><p>The Qdot@655 antibody conjugation kit was purchased from Molecular Probes Inc. (Eugene, OR), which includes quantum dots (QDs, CdS@ZnS), succinimidyl trans-4-(N-maleimidylmethyl) cyclohexane-1-carboxylate solution, dithiothreitol (DDT) solution, dye-labeled marker for antibody elution, mercaptoethanol, separation media, and exchange buffer. BioMag® magnetic immobilization kit was obtained from Polysciences Inc. (Warrington, PA), with amine-terminated amorphous magnetic particles (MPs, 50 mg/mL), glutaraldehyde (GLU) coupling reagent, and glycine quenching solution. Human acetylcholinesterase (AChE), polyethylene glycol (PEG, MW 10 KD), bovine serum albumin (BSA), hydroxylamine, phosphate buffer saline (PBS), Tris-HCl stock buffer (1.0 M) and Tween-20 were the products of Sigma-Aldrich. Anti-phosphoserine polyclonal antibodies (Ab1) and their horseradish peroxidase (HRP) conjugate, and anti-human monoclonal AChE antibodies (Ab2) were purchased from Abcam Inc. (Cambridge, MA). Paraoxon was bought from Chem Service, Inc. (West Chester, PA). The BSA-PEG blocking agent consists of 3 % BSA and 1 % PEG in Tris-HCl buffer (0.02 M), which pH was adjusted to 7.4 using 0.1 M HCl. The washing buffer was prepared with 0.02 M Tris-HCl buffer containing 0.1 % Tween-20, 0.5 % BSA and 0.15 M NaCl. All stock and buffer solutions were prepared with Nanopure-purified water. Other reagents were of analytical reagent grade.</p><!><p>MCB 1200 Biomagnetic Processing Platform was the product of Dexter Magnetic Technologies (Sigris, CA). Square-wave voltammetric (SWV) measurements were performed using an electrochemical analyzer CHI 660 (CH Instruments, Austin, TX), which is connected to a personal computer. Disposable screen-printed electrodes (SPEs) consisting of a carbon working electrode, a carbon counter electrode, and an Ag/AgCl reference electrode, were purchased from Alderon Biosciences Inc. (Durham, NC). A sensor connector allows for connecting the SPE to the CHI electrochemical analyzer. Transmission electron microscope (TEM, Hitachi H-7000, Japan) was utilized to characterize the sample suspensions, each of which was dropped onto the carbonate film-coated copper grids (3 mm-diameter, 200-mesh) to be dried at room temperature and then measured at 75 kV. Microplates for enzyme-linked immunoassay (ELISA) were purchased from Becton (Franklin lakes, NJ). Disposable PD-10 desalting columns packed with Sephadex G-25 medium (Amersham Bioscience Corp.) were used to purify the protein solutions. Centrifugation was performed using a Sorvall RC 26 plus (Kendro Laboratory Product). Vortex mixer with touch-on-off mixing functions (Barnstead International, Iowa) was used for sample and reactant mixing.</p><!><p>The MP-Ab1 conjugates were prepared following the modified protocol available in the BioMag® magnetic immobilization kit. Typically, 1.0 mL of BioMag amorphous MP suspension terminated with amine groups was transferred to a 2.5-mL centrifuge tube to be washed three times with 0.5 mM PBS (pH 7.4), then magnetically separated on the Biomagnetic processing platform. The supernatant was aspirated out, leaving the BioMag packed as a wet cake on the tube wall. Two mL of GLU (5 %) was then added to the BioMag to be vortexed, and then rotated for 3 h at room temperature. Furthermore, magnetic separation was conducted to remove the unreacted GLU. After being washed four times using 0.5 mM PBS buffer, 1.0 mL of 0.5 mg/mL Ab1, which were prepared in 0.5 mM PBS were then added to the GLU-activated BioMag particles. The mixture was vortexed and further rotated overnight at room temperature, followed by magnetic separation and twice washings. Subsequently, 5.0 mL of glycine solution was introduced into the coupled BioMag to be further rotated for 30 min at room temperature so as to quench the activated BioMag possibly left, and then magnetically separated and washed three times. In the immunoassays, the MP-Ab1 suspension was diluted by the BSA-PEG blocking agent to 1.0 mg/mL of MP particle concentration, which was defined as the stock suspension of MP-Ab1 conjugates.</p><!><p>The QD-Ab2 labels were prepared following the protocol from the Qdot@655 antibody conjugation kit. Basically, 0.5 mg/mL Ab2 was firstly reduced by DDT to obtain Fab' fragments with free sulfhydryls via unfolding the disulfide bonds at the Fc region of antibody. The amine-terminated QDs (TEM image shown in the top right of Figure 2) were pre-activated by succinimidyl trans-4-(N-maleimidylmethyl) cyclohexane-1-carboxylate, and then added to the resultant antibody solution to be mixed and reacted for 1 h at room temperature. After being quenched by mercaptoethanol, the mixture was concentrated by ultra-filtration and purified using the size-exclusion chromatography. The QD-Ab2 suspension was orange-red-colored. Each QD nanocrystal was estimated to load about eight antibody fragments or four intact antibodies,24 with well-retained activity of binding antigens. In the immunoassays, the QD-Ab2 suspension was further diluted by the BSA-PEG blocking agent at the optimized dilution ratio of 1/120 (v/v), which was defined as the stock suspension of QD-Ab2 labels.</p><!><p>OP-AChE was prepared by the incubation of AChE with paraoxon as a model of OPs in 50 mM PBS buffer solution overnight. Briefly, 1.0 mL of a PBS buffer solution containing 240 µg human AChE was mixed with 50.0 µL of acetone containing 34 µg paraoxon to be incubated overnight. The resulted solution was purified with PD-10 column to remove unbound paraoxon and solvent acetone. The enzyme activity of the product was further determined using the Ellman assay till being completely inhibited.13 Moreover, the resultant OP-AChE was concentrated by using ultra-filtration to a final volume of ∼ 1.0 mL, and stored at −20 °C for future use. The protein concentration of OP-AChE stock solution was determined to be ∼ 0.2 mg/mL by spectrophotometry at 280 nm. Of note, all of OP-AChE in the experiments referred to those that were reductively unfolded by 5.0 mM DDT for 15 min prior to immunoassays, unless otherwise indicated.</p><!><p>The immuno-affinities of Ab1 and Ab2 to OP-AChE were examined by ELISA using HRP-conjugated Ab1 and Ab2, where the comparable assays for non-DDT-unfolded OP-AChE, AChE and BSA were conducted in parallel as the controls. Fifty µL aliquot of Ab2 solution (1.0 µg/mL in the blocking solution) was first added into each well of the microplate with three replicates to be incubated overnight at 4 °C. After rinsing the plate wells three times with the washing buffer, 250 µL aliquot of the BSA-PEG blocking agent was introduced to each well to be incubated for 1.5 h at 37 °C. Then, the plate was washed three times. Furthermore, 50 µL aliquot of samples with the desired concentrations was added into each well to be incubated for 1 h at 37 °C, and plate washing was conducted for three times. Following that, 50 µL aliquot of HRP-conjugated Ab1 solution (1.0 µg/mL) was added to each well, and the immunoreaction proceeded for 1 h at 37 °C. After the wells were rinsed four times, 100 µL aliquot of the color substrate was added, and the enzyme catalyzed reaction progressed for 10 min at room temperature. Subsequently, the enzymatic reaction was ceased by adding 10 µL aliquot of 1.0 M HCl per well. The plate was immediately measured at 450 nm on a microplate reader and the absorbance of each well was recorded separately.</p><!><p>Twenty five µL aliquot of samples, with the desired OP-AChE concentrations in Tris-HCl buffer (pH 7.4) or human plasma, were added separately into 1.5 mL plastic centrifuge tubes that were pre-washed twice with the washing buffer. Twenty five µL aliquot of MP-Ab1 conjugate suspension (1.0 mg/mL) was then introduced into each tube to be vortexed by the touch-on-off mixer, and further rotated on the Biomagnetic processing platform for 45 min at room temperature. The mixtures were magnetically separated simply by upraising the magnet at the platform, and then rinsed twice with the washing buffer. Following the same procedure above, 50 µL aliquot of QD-Ab2 labels (1/120, v/v) was injected into each tube to be vortexed and rotated for 30 min at room temperature. The resulted mixtures were rinsed twice (the formed immuno-complex was diluted in Tris-HCl buffer for TEM measurements, with the images shown in Figure 2). Moreover, 10 µL aliquot of 1.0 M HCl was added into each tube to release cadmium ions from the captured QD labels, followed by the addition of 50 µL aliquot of 0.20 M acetate buffer (pH 4.6) containing 10 µg/mL Hg and 0.5 µg/mL Bi. After mixing for 2 min, the BioMag were magnetically precipitated resulting in a "wet cake" formed on the tube walls. 50 µL aliquot of the supernatants was then drawn to be separately transferred to the surface of the SPEs, which were pretreated electrochemically by cyclic voltammetric scanning for 10 times at potential range of 0 – 1.5 V in 50 mM PBS (pH 7.4). Subsequently, electrochemical SWV measurements were carried out by using an in situ-plated Hg/Bi film formed on the SPEs by a 2-min accumulation at −1.4 V. The analysis parameters mainly included the applied potential range of −1.0 to 0.5 V, step potential of 4 mV, amplitude of 25 mV, and frequency of 15 Hz. A baseline correction of the resulting voltammogram was performed using CHI 660A software (Note: the waste after each immunoassay should be collected to a bottle to be disposed safely).</p><!><p>It is recognized that OPs have the same general structure and toxicological mode of action.28 In this work, paraoxon was utilized as a model of OP nerve agent and pesticide to prepare the OP phosphorylated AChE (OP-AChE). Herein, the biochemical mechanism of OP inhibition of AChE has been well documented and understood, which process is initiated by precursory phosphorylation at the catalytic serine residue.28–30 The phosphorylation of AChE by paraoxon is synchronous with the release of p-nitrophenoxy and further goes through an aging process to produce a stable, covalent phosphoserine ester bond or an OP-AChE conjugate.30 The so formed OP-AChE can quantitatively correlate to the original OP dose, thus potentially serving as a selective OP biomarker. However, the detection of this biomarker may be problematic if specific antibodies cannot detect the modified protein. Moreover, in addition to binding with AChE at its peripheral anionic site,34, 35 OP (i.e., paraxon) is recognized mostly to phosphorylate the serine residues at the catalytic active center locating at the base of a deep gorge within the proteins,30, 36 of which phosphoserine moieties are thought to be inaccessible to many traditional forms of analysis.</p><p>Inspired by the fate of OP-AChE formed with structurally precise phosphoserine esters, herein, we attempted to utilize two kinds of commercially-available antibodies, Ab1 and Ab2, to bind the phosphoserine and AChE moieties of the OP-AChE, respectively, achieving the specific recognition of the meaningful adducts. It is well established that ChE (i.e., AChE) is made up of subunits structurally containing inter- and intra-molecular disulfide bonds,36–38 which can be cleaved by DDT serving as a reducing agent.37, 38 Accordingly, DDT was employed to reductively unfold OP-AChE so as to expose its phosphoserine moieties for more accessible immuno-recognition. An electrochemical immunoassay method was thereby developed by combining magnetic separation with electrochemical SWV analysis using QDs as signal amplification tags. Figure 1 schematically illustrates the process of electrochemical immunoassays for OP-AChE in a sandwich detection format, which was performed using centrifuge tubes and disposable SPEs. OP-AChE is first captured by MP-Ab1 through binding the phosphoserine moieties to be magnetically separated from the sample matrixes. QD-Ab2 is then introduced to selectively recognize the captured OP-AChE. The resultant immunocomplex is subsequently treated by acid to release cadmium ions from the bound QDs for SWV measurements on the SPEs. The yielded SWV signals are proportional to the OP-AChE concentrations in the samples. Figure 2 presents the typical TEM images of as-formed immuno-complex and QDs originally used (top right). One can note that amine-terminated MPs are amorphous, and the QD tags are uniform in size and short-rod shaped with dimensions of about 4 nm × 12 nm. Upon the immuno-recognition of targeting OP-AChE, QDs are attached onto the MP surface forming the sandwich immunocomplex, as clearly shown in the high-magnitude TEM image (bottom right of Figure 2).</p><p>In this protocol, use of amorphous MPs can afford higher surface-to-volume ratio for loading larger amount of antibodies. The magnetic separation of OP-AChE can allow for the enrichment of the analytes from the samples to facilitate the electrochemical SWV detections conducted on the SPEs, which might largely avoid the formidable interferences from the complex sample media. Moreover, SWV using mercury film is recognized as an ultra-sensitive technique for determination of heavy metals due to its unique ability of pre-concentrating target species in combination with pulse measurements that yields a high signal-to-background ratio. Additionally, QDs were coated with a thin layer of functionalized polymer providing terminal amine groups for antibody conjugation and spacing between the antibody and QD, which greatly increases the flexibility of antibodies on the surface of QDs. Use of QD tags for the SWV analysis may further benefit from the amplified response signal, achieving enhanced detection sensitivity of the immunoassays.</p><!><p>The manufacturer instructions state that anti-phosphoserine polyclonal antibodies are specific to free and conjugated phosphoserine as well as phosphoserine in modified cellular proteins, without crossing reaction with non-phosphorylated serine. However, it is not clear whether both of Ab1 and Ab2 can specifically recognize the phosphorylated AChE that was reductively unfolded by DDT.</p><p>We first studied the immuno-affinity of Ab1 and Ab2 to OP-AChE by the classic ELISA using HRP-conjugated Ab1 and pure Ab2. DDT-unfolded OP-AChE was employed as the test targets, with non-DDT-unfolded OP-AChE, non-phosphorylated AChE and BSA as the protein controls. Figure 3 displays the ELISA results. Obviously, the response to non-DDT-unfolded OP-AChE (a) is smaller than that to DDT-unfolded ones (b). Moreover, the responses obtained from the control experiments using AChE and high-concentration BSA are > 2.1 times lower than that from OP-AChE. The ELISA data demonstrate that use of Ab1 and Ab2 could present good immuno-affinity and specificity to the phosphorylated AChE treated by DDT.</p><p>Furthermore, the immuno-affinities of MP-Ab1 and QD-Ab2 to OP-AChE were investigated by electrochemical measurements. Here, non-DDT-unfolded OP-AChE, non-phosphorylated pure AChE, BSA and human plasma were simultaneously examined as the challenging protein rivals and sample background. The typical electrochemical responses were shown in Figure 4. It is noted that the current response for DDT-unfolded OP-AChE (curve a) is higher than that of non-DDT-unfolded ones (curve b), indicating the effective role of DDT in reductively unfolding the phosphorylated enzymes for maximizing exposure of their phosphoserine moieties. In contrast, negligibly low signals were observed in the control experiments for AChE (curve c), plasma (curve d) and BSA (curve e). Signals from the control experiments are presumably ascribed to the nonspecific adsorptions of QD-Ab2 to the magnetic particles and centrifuge tubes used. Such a phenomenon may make it necessary to block both of MP-Ab1 conjugates and QD-Ab2 labels as well as the plastic centrifuge tubes, which is discussed afterwards. The signal differences between OP-AChE and these controls indicate that the possibly co-existing AChE and plasma background can have no significant interference with the magnetic-aided electrochemical immunoassays for OP-AChE. The synergistic utilization of MP-Ab1 conjugates and QD-Ab2 labels can achieve desirably specific immuno-recognition to OP-AChE.</p><!><p>Nonspecific adsorption is one of the important issues to be addressed in the development of magnet-aided immunoassays with high selectivity and sensitivity. In the current study, we found that the immunoassays for OP-AChE could have serious nonspecific adsorptions, especially for probing the analytes in human plasma. To minimize such a nonspecific adsorption, we tried to separately introduce 3 % BSA and the BSA-PEG mixture as the blocking agents to pre-treat MP-Ab1 conjugates and QD-Ab2 labels prior to immunoassays. The minimization effectiveness of two kinds of blocking agents was evaluated with the same OP-AChE concentration together with non-phosphorylated AChE and plasma as the controls. The test results are summarized in Table 1. When no blocking step was carried out for two antibody conjugates, the current responses showed no significant difference between OP-AChE, AChE and plasma samples. However, when both antibody conjugates were separately blocked by 3 % BSA, non-specific adsorptions could be largely depressed. Herein, the current response for OP-AChE changed to 3.4 × 10−7 A, while the current responses for AChE and plasma were sharply down to 5.1 × 10−8 A and 3.3 × 10−8 A, respectively. Moreover, non-specific adsorptions could be further depressed when the BSA-PEG agent was utilized to block MP-Ab1 conjugates and QD-Ab2 labels. As is noted from Table 1, negligible current signals were observed for the protein rival (3.0 × 10−8 A) and plasma background (1.7 × 10−8 A), in contrast to the adduct sample (2.9 × 10−7 A). The above results imply that use of the BSA-PEG blocking agent can achieve desirable minimization of nonspecific adsorptions in the immunoassays for OP-AChE. In addition, a pre-treatment of the plastic centrifuge tubes with the BSA-PEG agent could be helpful for avoiding any nonspecific adsorption of QD tags onto the tube walls (data not shown).</p><p>Such an excellent limitation of nonspecific adsorptions may be attributed to the synergetic blocking effects of BSA and PEG that were simultaneously applied for MP-Ab1 conjugates and QD-Ab2 labels. BSA, a kind of small inert molecules of protein, has been well recognized and commonly applied as a blocking agent in varying immunoassays. PEG as a water-soluble and non-immunogenic polymer can possess the unique ability of depressing some nonspecific protein adsorptions and cell adhesions.39, 40 Many surfaces are accordingly derivatized with hydrophilic PEG coatings to improve their biocompatibility.41, 42 PEG layers in water, with rapidly moving hydrated anchored chains and a large excluded volume, tend to repel protein molecules approaching the meaningful surfaces.40, 42 Additionally, PEG is also widely utilized as reaction rate accelerator and detection sensitivity promoter in immunoassays.43, 44 The mechanism regarding the steric exclusion effects is described elsewhere.45 Therefore, the minimization of nonspecific adsorptions by use of the BSA-PEG blocking agent could additionally endow the immunoassays better analysis performances for quantifying OP-AChE in terms of detection rate, sensitivity and selectivity.</p><!><p>The dependence of current signals of immunoassays on the amount of QD-Ab2 labels was examined using different dilutions of the stock QD-Ab2 suspension. The experimental results are described in Figure 5, in which the current responses to the plasma are shown as the control. As can be seen from Figure 5, electrochemical SWV responses to the samples and the control decrease with greater dilution ratios of QD tags. In this regard, an excess of QD-Ab2 labels might cause an increasing nonspecific adsorption, indicating the need to obtain a maximal response while using a minimum amount of QD-Ab2 labels. Accordingly, the optimal amount of QD-Ab2 labels in the reactant solution was determined to be 1/120 dilution, at which the biggest signal-to-noise ratio was observed as shown in the inset of Figure 5.</p><p> Figure 6 displays the SWV response currents that could be affected by the amount of MP-Ab1 conjugates used, in which human plasma was determined in parallel as the control for different concentrations of MP-Ab1 conjugates. The current responses for the same OP-AChE concentration can increase with MP-Ab1 particle concentrations raising from 0.25 mg/mL to 2.0 mg/mL, so do the SWV responses to the plasma (noise). Obviously, high MP-Ab1 concentrations may result in high noise responses to the plasma. As is shown in the insert of Figure 6, however, their signal-to-noise ratios may peak at 1.0 mg/mL of MP particle concentration, after which the ratios may start to decrease. Therefore, 1.0 mg/mL MP-Ab1 is selected in the experiments by compromising between high sample responses with low background interference from the plasma.</p><p>Reaction times for both magnetic capture and QD recognition of OP-AChE are considered to be two vital factors largely affecting the detection performances of the proposed immunoassays. On the one hand, we have checked the effects of the adduct capture time on magnet-aided immunoassays using MP-Ab1 conjugates and high-concentration OP-AChE (Figure 7, curve a). It was found that longer reaction time could bring about bigger current response, which might result from more complete capture of OP-AChE from the sample. From Figure 7 (curve a), we can see that the current responses can increase with increasing capture time till 45 min, after which the responses reach a plateau. Accordingly, the time for the capturing immunoreaction between MP-Ab1 conjugates and OP-AChE is chosen as 45 min. It should be pointed out that the reaction time needed for magnetic capture of phosphorylated AChE may additionally depend on the analyte concentration and the physiochemical property of the sample media. For example, high viscosity of sample may lead to high transferring resistance for the reactants, resulting in relatively long capture time for targeting adducts by the magnetic conjugates. On the other hand, we have investigated the reaction time for the recognition of OP-AChE by QD-Ab2 labels, with the data manifested in Figure 7 (curve b). One can observe that the SWV responses may show no increase after 30-min reaction. That is, the immunoreaction time of 30 min may be enough for the recognition between the analytes and QD-Ab2 labels, which is thereby recommended as the secondary recognition time of OP-AChE in the immunoassays.</p><!><p>To further verify the application feasibility of the developed immunoassay, OP-AChE with different plasma concentrations were probed under the optimal experimental conditions. Figure 8 A displays typical characteristics of electrochemical SWV responses for increasing OP-AChE concentrations in plasma (curve a-j is from 0.01 to 600 ng/mL). Well-defined voltammetric peaks of cadmium were observed at −0.76 V, and the peak current intensities increase with increasing OP-AChE concentrations. Figure 8 B exhibits the resulting calibration curve between current versus [OP-AChE] plotted on a semi-log scale. As is shown in Figure 8 B, linear responses are obtained over the concentration range of 0.3 – 300 ng/mL OP-AChE with a detection limit of 0.15 ng/mL, as estimated by the "S/N = 3" rule. The top inset of Figure 8 B manifests a big difference between the "as-received" SWV responses to 0.15-ng/mL and 0-ng/mL (control) of OP-AChE. A trivially low current signal was observed in the control. Such a noise-depressed behavior is ascribed to the utilization of the BSA-PEG blocking agent as mentioned above. Moreover, a series of measurements of 300-ng/mL OP-AChE in plasma could yield reproducible electrochemical SWV responses with an RSD of 11.5 %, indicating that the sensitive and selective response of the magnet-aided immunoassays can be accompanied with favorable detection reproducibility.</p><p>This work aims at bio-monitoring of low-dose OP exposures, which are defined as < 15% inhibition of plasma cholinesterase at which the victims will not show acute symptoms but may have a harmful biological effect. Since the average AChE level in human plasma is around 8.0 ng / mL,46 the OP-AChE level in human plasma is estimated to be around 1.2 ng/mL for the victims of low-dose OP exposures. Accordingly, the developed immunoassay can have enough sensitivity for bio-monitoring of low-dose exposure to OPs, and holds great promise for analyzing real samples in clinic.</p><!><p>The current evaluation methods of potential exposures to nerve agents and pesticides are generally established by quantifying free OPs or their metabolites or cholinesterase activities, which may in a way have some intrinsic disadvantages. In this paper, a new electrochemical immunoassay method has been developed to assess the formation of phosphorylated AChE. This immunoassay protocol incorporates magnetic separation with QD-based electrochemical SWV analysis. It possesses some merits over the common methods for biological monitoring of OP exposure. First, two different antibodies with high immuno-affinities to the phosphoserine moieties and antigenic AChE, respectively, were employed in a synergetic way, achieving specific recognition of the formidable enzyme adducts towards selective quantification; Second, the enzyme adducts were reductively unfolded by DDT so as to maximize the exposure of their phosphoserine moieties, resulting in enhanced immuno-recognitions; Third, use of magnetic separation for capturing OP-AChE can largely avoid the interferences from the complex sample matrixes, facilitating the direct analysis of phosphorylated AChE in plasma without complex purification steps; Forth, metal ion-based SWV measurements using QDs as signal-amplifying tags could ultra-sensitively probe the enzyme adducts with plasma concentration as low as 0.15 ng/mL, which is comparable to that of mass spectrometric analysis for phosphylated butyrylcholinesterase in human serum;16 Fifth, introduction of the BSA-PEG blocking agent with protein-repelling behavior can significantly minimize nonspecific adsorptions in the immunoassays, making a selective and sensitive immunoassay for OP-AChE; Finally, the utilities of the new immunoassay may include centrifuge tubes, Biomagnetic processing platform, and disposable SPEs, which are truly portable, cheap, and especially convenient for field bio-monitoring and point-of-care diagnosis of OP exposures, if additionally coupled with a miniaturized electrochemical analyzer. Overall, this new electrochemical immunoassay is demonstrated to be a simple, selective, sensitive and field-deployable alternative tool for bio-monitoring and diagnosis of OP exposures to nerve-agents and pesticides. Such kind of detection format may pave the way towards the development of immunoassays for other formidable biomarkers, such as phosphated or nitrated proteins, with low exposure levels in complex biological systems.</p>

PubMed Author Manuscript

NMR insight into the multiple glycosaminoglycan binding modes of the Link module from human TSG-6

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA) binding protein that is essential for stabilizing and remodelling the extracellular matrix (ECM) during ovulation and inflammatory disease processes such as arthritis. The Link module, one of the domains of TSG-6, is responsible for binding hyaluronan and other glycosaminoglycans (GAGs) found in the ECM. In this study, we used a well-defined chondroitin sulfate (CS) hexasaccharide (\xce\x94C444S) to determine the structure of the Link module, in solution, in its chondroitin sulfate bound state. A variety of NMR techniques were employed, including chemical shift perturbation, residual dipolar couplings (RDCs), NOEs, spin relaxation measurements, and paramagnetic relaxation enhancements (PREs) from a spin-labeled analog of \xce\x94C444S. The binding site for \xce\x94C444S on the Link module overlapped with that of HA. Surprisingly, \xce\x94C444S binding induced dimerization of the Link module (as confirmed by analytical ultracentrifugation), and a second weak binding site that partially overlapped with a previously identified heparin site was detected. A dimer model was generated using chemical shift perturbations and RDCs as restraints in the docking program HADDOCK. We postulate that the molecular cross-linking enhanced by the multiple binding modes of the Link module may be critical for remodeling the ECM during inflammation/ovulation and may contribute to other functions of TSG-6.

nmr_insight_into_the_multiple_glycosaminoglycan_binding_modes_of_the_link_module_from_human_tsg-6

<!>Expression, purification and refolding processes of Link_TSG6<!>Preparation of hexasaccharides of chondroitin sulfate<!>Synthesis of \xce\x94C444S-TEMPO<!>NMR spectroscopy<!>Analytical Ultracentrifugation (AUC)<!>NMR solution structure calculation<!>HADDOCK<!>NMR-monitored titration of Link_TSG6 with CS<!>Binding of Link_TSG6 with a TEMPO derivative of \xce\x94C444S<!>Link_TSG6 forms a dimer in the presence of \xce\x94C444S<!>Link_TSG6:\xce\x94C444S solution NMR structure<!>Docking of \xce\x94C444S-TEMPO to the primary site in the Link_TSG6<!>DISCUSSION

<p>Link modules are domains found in proteins that are known primarily for their interaction with the non-sulfated glycosaminoglycan (GAG) hyaluronan (HA), a large polysaccharide composed of glucuronic acid (GlcA) and N-acetyl glucosamine (GlcNAc) ubiquitously present in the extracellular matrix of vertebrates1, 2. In many cases HA-protein interactions contribute to stabilization of the matrix and thus tissue homeostasis3, 4; examples include the massive complexes formed between HA and chondroitin sulfate proteoglycans that provide load bearing properties to cartilage, skin and brain via hydration/extension of the chondroitin sulfate chains1, 2. However, synthesis of new matrices and matrix remodeling also occurs during certain physiological processes, such as ovulation, as well as in response to tissue injury, inflammation and disease4–6. In these cases, HA along with the sulfated GAGs, chondroitin sulfate (CS; a copolymer of GlcA and N-acetyl galactosamine (GalNAc)) and heparan sulfate (HS; a copolymer of GlcA or iduronic acid (IdoA) and GlcNAc), are known to play a key role in dictating tissue structure, cell fate and availability of extracellular signaling molecules in the extracellular matrix7–10. However, the structural basis for this expanded set of roles, particularly with respect to interactions of certain Link modules with GAGs other than HA, is less defined. Here we provide some of that basis using NMR methodology to study the interaction of the Link module, from human tumor necrosis factor-stimulated gene-6 (TSG-6), with a CS hexasaccharide.</p><p>TSG-6 is a multifunctional GAG-binding protein that has been implicated in matrix remodeling11–13; it contains a single Link module that, unlike other members of this superfamily1, binds to a wide range of sulfated GAGs (CS, dermatan sulfate, heparin and HS) in addition to HA14. The interaction of TSG-6 with HA leads to the direct crosslinking and structural reorganization of the polysaccharide via HA-induced dimerization of TSG-6, which promotes the interaction of HA with its cell surface receptor CD44. Heparin can also dimerize the isolated Link module domain from human TSG-6, where this may regulate the activity of plasmin and thus the turnover of matrix15; moreover, TSG-6 can mediate the crosslinking of structural proteins in the matrix16.</p><p>TSG-6 is not constitutively expressed in most adult tissues, but is secreted in response to inflammatory signals and during ovulation11, 12, 17–20. In both of these contexts TSG-6 has been found to catalyze the covalent transfer of heavy chains (HC) from the CS proteoglycan, inter-α-inhibitor (IαI), onto HA21, 22; IαI has an usual structure in that its two HCs are covalently linked via ester bonds to the CS chain of the bikunin core protein23. The TSG-6 mediated formation of HA•HC complexes, which occurs via a covalent HC•TSG-6 intermediate21, is essential for murine ovulation24–26; here attachment of HCs to HA facilitates the crosslinking of HA chains via their binding to pentraxin-316, allowing the formation of a viscoelastic matrix around the oocyte just prior to its release into the oviduct. HC•HA complexes are also formed at sites of inflammation (e.g. in the synovial fluids from arthritis sufferers27), where the HC transferase activity of TSG-6 has been found to be predictive of osteoarthritis severity and the need for joint replacement28. The transfer of HCs onto HA likely involves the interaction of TSG-6 with the CS chain of IαI, i.e. during the formation of the HC•TSG-6 intermediate22, 29.</p><p>The Link module of human TSG-6 (Link_TSG6) is well studied and has become a model for Link module interactions in other HA-binding proteins30–34. Link_TSG6 is a small domain comprised of approximately 95 amino acids, and X-ray crystallography and solution NMR structures exist35, 33, 36. Its interaction with hyaluronan (HA) has been extensively characterized by a combination of NMR and site-directed mutagenesis and likely involves residues K11, Y12, H45, V57, Y59, P60, I61, K63, F70, I76, Y78, R81 and W8830–36. Heparin/HS binds to a site different from that of HA, namely one comprising residues K20, K34, K41and K5415, 33. Less information is known about where CS binds on TSG-6 Link module14, 37–39. However, competition assays between CS and HA and binding assays with chimeric chondroitin and HA oligosaccharides (that are non-sulfated) suggest that CS might interact with the HA rather than the HS binding surface34, 37, 39; it should be noted, however, that HA and heparin also compete for binding despite interacting at distinct sites, but this is believed to be mediated by an allosteric mechanism15, 33. Therefore, it is unclear where CS binds and indeed whether a sulfated region of a CS chain could be accommodated in the Link module HA-binding groove without steric interference.</p><p>Sulfated GAGs are extremely heterogeneous materials; for CS and HS this is primarily due to the possibility of sulfation at a number of distinct positions on the constituent sugar rings7, 10. HS can be N-sulfated, with the sulfate replacing the acetyl groups on the initial GlcNAc residues, as well as O-sulfated on the 3 and 6 positions of GlcNAc and the 2 position of IdoA residues. CS is less heterogeneous than HS with possibilities for sulfation at the 4 and 6 positions of the GalNAc residue in vertebrates.</p><p>A hexasaccharide of CS is an ideal way to study the interaction with Link_TSG6 since it is large enough to fill known binding sites and small enough to allow isolation or synthesis of a single well-defined oligosaccharide. However, even a hexasaccharide of CS has 64 possible sulfation patterns. Isolating a specific oligomer was overcome here by choosing a polymer that is rich in 4-O-sulfation (CS-A), digesting it with lyase or hydrolase enzymes, and purifying digests using size separation and ion exchange chromatography. This method allowed for the isolation of four CS candidates: ΔC444S, C444S, ΔC664S and C664S (in which the Δ indicates the unsaturated lyase product and the numbers indicate the sites of sulfation on the three GalNAc residues, numbered from the non-reducing end). ΔC444S was selected for structural studies of the Link module since it was available in larger quantities and 4-sulfated CS binds with a higher affinity than the 6-sulfated hexamers (see Supplementary data), i.e. with a similar affinity to HA37, 38.</p><p>NMR provides a reasonable approach to the structural characterization of protein-ligand complexes such as that of ΔC444S with Link_TSG6. Measuring chemical shift perturbations in 1H-15N HSQC spectra of 15N-labeled proteins provides one means of identifying amino acids that are potentially involved in binding. The structural information is very qualitative40. However, once NMR resonance assignments are made, data are easily collected on relatively small amounts of sample, and analysis of shifts as a function of ligand concentration can provide binding constants. Here we supplement such chemical shift data with paramagnetic relaxation enhancement (PRE) data using a tagged version of the ΔC444S oligomer carrying a TEMPO group at the reducing end. The structural data is more quantitative and provides a definitive location for the reducing terminus of the hexasaccharide. Residual dipolar couplings (RDCs) between pairs of magnetic nuclei such as 1H-15N pairs on amide groups of a protein backbone can also be used to provide quantitative data about changes in protein conformation through their dependence on the angles internuclear vectors make with the NMR magnetic field in partially ordered media41, 42; they also provide information on the structure of dimers if these exist in solution. Here we used RDCs and chemical shift perturbation, along with computer modeling in order to produce a structure for the Link_TSG6:ΔC444S complex. Our studies employed the docking program, HADDOCK, which incorporates a variety of quantitative and qualitative data in a simulated annealing search for a best structure for the complex43, 44.</p><p>We will show below that the Link module from human TSG-6 has a primary CS interaction surface that utilizes the same binding groove as HA. We also show that ligand binding induces a conformational change in the Link module, that a dimer is formed (confirmed by analytical ultracentrifugation) and that a second CS-binding site partially overlaps the heparin-binding site.</p><!><p>Expression and refolding followed published procedures45, 46. More specifically, the engineered gene of Link_TSG6 in the pRK172 expression vector was transformed into expression host BL21(DE3)pLysS. 10 ml starter cultures were grown overnight in LB medium containing 100μg/ml ampicillin. Grown cells were then transferred to 1 L M9 medium containing isotopes, 15NH4Cl, 13C-glucose from CIL (Cambridge Isotope Laboratories) and protein expression was induced by adding IPTG to 0.1 mM when the OD600nm reached 0.4. Cells were harvested 4 h after induction by centrifugation for 15 min at 5000g and stored at 20 °C in lysis buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 1mM EDTA). Cells were lysed by three passages through a French press and cells and inclusion bodies were gathered by centrifugation for 45 min at 20000g.45 Inclusion bodies were solubilized in 6M Guanidine-HCl containing 50mM Tris-HCl, pH 8.0, and 100 mM DTT and the sample was loaded onto a Superdex 75 (16 × 260 mm, Pharmacia) exclusion column, equilibrated and run with a flow rate at 1ml/min in the same buffer without DTT. Fractions containing the target protein were injected onto a C4 column (10 × 250 mm, YMC America Inc), equilibrated with H2O containing 0.01% TFA. After 5 min of washing with the same solvent at 3 ml/min, the protein was eluted over 40 min using a linear gradient from 0 to 80% acetonitrile containing 0.1% TFA. The eluent was monitored at 220 nm continuously. Eluent containing the denatured Link module was collected and lyophilized.</p><p>To accomplish the folding of Link_TSG6, the protein was re-suspended in 50 mM ammonium acetate at pH 6.0 at a concentration of 500 μg/ml (40 ml final volume). A 100-fold molar excess of β-mercaptoethanol was added, and the refolding solution was incubated at 25 °C for 2 days under aerobic condition without stirring. After 2 days the refolding solution was stored at the 4 °C for 5 additional days. The folded Link_TSG6 protein was purified by HPLC in the same manner as described above. The overall yield was approximately 10-20 mg/L.</p><!><p>The method described below is based on the works described in Pomin et al.47. 150 mg of CS-A from bovine trachea (btCS-A) was incubated with either 5 IU of chondroitin C lyase from Flavobacterium heparinum or 0.33 IU of chondroitin ABC lyase from Proteus vulgaris in 5 ml digestion buffer (50 mM Tris-HCl, pH 8.0, 150 mM sodium acetate, 100 μg/ml of BSA) at 37 °C for 36 h and 150 min, respectively. Hyaluronidase digestion of btCS-A was carried out by incubation of 150 mg of btCS-A with 10 mg of enzyme in 3 ml of digestion buffer (50 mM sodium phosphate, 150 mM NaCl, pH 6.0) at 37 °C for 48 h. The digested samples were heated to quench enzyme activities and subjected to separation on a Bio-Gel P-10, size exclusion column (15 × 1200 mm, Bio-Rad Life Science) with elution buffer (1 M NaCl, 10% ethanol) at a flow rate of 1.7 ml/15 min/fraction. Eluent was monitored at 232 nm and separated peaks in the chromatogram were desalted on a Sephadex G-15 column (10 × 500 mm, Sigma-Aldrich Co) and examined by mass spectrometry (MS) to identify those peaks having molecular weights corresponding to hexasaccharides. To eliminate complexities from anomeric equilibrium at the reducing end, most hexasaccharides were reduced in the presence of one equivalent of sodium borohydride (NaBH4) in 1 ml of water for 3 h and the reaction was quenched by adding a molar equivalent of acetic acid for 1 h in an ice-bath, followed by desalting. Hexasaccharides were then purified on a strong anion exchange column, SAX (2.5 × 10 mm, 5 μm, Waters Corporation) using a linear gradient from 25 to 45% of 2 M NaCl, 25 mM phosphate, pH 4.5, for 50 min at a flow rate of 3 ml/min. Each peak from the SAX chromatography was desalted and lyophilized to yield 1 mg for ΔC664S, 6.8 mg for C664S, 4.3 mg for C444S and 10.6 mg for ΔC444S. Structures were identified by NMR spectroscopy and judged to be approximately 90% homogeneous.</p><!><p>4-amino-TEMPO (4-amini-2,2,6,6-tetramethylpiperidine-1-oxyl) was purchased from Acros Organics. The reductive amination reaction was carried out on a 400 μl sample containing 1 mg non-reduced ΔC444S, 73 mM of 4-amino-TEMPO and 250 mM sodium cyanoborohydride in 80% MeOH at 65 °C for 3h. The sample was desalted and further purification was performed using SAX-HPLC monitored at 232nm. The yield was approximately 0.5 mg.</p><!><p>NMR spectra for titrations of protein with several isomers of CS were recorded at 25 °C on Varian 800- and 900-MHz spectrometers and processed and analyzed with NMRPipe and Sparky48, 49. Titration experiments were carried out combining 0.2–0.5 mM uniformly 15N-labeled Link module with increasing amounts of CS oligomers (ΔC664S, ΔC444S, C664S, and C444S) in 50 mM MES buffer, pH 6.0, 0.02% NaN3, 10% D2O. A series of 1H-15N HSQC spectra were recorded after adding sugar at 0.096, 0.192, 0.29, 0.38, and 0.48 mM to the protein sample. The degree of chemical shift change for amide proton and nitrogen resonances was calculated using the empirical formula, Δδ= [(ΔδHN)2 + (ΔδN × 0.12)2]1/2) where ΔδHN and ΔδN are the observed chemical shift changes for 1H and 15N, respectively. The weighting factor of 0.12 reflects the difference in chemical shift dispersion of 1H and 15N in folded proteins50. For relaxation experiments using a TEMPO labeled version of ΔC444S, a 0.17 mM 15N-labeled Link_TSG6 sample was prepared and spectra were recorded at 25 °C on a Varian 800-MHz spectrometer.</p><p>RDCs were measured on a 0.5 mM 13C and 15N labeled Link_TSG6 sample in the presence of 1 mM ΔC444S aligned in 5 % (w/v) stretched neutral polyacrylamide gel. Gels were cast initially in a 4.5 mm diameter glass tube overnight for polymerization. The polymerized gels were washed for two or three cycles in deionized water overnight, followed by a washing with protein buffer to equilibrate the pH. Finally, the gels were washed again with deionized water to remove residual protein buffer. Swollen gels were then trimmed to a length of 26 mm and dried at room temperature for 2 days. The dried gel was placed in the upper stage of an NMR tube and protein sample was added to cover the gel and let it swell for 2 days. Spectra were recorded using a two-stage NMR tube as described in Liu et al.51. RDC values were measured using a 1H-15N HSQC IPAP (in-phase and anti-phase) experiment under both isotropic and anisotropic conditions52.</p><p>Complete backbone and side chain resonance assignments were obtained with 0.5 mM 13C and 15N enriched Link_TSG6 in the presence of 0.6 mM ΔC444S using the following heteronuclear 2D and 3D experiments: 1H-15N HSQC, CBCA(CO)NH, HNCACB, HNCO and HCCH-TOCSY53, 54. All spectra for assignment were collected at 25 °C on Varian 600- and 900-MHz spectrometers. 15N, and 13C-edited NOESY-HSQCs were collected with 150 and 140 ms mixing time, respectively, on the same sample at 800-MHz and used for structure determination55, 56.</p><p>In order to measure 15N T1 and T2 relaxation rates and evaluate the level of dimerization, spectra were recorded at 25 °C on a Varian 800-MHz spectrometer using standard sequences from BioPack (Varian/Agilent). 15N T1 values were measured from the spectrum with different delay times, T = 10, 20, 30, 40, 50, 70, 90, 110, 150, 200, 250, 300, 400, and 500 ms. T2 values were determined from spectra recorded with delays T = 10, 30, 50, 70, 90, 110, and 150 ms. T1 and T2 values were extracted from log plots of peak intensities in 2D spectra as a function of delay duration.</p><!><p>All experiments were performed in 50 mM MES (pH 6.0) using an XL-A ultracentrifuge with an An60Ti -4 -hole rotor fitted with a six-sector epon-filled centerpiece with quartz glass windows. Equilibrium sedimentation was performed at 20°C, using rotor speeds of 22,000, 30,000 and 36,000 rpm scanning at 290 nm after equilibrium was reached (at 18 hours). Association kinetics were performed by using Link_TSG6 at 50 μM and varying the concentration of chondroitin sulfate (ΔC444S) between 50 and 1000 μM. Analysis of the data was performed with non-linear regression using the Heteroanalysis program57 using either an ideal non-associating model or monomer-dimer self-association model. For self-associating system analysis monomeric molecular weight of Link_TSG6 was fixed at 10.922 kDa and a molar extinction coefficient of 11976 (A290).</p><!><p>All NOESY crosspeaks were picked using Sparky and assigned manually followed by several rounds of automatic assignment. 1329 NOEs and 135 backbone torsion angle constraints derived from TALOS58 using the assigned chemical shifts of HA, CA, CB, CO, and N were used to calculate an initial solution structure. CYANA version 3.0 calculations were performed and 20 structures with the lowest energies were selected for analysis and further refinement59, 60. In the refinement stage, residual dipolar couplings were incorporated starting with Da and Rh values calculated from principle order parameters determined in REDCAT61 (−12.15 and 0.433, respectively). Structural refinement was then carried out using NOE distances, dihedral angles, and RDC values with Xplor-NIH62. Final ensemble contained 20 structures.</p><!><p>HADDOCK version 2.1 was used for protein-protein and protein-ligand docking63. The newly determined NMR structure of the protein was used for docking. The C444S structure was built in Glycam Biomolecular Builder64 (also http://dev.glycam.org) and the TEMPO group was added manually using UCSF Chimera65. Residues H4, R5, E6, A7, Y12, H45 and H96, were set as highly ambiguous interaction restraints (AIRs) for protein-protein docking. Residues, C47-A49, G58, K63-G65, and C68-I76, were used as AIRs for dimer-ΔC444S-TEMPO docking. In addition, PRE data from spin labeled ligands were used as unambiguous interaction restraints for dimer-ΔC444S-TEMPO docking. Initially 1,000 structures were determined by rigid-body docking. Then simulated annealing (SA) was carried out with the 200 lowest-energy structures using default force field parameters. Specified residues were allowed to be fully or semi flexible during the final stages of simulated annealing. For ligand docking ΔC444S-TEMPO was allowed to be fully flexible and residue segments, of the protein 45–49, 63–65, and 68–76, were allowed to be semiflexible. For monomer-monomer docking to produce a dimer protein, residues 9–12 near the dimer interface were specified as semiflexible during the simulated annealing. Semiflexible residues were allowed to have side chains move during the SA. Fully flexible residues were allowed to be fully flexible throughout the entire docking protocol except for the rigid body minimization. RDC restraints were given during docking simulations and a tensor was included in the structures calculations. Finally the structures were subjected to a water refinement stage. Models were clustered based on similarity of poses and ranked based on total score. The top five in each cluster were chosen for display and the lowest energy model of the top three scores was chosen for more detailed analysis. The best structures were chosen from the refined structures based on lowest total energy and docking score.</p><!><p>The isomers of the CS hexasaccharide (GlcA-GalNAc-GlcA-GalNAc-GlcA-GalNAc-ol; ol stands for reduced sugar with open ring at the reducing end) selected for our studies included, ΔC444S, C444S, ΔC664S and C664S. CS binding sites on Link_TSG6 were mapped using chemical shift perturbations in 1H-15N HSQC spectra as a function of ligand concentration up to a twofold molar excess over protein concentration. Figure 1 shows the superimposition of the 1H-15N HSQC spectra of 0.24 mM Link_TSG6 in the absence and presence of 0.096, 0.192, 0.29, 0.38, and 0.48 mM of the ΔC444S hexasaccharide. Assignments of crosspeaks in the HSQC spectra were made using triple resonance experiments on a 15N, 13C labeled sample; assignments for the most significantly perturbed crosspeaks are indicated on Figure 1.</p><p>The chemical shift changes involved can be conveniently summarized by combining shifts in the two dimensions, 1H and 15N and plotting the maximum variations as a function of residue number (Figure 2). The residues with the largest shift changes are those most likely to be involved in ligand binding, and the extremes in chemical shift for these residues can be associated with shifts in uncomplexed and complexed states. An equivalent plot has been generated for 13Cα and 13Cβ resonances (Figure S1 in Supplementary Data), and this shows large perturbations for a similar set of residues. Essentially identical behavior was observed in 1H-15N HSQC spectra on titration with other hexasaccharides (Figures S2 and S3 in Supplementary Data).</p><p>Three distinct types of chemical shift change were observed; these correspond to exchange between complexed and uncomplexed states on slow, intermediate and fast timescales relative to the reciprocal of the chemical shift change in Hz. The color-coded bars in Figure 2A represent the residues involved in slow and fast exchange and the residue positions are indicated on the ribbon diagram of the Link_TSG6 structure in Figure 2B. In the first case, when ligand is added, crosspeaks progressively decrease in intensity as other peaks at nearby positions progressively increase in intensity. Crosspeaks belonging to residues H4, L14, H45, C47, A48, A49, G58, K63, C68, K72, G74, I76, and D89 fall into this category. A few crosspeaks belonging to G65, F70, and G71 also exhibited similar behavior (data not highlighted); in this case, the crosspeaks disappeared but could not be correlated with newly appearing peaks due to overlap or additional broadening in the complexed state.</p><p>Since intensity changes in the limit of slow exchange reflect the population of complexed and uncomplexed states, they can be used to determine a dissociation constant. Figure 3A shows a fit to the normalized average intensity changes for residues A48, G58, and G74. Equation 1 below describes the intensity change in terms of the normalized concentration of the complex ([C]/[PT]). This depends on the binding constant, K1, the total protein concentration, [PT], and the total ligand concentration, [LT].</p><p>[PT]−[C])/[PT] gives the corresponding curve for free protein, [P]/[PT], which is also shown in Figure 3A. In the limit of slow exchange the amplitude of a particular cross peak is proportional to [P] or [C]. However, lifetime broadening also occurs as exchange rates (KE) increase, leading to an additional reduction in amplitude at low concentrations of [C] or [P] in proportion to KE, the effective exchange rate at the titration midpoint in s−1. This effect is easily added to the simulation by scaling amplitudes by the ratio of the initial line width (20 Hz) to the linewidth in the presence of lifetime broadening, more specifically the initial line width plus KE/π times the fraction in complexed or uncomplexed states. We have also compensated for the linewidth increase due to dimerization (see below) by scaling up amplitudes of experimental complex peaks by a factor of 1.4, which is based on the observed linewidth for those peaks.</p><p>Simulations of [C]/[PT] and [P]/[PT] as a function of [L] were done for a grid of K1 and KE parameters, and the best fit simulations are plotted in Figure 3A. While amplitude changes depart somewhat from ideal behavior due to factors such as intensity losses during HSQC transfers due to broadened peaks, which are not included in the simulation, it was possible to a find reasonable fit with a binding constant, K1 of 7.5 × 104 M−1, or equivalently, a dissociation constant, Kd, of 13 μM, and a KE of 30 s−1 for the ΔC444S-Link_TSG6 interaction. The fittings to the normalized average intensity changes for ΔC664S and C664S are provided in Figure S4 of Supplementary Data.</p><p>For a second class of perturbed peaks, crosspeaks displayed some chemical shift change and signal broadening as the ligand concentration increased. In most cases, this resulted in the disappearance of crosspeaks when the total ligand concentration was approximately one-half of the total protein concentration. These crosspeaks then reappeared at much higher ligand concentration. This is characteristic of an exchange process occurring on an intermediate timescale with respect to the reciprocal of chemical shift changes. Residues E18, G50, N67, I75 and W88 exhibited this behavior. The changes in chemical shifts were, in general, smaller than those for residues showing slow exchange, making them more susceptible to exchange broadening. The residues are in the same structural area where slow exchanging peaks are found. Hence, they are likely involved in the same ligand binding process.</p><p>A third type of behavior is characterized by peaks exhibiting small changes in chemical shift and line width at lower ligand concentrations, followed by larger continuous chemical shift changes at higher ligand concentrations. Crosspeaks for K11, E24, H29, L30, Y33, W51, A53, Y91 and Y93 belong in this category. Their behavior is a special case of fast exchange with the onset of this second weaker binding event occurring only after binding at the high affinity site is completed. Under rapid exchange, chemical shifts are population-weighted averages of complexed and uncomplexed states. The set of equations 2–4 below describe the change in concentrations of various species as a function of ligand concentration. They are based on the assumption that only the products of slow exchange are capable of participating in the fast exchange process and the fast exchange process involves binding to a dimer of the initial complex (see results section to follow).</p><p>Here [C2] is the concentration of the second complex. [PT] and [LT] are total concentration of protein and ligand, respectively. K1 represents a binding constant for slow exchange process, K3 is a protein dimerization constant, and K2 represents a binding constant for the second binding event. K1 was set to 7.5 × 104 M−1 based on the fit to slow exchange data, K3 was set to 104 M−1 based on an AUC experiment (see below) and [PT] was set to 0.24 mM. The chemical shift change is proportional to the fraction of protein in the second complex ([C2]/[PT]). The system of equations was solved using the Maple program, and simulations of the scaled chemical shift change were carried out with a series of binding constants, K2, in order to find the best fit to the experimental data. The resulting best curve is shown along with experimental data in Figure 3B. The binding constant extracted is 2 × 104 M−1 (i.e. a Kd of 50 μM).</p><!><p>In order to better define the position of the bound ΔC444S, a paramagnetic version of ΔC444S (ΔC444S-TEMPO), which includes a nitroxide carrying TEMPO group attached to the reducing end, was used to generate additional data (sugar sequence; 1GlcA-2GalNAc4S-3GlcA-4GalNAc4S-5GlcA-6GalNAc4S1N-TEMPO). Figure 4 shows a comparison of a sample of 0.17 mM Link_TSG6 with 0.34 mM ΔC444S-TEMPO in oxidized (4A, paramagnetic) and reduced (4B, diamagnetic) states; the reduced state was achieved by the addition of twelve equivalents of ascorbic acid66. With few exceptions, chemical shift perturbations were identical for ΔC444S with and without the attached TEMPO group suggesting that similar modes of binding exist. The time scales of exchange also appear to be similar. For example, A48 still shows slow exchange when it interacts with ΔC444S-TEMPO.</p><p>In the oxidized state certain crosspeaks lose intensity due to the paramagnetically enhanced decay of transverse magnetization during the transfer and refocusing periods of the HSQC experiment. The changes in intensity between reduced and oxidized TEMPO derivatives can be converted to distances between the nitroxide oxygen and a residue of interest using equation 5 below67–69.</p><p>Here, Ired and Iox are the peak intensities of the crosspeaks in the presence of reduced and oxidized TEMPO derivatives, f is the fraction of the protein bound to ligand, and t is the total time during INEPT and the refocusing periods of the 1H-15N HSQC pulse sequence (t=9.78 ms). K is a constant related to spin properties of the system (K=1/15*S(S+1)γ2g2β2=1.23 × 10−23 cm6S−2), r is the distance between the nitroxide and amide proton of the crosspeak of interest, τc is the correlation time for tumbling of the protein-ligand complex, and ωH (800 MHz * 2π) is the precession frequency for the amide proton. Given all values, and taking f and τc to be 1 and 10.15 ns, respectively, the distances shown in Table 1 were obtained.</p><p>The distances relate only to the positioning of the terminal TEMPO group. The amides of residues G79, I80, and N83 are most affected by the presence of the TEMPO group, with the crosspeak for I80 completely disappearing. Even though a direct correlation with residues showing chemical shift perturbation was not necessarily expected, G79 and I80 are near the 74–76 segment showing slow exchange, suggesting that distances involving these residues would be useful for positioning a ligand in the slow exchange binding site. Given that the TEMPO is attached to the reducing end of the hexasaccharide, data are consistent with the findings by Blundell et al., 2003 and Higman et al., 2014, showing the reducing end of bound HA to be close to Y78 and R8134, 36.</p><!><p>It is possible that some of the observed chemical shift perturbations arise from interactions that do not directly involve the ligand, but are interactions in remote parts of the protein that are induced by ligand binding. One possibility is ligand induced dimer formation with perturbed residues being involved in a dimer interface. NMR spin relaxation measurements provide one way of assessing the possibility of dimer formation. R1 and R2 relaxation rates for 15N nuclei in amide groups were measured in free and ΔC444S-bound states. The ratio of R2/R1 yields a rotational correlation time for a macromolecule, provided the amide sites measured do not experience substantial internal motion. R1, R2, and R2/R1 ratios are reported in Figure 5. Basic equations for this ratio70 can be reduced to an approximate relation that can be solved for a correlation time, R2/R1 −7/6 ≈ 2/3 (τc × ωN)2 where ωN is the precession frequency for 15N. Analysis yields correlation times of 6 and 11 ns for uncomplexed and complexed proteins respectively. At 25 °C in dilute aqueous solution the correlation times in ns derived from sites in rigid parts of the protein are expected to approximately equal half the molecular weight in kDa (derived from the Stokes formula for rotational diffusion). Hence, the value of 6 ns for Link_TSG6 at a concentration of 0.26 mM in the absence of ligand is in reasonable agreement with that expected for a monomer of 10.9 kDa. In the presence of ΔC444S, the rise to 11 ns, is much greater than that expected for the addition of ligand to the protein and is more consistent with dimer formation.</p><p>Analytical untracentrifugation analysis of Link_TSG6 in the absence and presence of ΔC444S provided further evidence for CS-induced dimerization. Link_TSG6 without any added ΔC444S gave a sedimentation equilibrium profile that fit well to an ideal single species model showing that in the absence of CS hexasaccharide the protein is monomeric; all experiments were conducted at three different rotor speeds; data at 30,000 rpm are illustrated in Table 2 and Supplementary Figure S5. In the presence of ΔC444S data were fit to a monomer-dimer self association model. The molecular mass of the protein species increased dose-dependently with increasing concentrations of the added hexasaccharide (at 1:1, 1:10 and 1:20 molar ratios; Table 2). Thus, this AUC analysis is consistent with dimerization of Link_TSG6 in the presence of ΔC444S; an apparent dissociation constant of 151 μM was derived by non-linear regression of the data at the 1:20 molar ratio.</p><p>Since ΔC444S addition induces dimer formation, it is likely that some of the ligand induced chemical shifts attributed to ΔC444S-protein interactions are the direct result of dimer formation. We therefore attempted to gain information about possible dimer contacts by examining 1H-15N HSQC spectra in the absence of ΔC444S, but at higher concentrations. Comparing chemical shifts at 0.1 mM and 0.5 mM there were a number of small but measureable changes, including H4, R5, E6, A7, Y12, H29, L30, H45, V46, Y91 and H96 (Figure S6 in Supplementary data); two of these, H29 and L30, were discounted since these were apart from other perturbed residues and the fact that histidine ionization can be altered with slight changes in pH and ionic strength as the samples are concentrated71, 72. The remaining perturbed resonances were deemed to be useful for the determination of a dimer structure; however, some of these perturbations do correspond to those seen with the addition of ΔC444S and therefore it will be necessary to eliminate the use of these in the determination of the ligand binding site.</p><!><p>The existence of a dimer and the possibility that ligand binding induces additional structural changes suggest that refinement of a structure in the presence of ΔC444S would be worthwhile. We, therefore, pursued solution structure determinations using conventional triple resonance assignment methods combined with NOE, dihedral angle, and RDC restraints. A statistical summary of the data is included in Table 3. The initial structure for a monomer of Link_TSG6 in the presence of ΔC444S was generated using CYANA version 3 with 1198 manually assigned NOEs out of 1329 total NOEs and 135 dihedral angles predicted from TALOS73. In addition, two disulfide bonds were created and restrained to normal bond distances, based on previous disulfide bond mapping and their existence in the crystal structures33, 45. The structure was then further refined using XPLOR-NIH with additional NOEs iteratively assigned based on initial structures from CYANA calculations, and with added RDC restraints. The final stage included refinement in the presence of discrete water molecules as solvent. 20 models having the lowest total energy were chosen and are represented in Figure 6. This model has two identifiable α-helices and two small β-sheet-like structures. Using default secondary structure parameters the terminal and central sheets have two and three antiparallel strands respectively. This topology is nearly identical to previously determined structures and secondary structure boundaries as previously defined have been added to Figure 636. One concern about the new structure is that some NOEs may have arisen from inter-monomer contacts in the dimer rather than intra-monomer contacts. After the generation of a dimer structure from the initial monomer structure (see below), inter-monomer contacts less than 5 Å for proton pairs were compared to NOE lists. 1736 contacts were found and 9 NOEs corresponding to those contacts were excluded in a second determination. The removal of these 9 NOEs caused no significant changes in the structure presented in Figure 6.</p><p>Comparing the 20 structures of Figure 6, significant variations among the structures were noted in the loop region between 63–73. This might be due to missing backbone resonances for several residues (I61, V62, G65, G59, F70, and G71) and the small number of NOEs observed around the loop. The variations are also large for the C-terminal region. Here resonances and assignments exist but no NOEs are observed, consistent with a floppy C-terminal tail33. Assignments, NOEs and RDCs of the complex of Link_TSG6:ΔC444S have been deposited into the Biological Magnetic Resonance Data Bank (BMRB) with accession number 25663, and the structures have been deposited into the Protein Data Bank (PDB) 2N40.pdb).</p><p>After generating the initial monomer structure, we addressed the issue of a dimer structure. In principle, RDCs in combination with a reasonable monomer structure can be used to independently generate a set of possible structures for symmetric dimers, but this usually requires multiple alignment media74. In our case, working with the positively charged Link_TSG6 and negatively charged ΔC444S greatly restricted the choice of alignment media, allowing only the generation of a single set of RDCs. We therefore chose to use a docking program, HADDOCK 2.1, to generate a dimer model43, 44. We began with the lowest energy monomer structure from those shown in Figure 6. 46 RDC restraints and a symmetry restraint were used to guide dimer formation. Residues H4, R5, E6, A7, Y12, H45 and H96 on each monomer were identified for ambiguous interaction restraints based on concentration dependent chemical shifts to drive dimer formation. The monomer structures were held rigid during docking and refinement except for residues 9–12 which were made flexible to encourage development of the best interface. The resulting dimer is shown in Figure 7. A correlation plot of experimental versus back-calculated RDCs using the best dimer model from HADDOCK has a Q-factor was 0.18 with principal order parameters, 1.68 × 10−4, 7.28 × 10−4, −8.96 × 10−4. One of the axes, the Y axis, is parallel to the two-fold rotation axis of the dimer, as expected. The structure appears to have good stabilizing contacts with identifiable inter-monomer hydrogen bonds (Y3-R5, H4-H96, R5-E26, R5-N94, R8-E26, R8-Y93), four ion pairs (two from E26-R8 and two from R5-E26), several hydrophobic contacts (Y3-Y3, P95-R8, H4-H96), and a decrease in the solvent accessible surface (SAS) of 723.3 Å.</p><!><p>The structure determination of a Link_TSG6-dimer:ΔC444S-TEMPO complex was also carried out using HADDOCK. Based on the consistency of protein regions perturbed by paramagnetic effects and slow exchange chemical shift effects, data from both effects were combined to generate a model that provides insight into the higher affinity ΔC444S binding site. The following ambiguous interaction restraints were used: C47-A49, G58, K63-G65, and C68-I76 along with all distance restraints determined with ΔC444S-TEMPO. Distance restraints coming from PREs were used with lower limits set to 3 Å and upper limits set to the measured distance plus 3 Å. 46 RDC restraints on protein 15N-1H vectors were provided to allow additional adjustments of loop regions designated as flexible during final stages of docking (residues 45–49, 63–65, and 68–76). The top 10 scoring complexes from HADDOCK were selected and clustered. These are shown in Figures 7A and 7B. One cluster (Figure 7A) shows ΔC444S-TEMPO sitting predominantly at a single site on the protein surface involving residues 62–76 but with a broad range of conformations. The other cluster (Figure 7B) shows the ΔC444S-TEMPO docked into a shallow groove overlapping the HA-binding site (see below).</p><p>The average energies for the two clusters are similar, i.e. −6986.6 and −6940.4 kcal/Mol, respectively, but the second cluster makes better contacts with the perturbed resonances (H4, L14, H45, C47, A48, A49, G58, K63, C68, K72, G74, I76, and D89). Also, the model with the lowest molecular energy (sum of Etot, Ebond, Eangle, Eimproper, Edihed, Evdw, and Eelec) among the top 3 scoring models, is a member of cluster 2; it also shows the lowest electrostatic energy (Eelec). Therefore, this model was chosen for further analysis/refinement of specific interactions between the protein and ΔC444S because it provides a reasonable representation of the oligosaccharide sitting within its primary binding site (Figure 8). 6 potential hydrogen bonds (H…O distance < 2.5Å) were observed between the protein and ΔC444S-TEMPO (1GlcA-O5:H45-HE2, 2GalNAc4S-O3:H45-HE2, 3GlcA-O5:N67-HD21, 3GlcA-OE2:N67-HD21, 3GlcA-OE1H:C68SG and 5GlcA-O5:G69-NH). In addition, 115 atom pairs are within 0.2 Å of van der Waals contact distance across the protein-sugar interface. Residues K11, H45-A49, Y59, P64, N67-G59, F70 K72, I76 and Y78 were involved in these contacts. Sulfate groups on two GalNAc residues were in close contact with the side chain of positively charged amino acids (K11 and H45). The other sulfate group, which is in the opened ring produced on TEMPO attachment was located outside of the binding groove where it could make effective contact with water. The nitroxide on the TEMPO group showed distances to G79 (11.76 Å), I80 (12.58 Å), and N83 (9.39 Å) that are consistent with distance restraints from the PRE data.</p><p>The bound conformation of ΔC444S differs from that expected in solution. Sugar rings all retain the expected 4C1 chair conformation, however glycosidic torsion angles vary. The glycan forcefield parameters used in our docking procedure were not specifically optimized for glycans and this can contribute to the departures. Recently the use of an auxiliary energy function specific for glycosidic torsion angles has been proposed as a means to improve scoring of poses produced by docking programs that lack adequate treatment of glycan conformational energies75. Evaluation of this function for the central three glycosidic bonds in the five poses in our second cluster show the selected conformer described above to have the lowest score (proper energy functions do not exist for the terminal residues) with the total penalty being less than 4 kcal. Also, departures from perturbations in glycosidic bond angles from solution conformations have been noted in HA-protein complexes 34, 76. Interestingly, the position of the pronounced kink in the structure shown in Figure 8 is similar to that observed previously. The information obtained on the weaker, fast exchange site was not adequate to generate a detailed structural model for ΔC444S binding. However, the perturbed sites are shown on the dimer structure in Figure 8A. The positioning is clearly separate from the primary site, allowing the possibility of simultaneous interactions at the two sites.</p><!><p>The investigations of the interactions of chondroitin sulfate hexasaccharides allow us to make several general observations: Chondroitin sulfate oligomers such as ΔC444S bind to Link_TSG6 with moderate affinity and a preference for high 4-sulfate content. There are a least two different binding sites, one characterized by slow exchange and tight binding (Kd = 13μM) one by fast exchange and weaker binding (Kd = 50μM). And, the initial binding event promotes Link_TSG6 dimer formation. The studies have allowed the generation of a structural model for the dimer and the bound conformation of the ΔC444S hexasaccharide. Here we discuss this model in light of previous work on binding of HA and heparin/HS to Link_TSG6, and the biological implications of our model.</p><p>The similarity of the slow exchange (higher affinity) binding site to that previously observed for HA oligomers is striking34. The similarity can even be seen at the level of residues experiencing perturbed chemical shifts on addition of ΔC444S. The following residues were previously identified as being in proximity to HA (i.e. lining the binding groove): K11, Y12, H45, V57, Y59, P60, I61, K63, F70, I76, Y78, R81 and W8830–36; CH-π stacking interactions of saccharide rings against aromatic planes for Y59 and Y78 were suggested to play an important role32, 34, 36. The list of residues with slow and intermediate exchange rates encompasses five of the 13 residues listed above. If we count residues that are one or two removed in sequence, the list expands to 8 of the 13 residues. Another piece of evidence to show ΔC444S shares the HA binding site is the perturbation of C47 and C68 resonances. These residues, which form a disulfide bridge underlying the HA binding surface, have previously been suggested to change geometry on binding with a concomitant rearrangement of the β4-β5 loop (residue 61–74) leading to this perturbation36. If we add these to the list, 10 of 15 are encompassed. All of the perturbed residues for ΔC444S are highlighted in Figure 9B and compared to those residues believed to be involved in the HA binding in Figure 9A. Even though ΔC444S and HA share a binding groove, the residues perturbed by ΔC444S do differ in some cases. Positively charged amino acids, such as H4, and K72 (that do not interact with HA31, 72), in addition to H45 and K63 (that do34), are perturbed indicating that they may be associating with sulfate groups on the GalNAcs of ΔC444S.</p><p>The binding region of the protein also seems to experience some distinct structural adjustments on interaction with ΔC444S when compared to HA. The overall differences are modest; a comparison of Link_TSG6 in its ΔC444S- and HA-bound conformations yields an RMSD for backbone atoms of 1.34 Å. However, the large loop (residue 61–74) involved in binding ΔC444S and HA shows a larger difference. As illustrated in Figure 10, the binding groove appears to be closed more tightly when ΔC444S is present than when HA is present. Given the similar shift pattern seen with the C664S oligomers (Supplementary Figures S2 and S3), it is evident that CS hexasaccharides containing 6-sulfates can also be accommodated within this binding groove, albeit with lower affinity. The lower affinity is consistent with previous observations indicating that chondroitin-6-sulfate (C6S; also termed CS-C), unlike chondroitin-4-sulfate (C4S; also termed CS-A), is a poor competitor of HA37, 39. Based on the model for Link_TSG6: ΔC444S (Figure 8) it would appear that the presence of sulfates on the 6-position of GalNAc are unlikely to cause steric clashes with the protein. However, it is possible that 6-sulfates are not be able to make as favorable ionic interactions with positively charged residues in the protein, or perhaps the inherent differences in C4S and C6S chain flexibility77, 78 may disfavor the binding of C6S.</p><p>The second mode of interaction of ΔC444S with the TSG-6 Link module, that exhibited fast exchange and a higher Kd of approximately 50 μM, was more difficult to characterize. However, the residues perturbed in this interaction are near some of the residues previously identified as important for heparin binding (K20, K34, K41, and K54)15, 33. As shown in Figure 2, the residues perturbed in this weaker interaction are physically separated from those exhibiting slow exchange. However, it is possible that longer CS chains could bind simultaneously to the two binding sites, which would increase the affinity of the interaction for longer polymers. This would also be expected have a large effect on the conformation of the polysaccharide by wrapping it around two sides of the Link module (see Figure 8A). This would cause a condensation of a CS chain (as we have seen for HA13) and thereby reduce the overall domain size of a CS proteoglycan. As noted previously13, 34, such structural perturbation could drive matrix reorganization at inflammatory sites, where the TSG-6 protein is expressed. For example, TSG-6-mediated collapse of the CS chains of aggrecan could enhance the movement of this proteoglycan through the cartilage extracellular matrix aiding tissue maintenance and repair34.</p><p>An additional discovery is that binding of ΔC444S to the Link module induces dimer formation. The dimer structure has an interface distinctly different from the possible dimer contacts suggested by the crystal structure determined in the absence of ligands 33. However, the reduction in solvent accessible surface areas for the three possible dimers in the non-liganded crystal structure are not significantly different (625, 884, 612 Å2) from that observed here 723 Å2. Another aspect of the dimer structure worth noting is the positioning of the C-termini that must connect to a CUB domain in intact TSG-6. These extend upward from the cleft between monomers seen in Figure 7. A crystal structure of the CUB domain has recently been published79, and without altering the structure of either domain we have been able to dock a pair of CUB modules to these termini with a minimal clash of surface residues (Supplementary Figure S7). The CUB modules are in close proximity to one another allowing for possible additional interactions in the context of a TSG-6 dimer. Based on our modeling the N-terminal peptide of TSG-6 could also potentially be accommodated within the CS-induced dimer, however, given that there are no structural data available for this region, further work would be needed to assess this possibility.</p><p>In our model, primary ΔC444S binding sites exist on each monomer, nearly on opposite sides of the protein from the dimerization interface. Thus, the hexasaccharide ligands used here cannot play a bridging role in dimer formation. They could however lead to a reduction in repulsive electrostatic effects. Link_TSG6 is a positively charged domain (pI = 9.48; calculated using the compute PI tool in ExPASy). The binding of a negatively charged ligand would then reduce the repulsion that exists in the unliganded monomers and allow dimerization. One consequence of dimerization is that multiple binding sites for CS will be brought together (with two on each monomer). Thus, it is possible that a polymeric CS chain might bind to the Link module dimer in a number of different ways, utilizing several different combinations of the four potential interaction surfaces. Importantly, some of these combinations would be anticipated to lead to CS crosslinking via the binding of two CS molecules to the Link module dimer; for example, each CS chain could associate with the high and low affinity sites as suggested above. Crosslinking of CS by TSG-6 could have a pronounced effect on the structural and functional properties of tissues such as brain, cartilage and skin that have a high CS content. For example, rigidification of matrix could alter cell phenotype through changes in mechanical sensing80–82. The structural reorganization of CS could also modulate the binding of matrix-associated signaling molecules, such as the chemokine CCL583; interestingly, TSG-6 binds to CCL584 as well as CS, and therefore may be able to interfere with its presentation (e.g. on endothelial cells).</p><p>CS-induced dimerization of the TSG-6 Link module could also be of relevance to the covalent transfer of the heavy polypeptide chains of IαI onto HA; this is catalyzed by TSG-621 and occurs via two sequential transesterification reactions, where the first of these transfers a heavy chain from CS onto TSG-6 to form a HC•TSG-6 intermediate. Importantly, there is evidence that the binding of TSG-6 to CS is involved in HC•TSG-6 formation22, 29, 85. Thus, HC transfer might potentially involve dimerization of TSG-6 via its binding to CS facilitating a subsequent transfer of HC to HA bound to a second TSG-6 molecule. While this cannot be ruled out, it seems unlikely given the weak affinity for dimerization determined here by AUC (Kd = 150 μM), whereas HC•TSG-6 complexes can form readily when TSG-6 and IαI are present at low micromolar concentrations21, 22. In any case, the finding that TSG-6 interacts with CS through its 'HA binding groove' will be helpful in further refining our molecular understanding of this important biological process (e.g. in the context of inflammation and ovulation).</p><p>There is also a precedent for the dimerization of Link_TSG6/TSG-6 on binding to GAGs. Heparin, for example, is known to induce dimer formation of Link_TSG6 in a manner that may potentiate the anti-plasmin activity of IαI15. In this case, it has been suggested that heparin may act as a bridge between the two Link modules by binding to continuous surface on the dimer, i.e. based on crystal contacts in the Link_TSG6 X-ray structure33. This is clearly a different mechanism from that identified here for CS. More similar is the binding of HA to TSG-6, which induces dimerization of the full-length protein, where this leads to crosslinking and a dramatic collapse and stiffening of the HA network13, 16. However, Link_TSG6 is not dimerized by HA, nor is its interaction with HA cooperative (as is the case for full-length TSG-6); it is the CUB module that likely mediates the protein-protein interaction between TSG-6 molecules13. Thus, the mechanisms underlying GAG-induced dimerization of TSG-6, and its Link module domain, appear to be distinct for CS, HA and heparin, indicating that TSG-6 may be able to play diverse roles in tissue remodeling. However, specific information related to how TSG-6 interacts with polymeric CS and whether this does indeed lead to crosslinking within extracellular matrix requires additional studies.</p>

PubMed Author Manuscript

Interaction of Tamoxifen Analogs With the Pocket Site of Some Hormone Receptors. A Molecular Docking and Density Functional Theory Study

In this paper, the antiestrogenic properties of Tamoxifen analogs have been investigated and a theoretical report of its analogs interaction with the pocket site of some hormone receptors are presented. Analogs were generated by modification of the hydrophilic functional group of Tamoxifen by hydroxyl, amide, carboxyl, and sulfhydryl functional groups, in an attempt to improve their activity and selectivity. The analogs exhibit a negative binding energy in the estrogen and progesterone receptors, which indicates a spontaneous interaction between the analogs and the pocket site in the hormone receptors. The values of the molecular polar surface area indicate that the analogs have good permeability and are strong electrophiles. The couplings showed electrostatic interactions such as hydrogen bond and π-π interactions. According with the Lipinsky Rule of Five, the four analogs presented a good biodistribution, permeability, and pharmacological action on the hormone receptors. The analysis of the charge transfer suggests a limited enhanced oxidative damage in the estrogen receptor that not takes place with the progesterone receptor.

interaction_of_tamoxifen_analogs_with_the_pocket_site_of_some_hormone_receptors._a_molecular_docking

1. Introduction<!>2.1. Optimization, frontier molecular orbitals, and electronic structure calculations<!>2.2. Molecular docking<!>3.1.1. Geometry optimization, frontier molecular orbitals, and electrostatic potential surface<!><!>3.1.1. Geometry optimization, frontier molecular orbitals, and electrostatic potential surface<!><!>3.1.2. Reactivity parameters<!><!>3.1.2. Reactivity parameters<!>3.2. Analysis of the hormone receptors with the tamoxifen analogs<!><!>3.2.1. Docking analysis of the estrogen receptor<!><!>3.2.1. Docking analysis of the estrogen receptor<!><!>3.2.2. Reactivity parameters<!><!>3.2.2. Reactivity parameters<!>3.2.3. Charge transfer in the estrogen receptor<!><!>3.2.4. Docking analysis of the progesterone receptor<!><!>3.2.4. Docking analysis of the progesterone receptor<!><!>3.2.5. Reactivity parameters<!><!>3.2.5. Reactivity parameters<!>3.2.6. Charge transfer in the progesterone receptor<!><!>4. Conclusions<!>Author contributions<!>Conflict of interest statement

<p>Tamoxifen (TAM) is a drug widely prescribed as chemopreventive for women to prevent and to treat all stages of breast cancer (Jordan, 2007; Esteve-Romero et al., 2010). TAM is a Selective Estrogen Receptor Modulator (SERM) (Boyd and Coner, 1996; Jordan, 2003), which acts as a blockage for the effects of estrogen in the breast tissue by attaching to the estrogen receptors in breast cells.The targets for this drug are some hormone receptors [estrogen receptors (ER) and progesterone receptors (PR)]. This drug is a prodrug and can be metabolically activated to 4-hydroxytamoxifen (4OHTAM) (Jordan et al., 1977; Borgna and Rochefort, 1981) or alternatively can be metabolically routed via N-desmethyltamoxifen (NDTAM) to 4-hydroxy-N-desmethyltamoxifen also known as endoxifene (END) (Irarrazával, 2011; Sanyakamdhorn et al., 2016). The hydroxyl metabolites of tamoxifen have a high binding affinity for the ER (Jordan et al., 1977).</p><p>The recent exponential growth of computational resources has facilitated successful development of theoretical algorithms that can also be used to study the electronic effects (Brewerton, 2008). These algorithms can also be used to calculate other physical and chemical properties of ligands using semiempirical and Density Functional Theory (DFT) methods (Correa-Basurto et al., 2012). The theoretical results obtained with these methods have been successfully compared with experimental results (Ravna et al., 2007).</p><p>A huge amount of theoretical studies on TAM has already been carried out to describe its interaction with ER. Using calculations of molecular dynamics, semiempirical, and DFT in conformational analysis of TAM and Toremifene (TOR), it was predicted that TOR conformations were slightly different from those of TAM owing to the effect of the chlorine atom at chloroethyl group (Kuramochi, 1996). In a recent research, Landeros-Martinez et al. analyzed the molecular docking of TAM in ER and PR in which the active site of the hormone receptors were determined, as well as the charge transfer of the TAM to the residues of the active sites in the hormone receptors (Landeros-Martínez and Flores-Holguín, 2016). Other theoretical studies analyzed the metabolism of TAM using semiempirical (PM3) and DFT with B3LYP/6-31G* methods (Hariharan and Pople, 1973; Francl et al., 1982). Also a study of the molecular conformations and the vibrational NMR spectra of TAM performed with B3LYP/6-311(d,p) has been reported (Badawi and Khan, 2016). Another theoretical IR and ultraviolet-visible (UV-Vis) spectra of TAM drug were compared with the experimental data where the methodology that have been found in a better correlation with experimental data is M06/6-31G(d) (Landeros-Martínez et al., 2017).</p><p>On the other hand, the molecular docking is an operation in which one molecule is brought into the vacancy of another while calculating the interaction energies of the numerous mutual orientations and conformations of the two interacting species (Bultinck et al., 2003). This technique allows predicting the preferred conformations of a molecule, being bonded to another (Lengauer and Rarey, 1996), and it is widely used in drug design (Kitchen et al., 2004). Mathew et al. have employed a molecular docking procedure to estimate the analogs of TAM and Reloxifen (REL) with high affinity, which could be considered a possible lead molecule for drug design (Mathew and Raj, 2009).</p><p>The aim of this work is to modify the hydrophilic functional groups of the TAM by the hydroxyl, amide, carboxyl, and sulfhydryl functional groups to achieve better activity improvement and selectivity. These analogs were studied to determine the binding activity into the hormone receptor by molecular docking and DFT analysis that allowed to decide which analog generates more oxidative damage at the active site. Also, the study of the molecular polar surface area (PSA) permitted to quantify if Tamoxifen analogs (TAM-analogs) have good permeability in cell.</p><!><p>The optimized structures of the different TAM-analogs were calculated by means of the hybrid meta-GGA M06 density functional (Zhao and Truhlar, 2008a,b) developed by the Truhlar group from the University of Minnesota, combined with the 6-31G (d) basis set proposed by the Pople group (Hariharan and Pople, 1973; Francl et al., 1982) and the continuous polarizable solvent model (CPCM) (Tomasi and Persico, 1994) using water as a solvent. The latter was used to obtain the Highest Occupied Molecular Orbital (HOMO) and Lower Unoccupied Molecular Orbital (LUMO) of each of the analogs, respectively. These calculations were carried out using the Gaussian 09 suite of programs (Frisch et al., 2018). The energy calculations of the amino acids that make up the active site on the estrogen, progesterone and TAM-analogs as well as the chemical reactivity descriptors are calculated with the M06/6-31G(d) model chemistry and CPCM using water as a solvent. All calculations were performed using DFT (Hohenberg and Kohn, 1964; Kohn and Sham, 1965; Parr and Yang, 1989). The charge distributions for the amino acids and TAM-analogs were obtained through the Hirshfeld population analysis (Hirshfeld, 1977).</p><p>Density functional methodology provides an excellent framework to define a set of known chemical concepts such as ionization potential (I) (Foresman and Frisch, 1996; Lewars, 2003), electron affinity (A) (Foresman and Frisch, 1996; Lewars, 2003), chemical hardness (η) (Parr and Pearson, 1983; Parr and Yang, 1984), electronegativity (χ) (Parr and Pearson, 1983; Parr and Yang, 1984), and electrophilicity (ω) (Parr et al., 1999). These reactivity descriptors were obtained by means of energy difference calculations. The chemical hardness, electronegativity, and electrophilicity are defined as: (1)η=12(I-A)≈12(ϵL-ϵH) (2)χ=-μ=12(I+A)≈12(ϵL+ϵH) (3)ω=μ22η=(I+A)24(I−A)≈(ϵL+ϵH)24(ϵL−ϵH) where μ is the chemical potential (Parr and Pearson, 1983; Parr and Yang, 1984) and ϵH and ϵL are the energies of the HOMO and LUMO, respectively.</p><p>The overall interaction between the TAM-analogs and the amino acids that make up the active site on ER and PR can be quantified through the charge transfer between the chemical species. This parameter determines the behavior of the different molecular systems as a donor or as an acceptor system. In this case, the electrons were transferred from the TAM-analogs to the amino acids of the active site of receptors or vice versa. The global interactions between two constituents has been calculated using the charge transfer parameter (ΔN) which is given by Padmanabhan et al. (2007): (4)ΔN=μB−μA2(ηA+ηB) The molecular polar surface area (PSA) was obtained through Molinspiration, a free software readily available on the Web (Molinspiration, 2018). To obtain PSA, the TAM-analogs were encoded with SMILES (Simplified Molecular Input Line System), which is a chemical notation system designed for modern chemical information processing (Weininger, 1988).</p><!><p>The crystal structures of the estrogen and progesterone receptor were retrieved from the Protein Data Bank PDB: 1A52 and 1A28 respectively. The molecular docking was calculated with the specially tailored AutoDock 4.2 software with the Lamarckian Genetic Algorithm (LGA) (Morris et al., 2009) to explore how ER and PR bond with the TAM analogs. The water molecules in the receivers were eliminated and only the H-atoms polar were added. The docking area is selected by constructing a grid box of size 52 × 36 × 34 points, centered at x, y, and z coordinates of 89.304, 14.745, and 70.512, respectively for ER, and for PR, the grid box size 20 × 18 × 26 points was centered at x, y, and z coordinates of 36.999, 31.767, and 42.694, respectively, using in both receptors a grid spacing of 0.375 Å in AutoGrid (Morris et al., 2009). The docking parameters used for the LGA based conformational searches are docking trials: 150, population size: 150, maximum number of energy evaluations: 25000000, maximum number of top individuals to survive to next generation: 1, rate of gene mutation: 0.02, rate of crossover: 0.8: Mean of Cauchy distribution for gene mutation: 0.0, variance of Cauchy distribution for gene mutation: 1.0, and number of generations for picking the worst individual: 10.</p><!><p>The geometry optimization and frequency calculation of the TAM-analogs were performed to make sure that the molecules were at their lowest energy level. Figure 1 shows the optimized geometries of the studied molecules. The optimized TAM-analogs show a non-planar geometry due to the four dihedral angles in their structures as we can see in Table 1. A small difference in the dihedral angles has been observed in comparison with the TAM drug reported by Landeros-Martínez et al. (2017): there is an average difference of 0.68 degrees in DA, 1.021 degrees in DA2 and 0.01 degrees in DA3. Moreover, the dihedral angles DA4, DA5, DA6, and DA7 that were found on the opposite end of the TAM-analogs were 179.87 degrees, −179.77 , 179.06, and 178.99 degrees respectively. These dihedral angles have a greater differences compared to the TAM drug results (Landeros-Martínez et al., 2017). The values for the cartesian coordinates belonging to the optimized molecular structures of all the analogs are presented within the Supplementary Materials.</p><!><p>Optimized molecular structure of the Tamoxifen analogs at the M06/6-31G(d) level of theory: (A) TAM-Hydroxyl; (B) TAM-Amide; (C) TAM-Carboxyl; (D) TAM-Sulfhydryl.</p><p>Dihedral angles (°) of the Tamoxifen analogs determined at the M06/6-31G(d) level of theory.</p><!><p>The evaluation of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) in each of the ligands were carried out to identify the zone that is richer in electrons. This analysis of the molecular orbitals allowed to explore the pharmacophore of the analogs. Figure 2 shows the HOMO and LUMO for the different ligands. In all cases, the pharmacophore of the TAM-analogs remains in the same area (phenyl, ethyl, and alkene functional groups) reported for the TAM drug (Landeros-Martínez et al., 2016). This study was also used to explain which zone of the ligands has the recognition ability in the hormone receptors. Furthermore, the electrostatic potential surface (EPS) maps were adequate for analyzing the binding sites on the basis of the recognition of one molecule by another, which is very important for drug design (Li et al., 2013). The maps in Figure 3 show in red the region with the most electronegative electrostatic potential and blue region for the most positive electrostatic potential. It can be observed that atoms that are more electronegative in TAM-Hydroxyl, TAM-Amide, and TAM-Carboxyl are the oxygen atoms while in TAM-Sulfihydryl are the oxygen and sulfur atoms.</p><!><p>Highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) of the (A) TAM-Hydroxyl, (B) TAM-Amide, (C) TAM-Carboxyl, and (D) TAM-Sulfhydryl calculated at the M06/6-31G(d) level of theory.</p><p>Optimized structure and electrostatic potential map on the molecular surface for (A) TAM-Hydroxyl, (B) TAM-Amide, (C) TAM-Carboxyl, and (D) TAM-Sulfhydryl calculated at the M06/6-31G(d) level of theory. Color range oscillates between −2.200e-3 to 5.300e-2: blue, more positive; red, more negative.</p><!><p>Chemical reactivity parameters such as electron affinity, ionization potential, chemical hardness, electronegativity, chemical potential, and electrophilicity index obtained with energy differences approximation as well as HOMO-LUMO approximation are presented in Table 2. These values suggest that TAM-Amide has the greater ease to react in the presence of the hormonal receptors according to the chemical hardness in both approximations. Also, the electrophilicity index information allowed to determinate that all TAM-analogs are strong electrophiles with ω > 1.5 eV for both approximations in accordance with Domingo et al. (2016). In addition, the nucleophilicity index was calculated by (5)N(Nu)=EHOMO(Nu)(eV)−EHOMO(TCE)(eV) Tetracyanoethylene (TCE) was used as a reference for these scales of nucleophilicity because it presents the lowest HOMO energy in a large series of molecules previously studied, being the EHOMO of the TCE of −9.13 eV. The values of nucleophilicity of the TAM drug, TAM-Hydroxyl, TAM-Amide, TAM-Carboxyl, and TAM-Sulfhydryl are 3.42, 3.40, 3.43, 3.41, and 3.38 eV, respectively. All the molecules have a strong nucleophiliic character with N > 3.0 eV according to the scale proposed by Domingo et al. (2016).</p><!><p>Reactivity parameters of the TAM-analogs determined at the M06/6-31G(d) level of theory with energy differences and HOMO-LUMO approximations.</p><!><p>Another important result of the TAM-analogs is the molecular polar surface area (PSA), which allows the prediction of the transport properties of drugs through membranes. PSA consists of the sum of all polar atoms, including the oxygen, nitrogen and hydrogen attached to these atoms (Ertl, 2008). The results were 29.46 , 29.54 46.53 , and 9.63 Å2 for TAM-Hydroxyl, TAM-Amide, TAM-Carboxyl, and TAM-Sulfhydryl, respectively. According to Clark, the drugs with a value less than 90 Å2 are completely absorbed in the cell membranes, while those drugs with values greater than 140 Å2 are poorly cell permeable (Clark, 1999).</p><!><p>The binding modes of a series of TAM-analogs were estimated by means of molecular docking calculations. The value of the root mean square deviation (RMSD) was considered as a measure of the accuracy of the docking results. The optimal RMSD value must be lower than 2 Å (Samanta and Das, 2016). Figure 4 shows the alignments to the native co-crystallized structure TAM (gray) with each one TAM-analogs (blue). Therefore, the RMSD in the estrogen receptor obtained between TAM with TAM-Hydroxyl, TAM-Amide, TAM-Carboxyl, and TAM-Sulfhydryl are 3.03 , 1.824 , 19.67 , and 2.272 Å, respectively, while for the progesterone receptor the RMSD value between TAM and TAM-Hydroxyl, TAM-Amide, TAM-Carboxyl, and TAM-Sulfhydryl are 2.082 , 3.445, 0.148, and 0.949 Å.</p><!><p>Alignments between the co-crystal structure TAM (gray) and the TAM-analogs (blue) at their absolute positions in the binding pocket in hormone receptors for (A) TAM-Hydroxyl, (B) TAM-Amide, (C) TAM-Carboxyl, and (D) TAM-Sulfhydryl.</p><!><p>All the TAM-analogs were successfully docked into the binding pocket of ER. In this work, the attention has been focused on the estrogen receptor-ligand because this analysis allows to determine which of these analogs are the most or least active.</p><p>The binding energy of TAM-Hydroxyl, TAM-Amide, TAM-Carboxyl, and TAM-Sulfhydryl with the ER are −9.63 , −10.79, −10.80, and −10.23 kcal/mol, respectively. Figure 5 shows the optimal docking position and binding energy into the binding pocket of ER. It has been observed that each of the TAM-analogs is located at the pocket site of the ER. Furthermore, based on our previous experience, it can be said that in spite of the differences between the ΔG values in each case being small, the results of the binding energies are significant enough to assert that the TAM-Amide and TAM-Carboxyl species are the most active while the least active is TAM-Hydroxyl in the pocket site of the ER.</p><!><p>Optimal docking position and binding energy of the estrogen receptor with (A) TAM-Hydroxyl, (B) TAM-Amide, (C) TAM-Carboxyl, and (D) TAM-Sulfhydryl.</p><!><p>After successful analysis of the bonding mode of TAM analogs, the hydrogen bond and π-π interaction were analyzed in each of the couplings. TAM-Hydroxyl has one π-π interaction and one hydrogen bond between hydroxyl of the analog and the oxygen atom of Lys 529 (OH–O, 1.94 Å); TAM-Amide formed one hydrogen bond with the oxygen atom of the ligand and the NH of Lys 529 (O–NH, 2.078 Å); TAM-Carboxyl present one π-π interaction and one hydrogen bond between oxygen atom of the analog and NH of Lys 529 (O–NH, 1.845 Å) and finally TAM-Sulfhydryl has one π-π interaction and one hydrogen bond between sulfhydryl (SH) of the ligand and the oxygen atom of Asp 351 (SH–O, 1.759 Å). In all cases, the TAM-analogs analyzed follow the Lipinsky Rule of Five which is used to predict whether a compound has or not has a drug-like character (Leeson, 2012). Additionally, when there are five or fewer hydrogen bonds, it can be said that the drug have good absorption or permeation and will be more active (Lipinski et al., 2001). Figure 6 shows the TAM-analogs in ball and stick and the amino acids of the pocket site in tube. The hydrogen bonds are showed in green dots and π-π interactions are the area in yellow.</p><!><p>Hydrogen bond and π-π interactions at the active site of the estrogen receptor with (A) TAM-Hydroxyl, (B) TAM-Amide, (C) TAM-Carboxyl, and (D) TAM-Sulfhydryl.</p><!><p>The values of reactivity parameters calculated for each of the TAM-analogs and the amino acids of the pocket site of the ER were estimated using the vertical A and I and are given in Tables 3, 4 respectively. The chemical potential of the TAM-analogs varies from −3.23 to −3.44 eV. Meanwhile, for the active site in each of the couplings, the values range from −2.39 to −4.31 eV. For the amino acids of the active site, the electronegativity decreases in the order Leu 346-Thr 347 > Lys 529 > Leu 525 > Thr 347 > Leu 387-Met 388 > Arg 394 > Leu 428 > Ala 350 > Leu 428 > Gly 521 > Met 421 > Phe 404 > Leu 387-Met 388 > His 524 > Leu 349-Ala 350-Asp 351 >Trp 383 > Glu 353- Leu354 > Ala 350-Asp 351 > Glu 353 > Met 388. The maximum value of electronegativity of the TAM-analogs is for TAM-Carboxyl; therefore the maximum difference in electronegativity occurs between TAM-Carboxyl and Glu 353.</p><!><p>Reactivity parameters of the different TAM analogs in the pocket size of the estrogen receptor.</p><p>Reactivity parameters of the pocket site of the estrogen receptor.</p><!><p>Among TAM-analogs, the TAM-Amide has the lowest chemical hardness which means this molecule is more reactive in the presence of ER. The chemical hardness of the TAM-analogs are in the order: TAM-Amide > TAM-Hydroxyl > TAM-Sulfhydryl > TAM-Carboxyl. The chemical hardness of the active site of the four couplings are in the order: Glu 353- Leu 354 > Trp 383 > Met 421 > Ala 350-Asp 351 > Glu 353 >Met 388 > Leu 387-Met 388 > His 524 > Phe 404 >Leu391 > Leu 525 > Lys 529 > Leu 428 > Thr 347 > Leu 428 > Arg 394 > Ala 350 > Gly521 > Leu 346-Thr 347 > Leu 349-Ala 350- Asp 351. The electrophilicity index suggest that TAM-Amide has a greater capacity to accept electrons from the pocket site, whereas in the pocket site of the couplings decreases in the order Leu 346-Thr 347 > Lys 529 > Leu 525 > Met 421 > Thr 347 > His 524-Leu 525 >Leu 428 > Arg 394 > Leu 349-Ala 350- Asp 351 > Trp 383 > Phe 404 > Leu 428 > Leu 387-Met 388 > Glu 353-Leu354 > Gly 521 > Met 388 >His 524 > Ala 350-Asp 351> Leu391 > Glu 353.</p><!><p>The interaction between the TAM-analogs and the amino acids of the pocket site was calculated by means of the parameter ΔN which determines the fractional number of electrons transferred form a system A to a system B with ΔN described by Equation (4). In this formula, μA is for the TAM-analogs and μB is for the amino acids of the active site. ηA and ηB represent the chemical hardness of the TAM-analogs and the amino acids of the active site, respectively. Values of ΔN < 0 suggest that the charge transfer flows from A to B (A acts as an electron donor), and for values of ΔN > 0 charge flows from B to A (A acts as electron acceptor). In previous works, Kanvah et al. and Wan et al. have used the charge transfer concept to describe the oxidative damage in DNA bases (Wan et al., 2000; Kanvah and Schuster, 2005).</p><p>According to the results of Table 5, some amino acids of the pocket site act as electron donor for example: TAM-Hydroxyl with Glu 353 and Met 388, TAM-Amide with Leu 391, Phe 404, Glu 353-Leu354 and Trp 383, TAM-Carboxyl with Glu 353, and Trp 383 and finally TAM-Sulfhydryl with Glu 353, His 524, Trp 383, and Ala 350-Asp 351, while the rest of the amino acids act as electron acceptors. The oxidative damage in the active site decreases in the order TAM-Amide > TAM-Sulfhydryl > TAM-Hydroxyl >TAM-Carboxyl.</p><!><p>Charge transfer between TAM analogs and the estrogen receptor.</p><!><p>The binding energy values of progesterone receptor (PR) are: −8.61 kcal/mol for TAM-Hydroxyl, −8.41 kcal/mol for TAM-Amide, −7.73 kcal/mol for TAM-Carboxyl, and −9.50 kcal/mol for TAM-Sulfhydryl. Figure 7 shows the most favorable docking positions and binding energies into the binding pocket of PR. Here the situation is simpler to understand in comparison with the case of the estrogen receptor. According to the results of the binding energies, the TAM-Sulfhydryl is the most active while TAM-Carboxyl is the least active in the pocket site of the PR.</p><!><p>Optimal docking position and binding energy of the progesterone receptor with (A) TAM-Hydroxyl, (B) TAM-Amide, (C) TAM-Carboxyl, and (D) TAM-Sulfhydryl.</p><!><p>On the other hand, the analysis of hydrogen bond and π-π interaction in each of the couplings showed that TAM-Hydroxyl formed one hydrogen bond between hydroxyl of the analog and the oxygen atom of Asn 719 (OH–O, 1.971 Å); TAM-Amide has two hydrogen bonds with either of the ligand and the oxygen atom of Leu 715 (O–O, 3.046 Å) and Asn 719 (O–O, 2.449 Å); TAM-Carboxyl present two hydrogen bonds between either of the analog and oxygen of Leu 715 (O–O, 2.984 Å) and Asp 719 (O–O, 2.705 Å). Meanwhile TAM-Sulfhydryl has one hydrogen bond between sulfhydryl (SH) of the ligand and the oxygen atom of Asn 719 (SH–O, 1.886 Å). All TAM-analogs have good absorption or permeation according to the Lipinsky Rule of Five (Lipinski et al., 2001). Figure 8 shows the TAM-analogs in ball and stick and the amino acids of the pocket site in tube. The hydrogen bonds are showed in green dots.</p><!><p>Hydrogen bond and π-π interactions at the active site of the progesterone receptor with (A) TAM-Hydroxyl, (B) TAM-Amide, (C) TAM-Carboxyl, and (D) TAM-Sulfhydryl.</p><!><p>After having obtained the most stable structure of TAM-analogs in the pocket site, an analysis of the chemical reactivity of TAM-analogs and progesterone residues was performed by means of the reactivity descriptors. The results for these calculations are presented in Tables 6, 7, respectively.</p><!><p>Reactivity parameters of the different TAM-analogs in the pocket size of the progesterone receptor.</p><p>Reactivity parameters of the pocket site of the progesterone receptor.</p><!><p>The highest value of the electron affinity in the TAM-analogs is TAM-Amide and in the pocket site of each of the couplings is for the Leu 718-Asn 719 residue. The ionization potential results show that the greatest possibility of losing electrons in the TAM-analogs is TAM-Carboxyl and in the amino acids of the pocket site in each of the couplings is for the Leu 718-Asn 719 residue. The chemical hardness of the TAM-analogs are in the order: TAM-Amide > TAM-Sulfhydryl > TAM-Hydroxyl > TAM-Carboxyl. The chemical hardness of the pocket site in each of the couplings is in the order: Met 759-Val 760 > Tyr89- Cys 891 > Met 756 > Met 801 > Phe 905 > Met 909 > Phe 778 > Gly 722 > Arg 766 > Leu 718-Asn 719 > Leu 797 > Cys 891 > Gln 725 > Leu 715 > Leu 887. Among the TAM-analogs, the electronegativity decreases in the order TAM-Hydroxyl > TAM-Sulfhydryl > TAM-Carboxyl > TAM-Amide. As the minimum value of the electronegativity within the pocket site in the coupling of TAM-Hydroxyl is for Met 909. Therefore, the maximum difference in electronegativity occurs in this case between TAM-Hydroxyl and Met 909. The values for the electrophilicity index of the TAM-analogs indicate that TAM-Carboxyl have the greatest capacity to accept electrons of the pocket site, while the residues of each of the couplings decrease in the order: Leu 718-Asn 719 > Met 759-Val 760 > Met 756 > Phe 905 > Phe 778 > Gln 725 > Arg 766 > Leu 797 > Leu 715 > Met 756 > Cys 891 > Tyr89-Cys 891> Met 801 > Leu 887 >Gly 722 > Met 909.</p><!><p>The amount of charge transfer between the TAM-analogs and the amino acids of the pocket site was estimated with the parameter ΔN described with Equation (4). The values of ΔN are shown in Table 8. The analysis of the interaction of TAM-Hydroxyl with Met 801 and Met 909, TAM-Amide with Met 909, TAM-Carboxyl with Met 909, and TAM-Sulfhydryl with Met 801 and Met 909 for the charge transfer is positive indicating that these TAM analogs act as electron acceptors. Meanwhile, the one with the rest of amino acids acts as electron donor. The oxidative damage in the pocket site decreases in the order: TAM-Sulfhydryl > TAM-Hydroxyl > TAM-Amide > TAM-Carboxyl.</p><!><p>Charge transfer between TAM analogs and the progesterone receptor.</p><!><p>In this work, the replacement of polar groups, such as the hydroxyl, amide, carboxyl, and sulfhydryl in the hydrophilic zone of the TAM drug did not modified the pharmacophore. According to the PSA values, the permeability in cell of the TAM-analogs decreases in the order: TAM-Sulfhydryl > TAM-Hydroxyl > TAM-Amide > TAM-Carboxyl. The scale of electrophilicity of Domingo et al. allowed to classify all TAM-analogs as strong electrophiles.</p><p>The coupling of ER with each of the TAM-analogs showed that TAM-Carboxyl and TAM-Amide are the most active in the pocket site while TAM-Hydroxyl is the least active in the pocket site and in both cases the couplings have one hydrogen bond and one π-π interaction. According to the charge transfer descriptor, the coupling ER-TAM-Sulfhydryl and ER-TAM-Amide presented the greatest oxidative damage. In turn, the coupling of PR with the TAM-analogs showed that the most active analog is TAM-Sulfhydryl and the least active is TAM-Carboxyl, presenting in both cases one hydrogen bond. The charge transfer descriptor shows that the TAM-Sulfhydryl and TAM-Hydroxyl are more damage oxidative in the pocket site of the PR. The four TAM-analogs have a good biodistribution, permeability, and pharmacological action on the hormone receptors, according to the Lipinsky Rule of Five.</p><p>The values of the chemical hardness for TAM into the pocket site of ER and PR have been calculated earlier by us as being 2.40 and 2.33 eV, respectively. Thus, according with the chemical hardness values, TAM has a greater ease to react than the analogs in presence of both hormonal receptors. We can conclude that the activity has been not improved with any of the the TAM-analogs.</p><p>If we consider the selectivity or the degree to which the analogs acts in the active site, TAM-amide and TAM-carboxyl analogs improved the binding energy regarding with TAM in less than 0.5 kcal/mol for the case of the ER receptor for which was calculated as −10.38 Kcal/mol. In turn, for the PR case, there is an improvement in the binding energy exclusively with TAM-Sulfhydryl with −9.50 kcal/mol compared with −9.38 Kcal/mol of TAM calculated previously. However, due to the small difference between the two values, it can be concluded that this is a rather limited improvement. The main conclusion is that a marked better activity and selectivity improvement is not achieved through the studied TAM-analogs.</p><p>The reasoning behind the election of the different radical groups for building the different TAM-analogs was based on the previous knowledge of the improvement in the binding energy of hydroxyl-TAM metabolites. Nevertheless, the improvement was not significant. We believe that this behavior can be related with the low number of H-bonds because the studied TAM-analogs have only one of these bonds with either of the receptors. For this reason, the future design of potential TAM-analogs should include radical groups that make easier the formation of these kind of bonds.</p><p>Moreover, it is of outermost importance to increase the electron donor ability of the ligands and this could be probably achieved by including radical groups containing a larger number of polar atoms.</p><p>Finally, although the number of π-π bonds need to be larger in order to improve the interaction of the receptors with the TAM-analogs, this is not a fundamental issue because that interaction takes place between the rings of the pharmacophore and the receptor and our intention is to modify only the hydrophilic functional group.</p><!><p>L-LL-M, NF-H, and DG-M conceived and designed the research and equally headed, wrote, and revised the manuscript.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>

PubMed Open Access

64Cu-Labeled 2-(Diphenylphosphoryl)ethyldiphenylphosphonium Cations as Highly Selective Tumor Imaging Agents

Radiolabeled organic cations, such as triphenylphosphonium (TPP), represents a new class of radiotracers for imaging cancers and the transport function of multidrug resistance P-glycoproteins (particularly MDR1 Pgp) by single photon emission computed tomography (SPECT) or positron emission tomography (PET). This report presents the synthesis and biological evaluation of 64Cu-labeled 2-(diphenylphosphoryl)ethyldiphenylphosphonium (TPEP) cations as novel PET radiotracers for tumor imaging. Biodistribution studies were performed using the athymic nude mice bearing subcutaneous U87MG human glioma xenografts to explore the impact of linkers, bifunctional chelators (BFCs) and chelates on biodistribution characteristics of the 64Cu-labeled TPEP cations. Metabolism studies were carried out using normal athymic nude mice to determine the metabolic stability of four 64Cu radiotracers. It was found that most 64Cu radiotracers described in this study have significant advantages over 99mTc-Sestamibi for their high tumor/heart and tumor/muscle ratios. Both BFCs and linkers have significant impact on biological properties of 64Cu-labeled TPEP cations. For example, 64Cu(DO3A-xy-TPEP) has much lower liver uptake and better tumor/liver ratios than 64Cu(DO3A-xy-TPP), suggesting that TPEP is a better mitochondrion-targeting molecule than TPP. Replacing DO3A with DO2A results in 64Cu(DO2A-xy-TPEP)+, which has a lower tumor uptake than 64Cu(DO3A-xy-TPEP). Substitution of DO3A with NOTA-Bn leads to a significant decrease in tumor uptake for 64Cu(NOTA-Bn-xy-TPEP). The use of DOTA-Bn to replace DO3A has little impact on the tumor uptake; but the tumor/liver ratio of 64Cu(DOTA-Bn-xy-TPEP)- is not as good as that of 64Cu(DO3A-xy-TPEP), probably due to the aromatic benzene ring in DOTA-Bn. Addition of an extra acetamido group in 64Cu(DOTA-xy-TPEP) results in a lower liver uptake; but tumor/liver ratios of 64Cu(DOTA-xy-TPEP) and 64Cu(DO3A-xy-TPEP) are well comparable due to a faster tumor washout of 64Cu(DOTA-xy-TPEP). Substitution of xylene with the PEG2 linker also leads to a significant reduction in both tumor and liver uptake. MicroPET imaging studies on 64Cu(DO3A-xy-TPEP) in athymic nude mice bearing U87MG glioma xenografts showed that the tumor was clearly visualized as early as 1 h postinjection with very high T/B contrast. There was very little metabolite (<2%) detectable in the urine and feces samples for 64Cu(DO3A-xy-TPEP), 64Cu(DOTA-Bn-xy-TPEP)- and 64Cu(NOTA-Bn-xy-TPEP). Considering both tumor uptake and T/B ratios (particularly tumor/heart, tumor/liver and tumor/muscle), it was concluded that 64Cu(DO3A-xy-TPEP) is a promising PET radiotracer for imaging the MDR-negative tumors.

64cu-labeled_2-(diphenylphosphoryl)ethyldiphenylphosphonium_cations_as_highly_selective_tumor_imagin

INTRODUCTION<!>Materials and Instruments<!>HPLC Methods<!>(4-(Bromomethyl)benzyl)(2-(diphenylphosphoryl)ethyl)diphenylphosphonium Bromide (TPEP-xy-Br)<!>(2-(Diphenylphosphoryl)ethyl)diphenyl(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)phosphonium Acetate (DO3A-xy-TPEP)<!>(4-((4,10-Bis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)(2-(diphenylphosphoryl)ethyl)diphenylphosphonium Acetate (DO2A-xy-TPEP)<!>(2-(Diphenylphosphoryl)ethyl)diphenyl(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethyl) phosphonium Tosylate (TPEP-PEG2-OTs)<!>(2-(Diphenylphosphoryl)ethyl)diphenyl(2-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)ethoxy)ethoxy)ethyl)phosphonium Tosylate (DO3A-PEG2-TPEP)<!>(4-((1,3-Dioxoisoindolin-2-yl)methyl)benzyl)(2-(diphenylphosphoryl)ethyl)diphenyl-phosphonium Bromide (TPEP-xy-PA)<!>(4-(Aminomethyl)benzyl)(2-(diphenylphosphoryl)ethyl)diphenylphosphonium (TPEP-xy-NH2)<!>(2-(Diphenylphosphoryl)ethyl)diphenyl(4-((2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)methyl)benzyl)phosphonium Acetate (DOTA-xy-TPEP)<!>(2-(Diphenylphosphoryl)ethyl)diphenyl(4-((3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)methyl)benzyl)phosphonium Acetate (DOTA-Bn-xy-TPEP)<!>(2-(Diphenylphosphoryl)ethyl)diphenyl(4-((3-(4-((1,4,7-tris(carboxymethyl)-1,4,7-triazonan-2-yl)methyl)phenyl)thioureido)methyl)benzyl)phosphonium Acetate (NOTA-Bn-xy-TPEP)<!>64Cu-Labeling<!>Dose Preparation<!>Solution Stability<!>Partition Coefficient<!>Tumor-Bearing Animal Model<!>Biodistribution Protocol<!>Calibration of microPET<!>MicroPET Imaging<!>Metabolism<!>Stability of 64Cu(DO3A-xy-TPEP) in Liver<!>Data and Statistical Analysis<!>Synthesis of TPEP Conjugates<!>Radiochemistry<!>Solution Stability<!>Biodistribution Data<!>Comparison with 99mTc-Sestamibi and 64Cu(DO3A-xy-TPP)<!>Impact of BFCs<!>Linker Effects<!>Pet Imaging<!>Metabolic Properties<!>Transchelation of 64Cu(DO3A-xy-TPEP) in Liver<!>DISCUSSION<!>CONCLUSIONS

<p>Alteration in the mitochondrial potential (Δψm) is an important characteristic of cancer (1-4). It has been demonstrated that the mitochondrial potential in carcinoma cells is significantly higher than that in normal epithelial cells (5-9). For example, the difference in Δψm between the colon carcinoma cell line CX-1 and the control green monkey kidney epithelial cell line CV-1 was approximately 60 mV (163 mV in tumor cells versus 104 mV in normal cells). The observation that the enhanced mitochondrial potential is prevalent in tumor cell phenotype provides the conceptual basis for development of the mitochondrion-targeting pharmaceuticals and imaging probes (1-3, 10-13).</p><p>Recently, we reported several 64Cu-labeled triphenylphosphonium (TPP) cations as radiotracers for tumor imaging by positron emission tomography (PET) in athymic nude mice bearing U87MG human glioma xenografts (14-16). We found that that 64Cu(DO3A-xy-TPP) and 64Cu(DO2A-xy-TPP)+ have relatively high tumor uptake with long tumor retention. The most striking difference between the 64Cu-labeled TPP cations and 99mTc-Sestamibi, the most successful radiopharmaceutical currently available for both tumor and myocardial perfusion imaging, is that they all have much lower the heart uptake (<0.6% ID/g) than 99mTc-Sestamibi (∼18 % ID/g) at >30 min postinjection (p.i.). Their tumor/heart ratios are >40x better than that of 99mTc-Sestamibi at 120 min p.i. The muscle uptake of 64Cu(DO3A-xy-TPP) was undetectable at >30 min p.i. while 99mTc-Sestamibi has very high muscle uptake (∼5 %ID/g) over the 2 h study period. Despite of their charge difference, 64Cu(DO3A-xy-TPP) and 64Cu(DO2A-xy-TPP)+ share very similar lipophilicity, tumor uptake, tumor/heart and tumor/lung ratios. However, the positive charge in 64Cu(DO2A-xy-TPP)+ results in a dramatic reduction in its liver uptake. As a result, its tumor/liver ratio significantly better than that of 64Cu(DO3A-xy-TPP) (16). Results from in vitro assays show that 64Cu(DO3A-xy-TPP) is able to localize in mitochondria of glioma cells. MicroPET imaging data show that the tumor could be visualized as early as 30 min p.i. in the tumor-bearing mice administered with 64Cu(DO3A-xy-TPP) (14). However, its high liver uptake remains a significant challenge for 64Cu(DO3A-xy-TPP) to be clinically useful as a PET radiotracer.</p><p>To further improve the tumor uptake and tumor/background (T/B) ratios, we prepared several 2-(diphenylphosphoryl)ethyldiphenylphosphonium (TPEP) conjugates (Figure 1) and their 64Cu complexes. We are particularly interested TPEP cation because the phosphoryl group (P=O) that may help improve the radiotracer hydrophilicity and excretion kinetics from the liver. Biodistribution and imaging studies were performed using athymic nude mice bearing subcutaneous U87MG glioma xenografts. The U87MG glioma cell line was chosen because it has no expression of multidrug resistance P-glycoprotein (particularly MDR1 Pgp) (17-20). This tumor-bearing animal model would allow us to evaluate the intrinsic tumor-targeting capability of 64Cu radiotracers and their excretion kinetics from non-cancerous organs, such as heart, liver, lungs and muscle. The main objective of this study is to explore the impact of linkers, bifunctional chelators (BFCs) and 64Cu-chelates on biodistribution characteristics and excretion kinetics of the 64Cu-labeled TPEP cations.</p><!><p>Chemicals were purchased from Sigma/Aldrich (St. Louis, MO) as received. DO3A(OBu-t)3 (1,4,7,10-tetraazacyclododecane-4,7,10-tris(t-butyl acetate)), DO2A(OBu-t)2 (1,4,7,10-tetraazacyclododecane-4,7-bis(t-butyl acetate)), DOTA(OBu-t)3-NHS (1,4,7,10-tetraazacyclododecane-1-(N-hydroxysuccinimide acetate)-4,7,10-tris(t-butyl acetate)) p-SCN-Bn-DOTA(2-(p-isothiocyanobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and p-SCN-Bn-NOTA (2-(p-isothiocyanobenzyl)-1,4,7-triaazacyclononane-1,4,7-triacetic acid) were purchased from Macrocyclics Inc. (Dallas, TX). NMR (1H, 13C and 31P) data were obtained using a Bruker DRX 300 MHz FT NMR spectrometer. Chemical shifts are reported as δ in ppm relative to TMS. ESI mass spectral data were collected using positive mode on a Finnigan LCQ classic mass spectrometer, School of Pharmacy, Purdue University. Elemental analysis was performed by Dr. H. Daniel Lee using a Perkin-Elmer Series III analyser, Department of Chemistry, Purdue University. 64Cu was produced using a CS-15 biomedical cyclotron at Washington University School of Medicine by the 64Ni(p,n)64Cu nuclear reaction.</p><!><p>Method 1 used a LabAlliance semi-prep HPLC system equipped with a UV/vis detector (λ = 254 nm) and Zorbax C18 semi-prep column (9.4 mm × 250 mm, 100 Å pore size). The flow rate was 2.5 mL/min. The mobile phase was isocratic with 90% solvent A (0.1% acetic acid in water) and 10% solvent B (0.1% acetic acid in acetonitrile) at 0 - 5 min, followed by a gradient mobile phase going from 90% solvent A and 10% solvent B at 5 min to 60% solvent A and 40% solvent B at 20 min. Method 2 used the LabAlliance HPLC system equipped with a UV/vis detector (λ = 254 nm), a β-ram IN-US detector and Vydac C18 column (4.6 mm × 250 mm, 300 Å pore size). The flow rate was 1 mL/min with the mobile phase being isocratic with 90% solvent A (10 mM ammonium acetate) and 10% solvent B (acetonitrile) at 0 - 5 min, followed by a gradient mobile phase going from 10% B at 5 min to 60% B at 20 min.</p><!><p>To a hot solution of α,α'-dibromo-p-xylene (528 mg, 2 mmol) in toluene (30 mL) was added slowly bis(diphenylphosphino)ethane monooxide (828 mg, 2 mmol). The mixture was heated to reflux for 20 h. The white solid was filtered, washed with toluene (20 mL) and diethyl ether (40 mL), and then dried under vacuum to give the expected intermediate product: 4-bromomethylbenzyl(diphenylphosphinoethane)phosphonium bromide (TPEP-xy-Br). The yield was 1.6 g (93 %). 1H NMR (CDCl3, chemical shift δ in ppm relative to TMS): 2.61(m, 2H, O=PCH2); 3.05(m, 2H, PCH2); 4.59 (s, 2H, CH2Br); 4.75 (d, 2H, PCH2, JPH = 13 Hz); 6.83 (d, 4H, C6H4); 7.52 - 7.91 (m, 20H, C6H5). ESI-MS: m/z = 598.5 for [M+ + H] (calcd. 598 for C34H32OP2Br+). TPEP-xy-Br was isolated as the bromide salt, and was used for the next step reaction without further purification.</p><!><p>To a solution of TPEP-xy-Br (67.8 mg, 0.1 mmol) and DO3A(OBu-t)3 (51.5 mg, 0.1 mmol) in dry DMF (3 mL) was added triethylamine (0.07 mL, 0.5 mmol). The reaction mixture was stirred at 50 °C overnight. After complete removal of volatiles, the residue was dissolved in 12 N HCl (2 mL). The resulting solution was stirred at room temperature for 30 min. Volatiles were completely removed under vacuum. The residue was then dissolved in water (3 mL), was then subjected to HPLC purification (Method 1). The fractions at ∼17.3 min were collected, combined, and lyophilized to give a white powder. The yield was 30.1 mg (33 %). 1H NMR (D2O, chemical shift δ in ppm relative to TMS): 1.89 (s, 3H, CH3COO-); 2.80 - 3.7 (m, 26H); 3.67 (s, 2H); 4.29 (d, 2H, PCH2, JPH = 13 Hz); 6.71(d, 2H, C6H4); 7.18 (d, 2H, C6H4); 7.38 - 7.67 (m, 20H, C6H5). ESI-MS: m/z = 863.2 for [M + H]+ (calcd. 863 for [C48H57N4O7P2]+). Anal. Calcd. for C48H57N4O7P2·CH3CO2·5H2O: C, 59.28; H, 6.96; N, 5.53. Found: C, 59.48; H, 6.85; N, 5.71.</p><!><p>To a solution of TPEP-xy-Br (67.8 mg, 0.1 mmol) and DO2A(OBu-t)2 (80 mg, 0.2 mmol) in dry DMF (3 mL) was added triethylamine (0.07 mL, 0.5 mmol). The mixture was stirred at room temperature overnight. After complete removal of volatiles, the residue was dissolved in 12 N HCl (3 mL). The solution was stirred at room temperature for 30 min. Volatiles were removed completely on a rotary evaporator. The residue was then dissolved in water (3 mL), and the product was separated from the mixture by HPLC purification (Method 1). The fractions at ∼11.8 min were collected, combined and lyophilized to give a white powder. The yield was 28.5 mg (33 %). 1H NMR (in CDCl3): 1.89 (s, 3H, CH3COO-); 2.42 (m, 4H); 2.70 - 3.01 (m, 20H); 3.40 (s, NCH2); 4.85 (d, 2H, PCH2, JPH = 13 Hz); 6.78 (d, 2H, C6H4); 7.30 (d, 2H, C6H4); 7.47 - 7.83 (m, 20H, C6H5). ESI-MS: m/z = 805.4 for [M + H]+ (805 calcd. for C46H55N4O5P2+). Anal. Calcd. for C46H55N4O5P2·CH3CO2·6.5H2O: C, 58.71; H, 7.29; N, 5.71. Found: C, 58.50; H, 6.89; N, 5.77.</p><!><p>TsO-PEG2-OTs was prepared using the literature procedure.16 TPEP mono-oxide (104 mg, 0.25 mmol) and TsO-PEG2-OTs (230 mg, 0.5 mmol) were suspended in dry toluene (10 mL). The mixture was heated to reflux for 20 h. Volatiles were removed under vacuum. The residue was dissolved in 50% DMF/H2O mixture. The resulting solution was subject to HPLC purification (Method 1). The fractions at ∼16.5 min were collected and lyophilized to give a white solid. The yield was 65.5 mg (30%). 1H NMR (in CDCl3): 2.33 (s, 3 H, CH3), 2.43 (s, 3 H, CH3), 2.75 (m, 2H, O=PCH2), 3.17-3.71 (m, 12 H), 4.09 (t, 2H, PCH2), 7.12 (d, 2 H, J = 7.8 Hz), 7.32-7.78 (m, 26 H). ESI-MS: m/z = 701.17 for [M]+ (calcd. 701 for [C39H43O6P2S]+).</p><!><p>TPEP-PEG2-OTs (24 mg, 0.027 mmol) and DO3A(t-Bu)3 (14 mg, 0.027 mmol) were dissolved in dry DMF (3 mL). After addition of Et3N (30 μL, 0.20 mmol), the solution was stirred at room temperature for 20 h. After removal of volatiles, the residue was dissolved in 12 N HCl (3 mL). The solution was stirred for 2 h. After complete removal of volatiles under vacuum, the residue was dissolved in water (3 mL). The resulting solution was subjected to HPLC purification (Method 1). The fractions at ∼10 min were collected, combined and lyophilized to give a white powder. The yield was 8.5 mg (30%). 1H NMR (in CDCl3): 2.21 (s, 3H, CH3), 2.39-3.63 (m, 38 H), 7.19 (d, 2H), 7.35-7.72 (m, 22 H). 31P NMR: 32.41 (d, J = 54.4 Hz), 42.84 (d, J = 54.4 Hz). ESI-MS: m/z = 875.01 for [M]+ (calcd. 875 for [C46H61N4O9P2]+). Anal. Calcd. for C46H61N4O9P2·CH3C6H4SO3·H2O: C, 59.76; H, 6.62; N, 5.71. Found: C, 59.48; H, 6.85; N, 5.62.</p><!><p>TPEP-xy-Br (340 mg, 0.5 mmol) and potassium pthalimide (110 mg, 0.6 mmol) were suspended in acetonitrile (10 mL). The suspension was heated to reflux for 24 h. The solid was filtered and the filtrate was dried under vacuum to give a light-yellow solid (300 mg, 0.40 mmol, 80%). 1H NMR (in CDCl3): 2.83 (m, 2 H), 3.14 (m, 2 H), 4.66 (s, 2 H, CH2N), 5.14 (d, 2 H, PCH2, JPH = 14.4 Hz), 6.80 (dd, 2 H, J = 8.1, 2.7 Hz), 6.93 (d, 2 H, J = 8.1 Hz), 7.45-7.90 (m, 20 H), 8.05-8.18 (m, 4 H). 31P NMR: 28.9 (d, J = 53.1 Hz), 33.0 (d, J = 53.1 Hz). ESI-MS: m/z = 664.03 for [M]+ (calcd. 664 for [C42H36NO3P2]+).</p><!><p>The TPEP-xy-PA intermediate (300 mg, 0.40 mmol) and 0.53 mL of aqueous hydrazine (55%, 88 mmol) were added into absolute ethanol (20 mL). The resulting solution was heated to reflux for 20 h. Volatiles were removed under vacuum. The light-yellow solid was dissolved in dichloromethane (20 mL). After filtration, the solvent was evaporated to get light-yellow solid. The yield was 230 mg (92%). 1H NMR (in CDCl3): 2.88 (m, 2 H), 3.10 (m, 2 H), 3.72 (s, 2 H, CH2N), 5.14 (d, 2 H, PCH2, JPH = 14.4 Hz), 6.85 (m, 4 H), 7.42-7.85 (m, 16 H), 8.05-8.15 (m, 4 H). 31P NMR: 28.8 (d, J = 51.7 Hz), 33.1 (d, J = 51.7 Hz). ESI-MS: m/z = 534.11 for [M]+ (calcd. 534 for [C34H34NOP2]+).</p><!><p>TPEP-xy-NH2 (10 mg, 0.01 mmol) and PF6 salt of DOTA(t-Bu)3-NHS (6.7 mg, 0.01 mmol) were dissolved in dry DMF (3 mL). Upon addition of triethylamine (44 μL, 0.32 mmol), the resulting mixture was stirred at room temperature for 20 h. After removal of volatiles, the residue was dissolved in the concentrated HCl (3 mL). The solution was then stirred at room temperature for 2 h. Volatiles were completely removed under vacuum, and the residue was dissolved in water (3 mL). The product was separated from the mixture by HPLC (Method 1). The fractions at ∼18 min were collected and lyophilized to give a white powder. The yield was 5.6 mg (56%). 1H NMR (in D2O): 1.88 (s, 3H, CH3COO-); 2.55-3.35 (m, 26 H), 3.59 (d, 2 H, NCH2), 4.12 (s, 2 H, CH2NH), 4.21 (d, 2 H, PCH2, J = 14.7 Hz), 6.60 (dd, 2 H, J = 7.8 and 2.2 Hz), 6.94 (d, 2 H, J = 7.8), 7.32-7.75 (m, 20 H). 31P NMR: 30.64 (d, J = 53.0 Hz), 43.09 (d, J = 53.0 Hz). ESI-MS: m/z = 920.1 for [M]+ (calcd. 920 for [C50H60N5O8P2]+). Anal. Calcd. for C50H60N5O8P2·CH3CO2·2H2O: C, 61.47; H, 6.65; N, 6.89. Found: C, 61.11; H, 6.53; N, 6.96.</p><!><p>TPEP-xy-NH2 (6 mg, 0.01 mmol) and p-SCN-Bn-DOTA (6.7 mg, 0.01 mmol) were dissolved in the equal volume mixture (2 mL) of DMF and water. The pH value in the mixture was adjusted to 8.5 with 1 N NaOH. The resulting mixture was stirred at room temperature overnight and then purified by HPLC (Method 1). The fractions at ∼17.8 min were collected. Lyophilization of the collected fractions gave a white powder. The yield was 6.3 mg (56%). 1H NMR (in D2O): 1.90 (s, 3H, CH3COO-); 2.35-3.82 (m, 29 H), 4.22 (d, 2 H, PCH2, JPH = 16.2 Hz), 4.49 (s, 2 H, CH2NH), 6.55 (dd, 2 H), 6.78 (m, 2 H), 7.16 (m, 2 H), 7.32-7.53 (m, 20 H), 7.69 (m, 3 H). 31P NMR: 30.62 (d, J = 52.4 Hz) and 42.84 (d, J = 52.4 Hz). ESI-MS: m/z = 1085.22 for [M]+ (calcd. 1085 for [C58H67N6O9P2S]+). Anal. Calcd. for C58H67N6O9P2S·CH3CO2: C, 62.92; H, 6.16; N, 7.34. Found: C, 62.80; H, 6.46; N, 7.39.</p><!><p>NOTA-Bn-xy-TPEP was prepared according to the same procedure for DOTA-Bn-xy-TPEP using TPEP-xy-NH2 (5 mg, 0.008 mmol) and p-SCN-Bn-NOTA (3.7 mg, 0.008 mmol). The fractions at ∼15.1 min (Method 1) were collected, combined and lyophilized to give a white powder. The yield was 4.6 mg (54%). 1H NMR (in D2O): 1.92 (s, 3H, CH3COO-); 2.35-3.70 (m, 23 H), 4.47 (s, 2 H, CH2NH), 4.21 (d, 2 H, PCH2, JPH = 14.1 Hz), 6.53 (d, 2 H), 6.86 (d, 2 H), 7.02-7.22 (m, 4 H), 7.30-7.56 (m, 18 H), 7.68(m, 2 H). 31P NMR: 30.38 (d, J = 52.6 Hz), 42.66 (d, J = 52.6 Hz). ESI-MS: m/z = 984.06 for [M]+ (calcd 984 for [C54H60N5O7P2S]+). Anal. Calcd. for C54H60N5O7P2S·CH3CO2: C, 64.42; H, 6.08; N, 6.71. Found: C, 64.29; H, 6.00; N, 6.83.</p><!><p>To a 5 mL vial were added 0.5 mL 0.1 M NaOAc buffer (pH = 6.9) containing 50 μg of the TPEP conjugate and 0.12 mL of 64CuCl2 solution (1.0 - 2.0 mCi) in 0.05 N HCl. The final pH was 5.0 - 5.5. The mixture was heated at 100 °C for 30 min. After cooling to room temperature, a sample of resulting solution was analyzed by radio-HPLC (Method 2). The radiochemical purity (RCP) was >90% for all 64Cu radiotracers with specific activity being ∼50 Ci/mmol. RCP data and HPLC retention times of 64Cu radiotracers are listed in Table 1.</p><!><p>All newly synthesized 64Cu radiotracers were purified by HPLC before being used for biodistribution studies. Volatiles in the HPLC mobile phases were completely removed under vacuum (< 10 mmHg) at 40 - 50 °C. The residue was dissolved in saline to ∼25 μCi/mL. The resulting solution was filtered with a 0.20 μ syringe-driven filter unit to eliminate any particles. Each animal was administered intravenously with ∼0.10 mL of the dose solution. For the imaging study, 64Cu(DO3A-xy-TPEP) was prepared and the resulting mixture was used without purification. The reaction mixture was diluted to ∼5 mCi/mL with saline. The injected dose for each tumor-bearing mouse was about 250 μCi of 64Cu(DO3A-xy-TPEP).</p><!><p>For solution stability, 64Cu(DO3A-xy-TPEP), 64Cu(DO2A-xy-TPEP), 64Cu(NOTA-Bn-xy-TPEP) and 64Cu(DOTA-Bn-xy-TPEP)- were prepared and purified by HPLC (Method 2). Volatiles in the HPLC mobile phase were removed under vacuum. The residue was dissolved in saline to ∼1 mCi/mL. Samples were analyzed by HPLC (Method 2) at 0, 1, 2, 4 and 12 h post purification. In the EDTA challenge experiment, the HPLC purified 64Cu radiotracers were dissolved in 25 mM phosphate buffer (pH = 7.4) containing EDTA (1 mg/mL) to 1 mCi/mL. Samples of the resulting solution were analyzed by radio-HPLC (Method 2) at 0, 1, 2, 4 and 12 h post purification.</p><!><p>All new 64Cu radiotracers were purified before being used for partition coefficient determination. HPLC purification is needed to eliminate potential interference from other radio-impurities. After complete removal of volatiles in the HPLC mobile phases under vacuum (< 10 mmHg) at 40 - 50 °C, the residue was dissolved in a mixture of 3 mL saline and 3 mL n-octanol. The mixture was stirred vigorously for 20 min at room temperature, and was then transferred to an Eppendorf microcentrifuge tube. The tube was centrifuged at 12,500 rpm for 5 min. Samples in triplets from n-octanol and aqueous layers were obtained, and were counted a Perkin Elmer Wizard - 1480 automatic γ-counter (Shelton, CT). The log P value was reported as an average of the data obtained in three independent measurements.</p><!><p>The ex-vivo biodistribution studies were performed using athymic nude mice bearing U87MG human glioma xenografts in compliance the NIH animal experiment guidelines (Principles of Laboratory Animal Care, NIH Publication No. 86-23, revised 1985). The animal protocol has been approved by Purdue University Animal Care and Use Committee (PACUC). Female athymic nu/nu mice (4 - 5 weeks of age) were purchased from Harlan (Charles River, MA). Each mouse was implanted with 5 × 106 the U87MG glioma cells into the upper shoulder flank. Three to four weeks after inoculation, the tumor size was in the range of 0.3 - 0.6 g, and animals were used for both biodistribution and imaging studies.</p><!><p>Twelve tumor-bearing mice (20 - 25 g) were randomly divided into four groups. The 64Cu radiotracer (∼2.5 μCi dissolved in 0.1 mL saline) was administered each animal via tail vein. Three animals were sacrificed by sodium pentobarbital overdose (100 mg/kg) at 5, 30, 60, and 120 min p.i. The blood sample was withdrawn from the heart. Organs were excised, washed with saline, dried with absorbent tissue, weighed, and counted on a Perkin Elmer Wizard - 1480 γ-counter (Shelton, CT). Organs of interest included tumor, brain, heart, spleen, lungs, liver, kidneys, muscle and intestine. The organ uptake was calculated as a percentage of the injected dose per organ (%ID/organ) and a percentage of the injected dose per gram of organ tissue (%ID/g).</p><!><p>Scanner activity calibration was performed to map between microPET image units and units of activity concentration. A pre-weighed 50 mL centrifuge tube was filled with solution containing 64CuCl2 (∼ 9.3 MBq as determined by the dose calibrator) was used to simulate whole body of the mouse. This tube was weighed, centered in the scanner aperture, and imaged for 30 min static image. From the sample weight and the density of 1 g/mL, the activity concentration was calculated as μCi/mL. Eight planes were acquired in the coronal section. A rectangular region of interest (ROI) (counts/pixel/s) was drawn on the middle of 8 coronal planes. The calibration factor was obtained by dividing the known radioactivity in the cylinder (μCi/mL) by the image ROI. This calibration factor was determined periodically and did not vary significantly with time.</p><!><p>MicroPET imaging of the tumor-bearing mice was performed using a microPET R4 rodent model scanner (Concorde Microsystems, Knoxville, TN). The tumor-bearing mice (n = 3) were imaged in the prone position in the microPET scanner. Each tumor-bearing mouse was injected with ∼250 μCi of 64Cu(DO3A-xy-TPEP) via the tail vein, then anesthetized with 2% isoflurane and placed near the center of the FOV where the highest resolution and sensitivity are obtained. Multiple static scans were obtained at 0.5, 1.0, 2 and 20 h p.i. The images were reconstructed by a two-dimensional ordered subsets expectation maximum (OSEM) algorithm. No correction was necessary for attenuation or scatter. At each microPET scan, the ROIs were drawn over the tumor and major organs on decay-corrected whole-body coronal images. The average radioactivity concentration within the tumor or an organ was obtained from mean pixel values within the multiple ROI volume, which were converted to counts/mL/min by using the calibration constant C. Assuming that the tissue density is 1 g/mL, the ROIs were converted to counts/g/min, and were then divided by the total administered activity to obtain an imaging ROI-derived percentage administered activity per gram of tissue (%ID/g).</p><!><p>Metabolic stability was evaluated in normal nude mice for 64Cu(DO3A-xy-TPEP), 64Cu(DOTA-xy-TPEP), 64Cu(DOTA-Bn-xy-TPEP)- and 64Cu(NOTA-Bn-xy-TPEP). Each mouse was administered with 100 μCi of 64Cu radiotracer via tail vein. Urine samples were collected at 30 and 120 min p.i. by manual void, and were mixed with equal volume of acetonitrile. The mixture was centrifuged at 8,000 rpm. The supernatant was collected and filtered through a 0.20 μm Millex-LG syringe-driven filter unit to remove the precipitate and large proteins. The filtrate was analyzed by radio-HPLC (Method 2). The feces samples were collected at ∼120 min p.i., and were suspended in a mixture of 50% acetonitrile aqueous solution. The mixture was vortexed for 5 - 10 min. After centrifuging at 8,000 rpm for 5 min, the supernatant was collected and passed through a 0.20 μm Millex-LG syringe-driven filter unit to remove the precipitate or particles. The filtrate was then analyzed by radio-HPLC (Method 2).</p><!><p>The liver tissue was harvested at 120 min p.i. from the mouse administered with 64Cu(DO3A-xy-TPEP) (∼100 μCi), counted in a γ-counter (Perkin Elmer Wizard - 1480, Shelton, CT) for total liver radioactivity, and was then homogenized. The homogenate was mixed with 2 mL of saline. After centrifuging at 8,000 rpm for 5 min, the supernatant was collected and counted on a γ-counter (Perkin Elmer Wizard - 1480, Shelton, CT) to determine the percentage of radioactivity recovery. After filtration through a 0.20 μm Millex-LG filter unit to remove foreign particles, the filtrate was then analyzed by radio-HPLC (Method 2).</p><!><p>The biodistribution data and T/B ratios are reported as an average plus the standard variation based on results from three tumor-bearing mice at each time point. Comparison between two different radiotracers was made using the two-way ANOVA test (GraphPad Prim 5.0, San Diego, CA). The level of significance was set at p < 0.05.</p><!><p>DO3A-xy-TPEP, DO2A-xy-TPEP and DO3A-PEG2-TPEP were prepared according to Chart I. They were designed to examine the impact of linkers (xy vs. PEG2) and molecular charge (64Cu-DO3A vs. 64Cu-DO2A) on organ uptake and excretion kinetics of the 64Cu radiotracers. DOTA-xy-TPEP, DOTA-Bn-xy-TPEP, NOTA-Bn-xy-TPEP were designed to examine the impact of BFCs (DO3A vs. DOTA, DOTA-Bn and NOTA-Bn) on biodistribution patterns of 64Culabeled TPEP cations. DOTA-xy-TPEP was prepared from the reaction of DOTA(OBu-t)3-NHS with TPEP-xy-NH2 (Chart II), followed by hydrolysis of DOTA(OBu-t)3-xy-TPEP in 12 N HCl. DOTA-Bn-xy-TPEP and NOTA-Bn-xy-TPEP were prepared by reacting TPEP-xy-NH2 with p-SCN-Bn-DOTA and p-SCN-Bn-NOTA (Chart II), respectively. All the TPEP conjugates, except DO3A-PEG2-TPEP, were obtained as acetate salts after lyophilization since the HPLC mobile phases contain 0.1% acetic acid. They all have been characterized by NMR, ESI-MS and elemental analysis. The purity of the TPEP conjugates were >95% before being used for 64Cu-labeling. Both TPEP-PEG2 and DO3A-PEG2-TPEP were isolated as tosylate salts, as evidenced by the proton signals of tosylate anion in their 1H NMR spectra, and the molecular ion at m/z = 171 in their negative mode ESI-mass spectra. It is not clear why TPEP-PEG2 and DO3A-PEG2-TPEP were obtained as tosylate salts while other TPEP conjugates were isolated as acetate salts after HPLC purification and lyophilization.</p><!><p>All 64Cu radiotracers were prepared by reacting 64CuCl2 with the TPEP conjugate in 0.1 M NaOAc buffer (pH = 5.0 - 5.5) at 100 °C for 30 min, and were analyzed using the same reversed-phase HPLC method (Method 2). The RCP for all new 64Cu radiotracers was >95% with the specific activity being >50 Ci/mmol. Their partition coefficients were determined in a 50%:50% (v:v) mixture of n-octanol and 25 mM phosphate buffer (pH = 7.4). Their log P values and HPLC retention times are listed in Table 1. All new 64Cu radiotracers are very hydrophilic with their log P values < -1.50. BFCs and linkers have a significant impact on lipophilicity of the 64Cu radiotracer. For example, 64Cu(DOTA-xy-TPEP) (log P = -2.45 ± 0.02) is more hydrophilic than 64Cu(DO3A-xy-TPEP) (log P = -1.69 ± 0.11) due to the replacement of DO3A with DOTA. Substitution of xylene with the PEG2 linker made 64Cu(DO3A-PEG2-TPEP) (log P = -3.06 ± 0.08) more hydrophilic than 64Cu(DO3A-xy-TPEP). Despite their charge difference, the lipophilicity of 64Cu(DO3A-xy-TPEP) (log P = -1.69 ± 0.11) was close to that of 64Cu(DO2A-xy-TPEP)+ (log P = -1.90 ± 0.10). 64Cu(NOTA-Bn-xy-TPEP) (log P = -1.75 ± 0.03) also shared the similar lipophilicity with 64Cu(DO3A-xy-TPEP) in spite of the extra aromatic phenyl group. Since NOTA-Bn has the same number of acetate chelating arms as DO3A, 64Cu in 64Cu-NOTA-Bn is most likely coordinated by six N3O3 donor atoms in the distorted-pseudo-prismatic coordination geometry as observed in the solid state structure of Na[Cu(NOTA)]·2NaBr·8H2O (21). Therefore, it is reasonable to believe that 64Cu(NOTA-Bn-xy-TPEP) exists as its Zwitterion form. In 64Cu(DOTA-Bn-xy-TPEP)-, however, 64Cu is likely coordinated by only six (N4O2) of its eight donor atoms (N4O4) in the distorted octahedral coordination geometry, as observed in the solid state structure of Cu(DOTA)2- with two acetate-O atoms remaining uncoordinated (22, 23). The extra acetate groups will results in the negative molecular charge under physiological conditions (pH = 7.4), 64Cu(DOTA-Bn-xy-TPEP)- (log P = -1.92 ± 0.03) is slightly more hydrophilic than 64Cu(DO3A-xy-TPEP).</p><!><p>The EDTA challenge experiment was used to study the solution stability of 64Cu(DO3A-xy-TPEP), 64Cu(DO2A-xy-TPEP)+, 64Cu(DOTA-Bn-xy-TPEP)- and 64Cu(NOTA-Bn-xy-TPEP). These four 64Cu radiotracers were selected because they represent four different types of BFCs (DO3A, DO2A, DOTA-Bn and NOTA-Bn) for 64Cu chelation. The purpose of these studies was to demonstrate that the 64Cu radiotracer remains intact before being injected into the tumor-bearing mice. Table 2 summarizes their solution stability data in the presence of excess EDTA (3 mM, pH = 7.4). On the basis of these data, it becomes quite clear that all four 64Cu-labeled TPEP cations are stable for >12 h in the presence of 3 mM EDTA (25 mM phosphate buffer, pH = 7.4).</p><!><p>Biodistribution characteristics of six new 64Cu radiotracers were evaluated in athymic nude mice bearing U87MG glioma xenografts. The Western Blot data (Figure SI) clearly demonstrated that there is no MDR1 Pgp expression on U87MG glioma xenografts. Selected biodistribution data and T/B ratios are listed in Tables 3 and 4, respectively. Detailed biodistribution data and T/B ratios are summarized in Tables SI-SVI. The main objective of these studies was to explore the impact of linkers and 64Cu chelates on biodistribution characteristics and excretion kinetics of 64Cu-labeled TPEP cations.</p><!><p>Figure 2 compares the selected organ uptake and T/B ratios between 64Cu(DO3A-xy-TPEP), 64Cu(DO3A-xy-TPP) and 99mTc-Sestamibi. This comparison allows us to demonstrate advantages of the 64Cu-labeled TPEP cations over 99mTc-Sestamibi with respect to tumor uptake and T/B ratios, and to assess the impact of "targeting moiety" (TPEP vs. TPP) on biodistribution patterns of 64Cu radiotracer. 99mTc-Sestamibi was used as the "control" since it has been clinically used for imaging tumors and monitoring the tumor MDR transport functions (25-33). Biodistribution data and T/B ratios for 64Cu(DO3A-xy-TPP) and 99mTc-Sestamibi were obtained from our previous report (14).</p><p>The most striking differences between 64Cu(DO3A-xy-TPEP) and 99mTc-Sestamibi are their uptake in the heart and muscle and their T/B (tumor/heart, tumor/liver, tumor/lung and tumor/muscle) ratios. For example, the heart uptake of 64Cu(DO3A-xy-TPEP) was <1% ID/g at >30 min p.i., while the heart uptake of 99mTc-Sestamibi was 19.22 ± 7.62 at 5 min p.i. and 19.19 ± 5.32 %ID/g at 120 min p.i. (14). The muscle uptake of 64Cu(DO3A-xy-TPEP) was almost undetectable at ≥60 min p.i. while 99mTc-Sestamibi had a high muscle uptake (4.84 ± 1.22 and 5.45 ± 1.24 %ID/g at 5 and 120 min p.i., respectively) over the 2 h period (Table 3). 64Cu(DO3A-xy-TPEP) had the tumor uptake that is significantly (p <0.05) higher than that of 99mTc-Sestamibi at all four time points. The tumor/heart ratio of 64Cu(DO3A-xy-TPEP) at 120 min p.i. was more than 50x better than that of 99mTc-Sestamibi. Its tumor/lung and tumor/liver ratios were much better than those of 99mTc-Sestamibi (Figure 3). 64Cu(DO3A-xy-TPEP) had a tumor uptake that is well comparable to that of 64Cu(DO3A-xy-TPP) at >30 min p.i.; but its liver uptake was significantly lower (p < 0.01) than that of 64Cu(DO3A-xy-TPP) at all four time points (Table 3). As a result, 64Cu(DO3A-xy-TPEP) has tumor/liver ratios >3x better than those of 64Cu(DO3A-xy-TPP). Thus, TPEP is better than TPP as the mitochondrion-targeting molecules.</p><!><p>Figure 3 compares the organ uptake (heart, liver and muscle) and T/B ratios of 64Cu(DO3A-xy-TPEP), 64Cu(DO2A-xy-TPEP)+, 64Cu(DOTA-Bn-xy-TPEP)- and 64Cu(NOTA-Bn-xy-TPEP). This comparison allows us to assess the impact of BFCs on biological properties of 64Cu radiotracers. 64Cu(DO3A-xy-TPEP) has the lipophilicity (log P = -1.69 ± 0.11) close to that of 64Cu(DO2A-xy-TPEP)+ (log p = -1.90 ± 0.10) despite their different charge. While the tumor uptake of 64Cu(DO3A-xy-TPEP) remained unchanged between 30 and 120 min p.i., there was a significant tumor washout for 64Cu(DO2A-xy-TPEP)+ over the 2 h period. The liver uptake of 64Cu(DO2A-xy-TPEP)+ was significantly (p < 0.01) lower than that of 64Cu(DO3A-xy-TPEP) at all four time points. Its tumor/liver ratio at 120 min p.i. was also significantly (p < 0.05) lower. The lipophilicity of 64Cu(DOTA-Bn-xy-TPEP)- (log p = -1.92 ± 0.03) is close to that of 64Cu(DO3A-xy-TPEP). Even though they share a similar tumor uptake, the uptake of 64Cu(DOTA-Bn-xy-TPEP)- in the heart, liver and muscle was significantly higher at >30 min p.i. As a result, the T/B ratios of 64Cu(DOTA-Bn-xy-TPEP)- was lower than that those of 64Cu(DO3A-xy-TPEP). 64Cu(NOTA-Bn-xy-TPEP) has the same overall charge as 64Cu(DO3A-xy-TPEP); but its tumor uptake and tumor/heart ratios were much lower than those of 64Cu(DO3A-xy-TPEP). Apparently, the BFCs and their 64Cu chelates have a significant impact on both tumor uptake and T/B ratios of the radiotracer.</p><!><p>Figure 4 illustrates a comparison of the tumor uptake and tumor/heart ratios between 64Cu(DO3A-xy-TPEP), 64Cu(DO3A-PEG2-TPEP) and 64Cu(DOTA-xy-TPEP). The addition of an extra acetamide group between DO3A and TPEP resulted in a significant reduction of liver uptake. However, the tumor/liver ratios (Table 4) of 64Cu(DOTA-xy-TPEP) and 64Cu(DO3A-xy-TPEP) were comparable within the experimental error, probably due to the faster tumor washout of 64Cu(DOTA-xy-TPEP). Substitution of xylene with PEG2 also leads to a significant reduction of the uptake in tumor and liver. As a result, 64Cu(DO3A-PEG2-TPEP) and 64Cu(DO3A-xy-TPEP) shared very similar tumor/liver ratios.</p><!><p>We performed a microPET imaging study on 64Cu(DO3A-xy-TPEP) using the athymic nude mice bearing U87MG glioma xenografts. Figure 5 shows microPET images of the tumor-bearing mouse administered with ∼250 μCi of 64Cu(DO3A-xy-TPEP) at 1, 4 and 24 h p.i. The tumor was clearly visualized as early as 1 h p.i. with very high T/B contrast. No significant radioactivity accumulation was detected in the brain, heart and muscle. After normalization, the tumor uptake of 64Cu(DO3A-xy-TPEP) was 2.57 ± 0.41 %ID/g, 3.51 ± 1.20 %ID/g, and 3.11 ± 1.44 %ID/g at 1, 4 and 24 h p.i., respectively. These data are completely consistent with that observed in the ex-vivo biodistribution study (Figure 3), and have clearly demonstrated that 64Cu(DO3A-xy-TPEP) is useful for imaging the MDR-negative tumors.</p><!><p>Metabolism studies were performed on 64Cu(DO3A-xy-TPEP), 64Cu(DOTA-xy-TPEP), 64Cu(DOTA-Bn-xy-TPEP)- and 64Cu(NOTA-Bn-xy-TPEP) using normal mice. These four 64Cu radiotracers were selected because of their difference in 64Cu chelate. Since they were excreted from the renal and hepatobiliary routes, we tried to collect both urine and feces samples from the normal mice administered with the 64Cu radiotracer. A reversed-phase HPLC method (Method 2) was used to analyze the collected urine and feces samples. Figure 6 shows representative radio-HPLC chromatograms of 64Cu(DO3A-xy-TPEP) (left) and 64Cu(DOTA-xy-TPEP) (right) in saline before injection, in urine at 30 and 120 min p.i., and in feces at 120 min p.i. There was very little metabolite (<2%) detectable for 64Cu(DO3A-xy-TPEP) in both urine and feces samples. Similar metabolic stability was also observed for 64Cu(DOTA-Bn-xy-TPEP)- and 64Cu(NOTA-Bn-xy-TPEP) (Figure SII). 64Cu(DOTA-xy-TPEP) is excreted without significant metabolism via renal route (Figure 6). However, only 15% of it remained intact in feces, and 85% of radioactivity appeared at 4 min. Thus, the extra acetamido group has a significant impact on metabolic stability of 64Cu(DOTA-xy-TPEP) during its hepatobiliary excretion while it had no effect on its metabolic stability during renal excretion.</p><!><p>We examined the in vivo stability of 64Cu(DO3A-xy-TPEP) in the liver. About 50% of total liver activity was recovered in the liver homogenate. The liver homogenate samples were analyzed using the reversed-phase and size-exclusion radio-HPLC methods. Figure SIII shows radio-HPLC chromatograms of the supernatant from liver homogenate. Using the size-exclusion HPLC method, we were able to identify four major radiometric peaks in its HPLC chromatogram (Figure SIII: bottom). While the identity of these radiometric peaks are not known, one thing is sure that 64Cu(DO3A-xy-TPEP) underwent extensive tranchelation in the liver during the 2 h study period.</p><!><p>For the new radiotracer to be successful as a tumor imaging agent by PET or SPECT (single photon emission computed tomography), it must show a high tumor uptake. Since most of high-incidence tumors occur in torso (namely lung, colorectal and breast cancers metastatic to the lymphatic system), it is highly desirable for the radiotracer to clear from the liver and lungs so that the high T/B ratios can be achieved and clinically useful tumor images can be obtained in a short period of time. Cationic radiotracers, such as 99mTc-Sestamibi and 99mTc-Tetrofosmin, have been clinically used for imaging tumors of different origin and the transport function of MDR Pgp by SPECT (25-33). However, their cancer diagnostic values are often limited due to their insufficient tumor localization and high uptake in the non-cancerous organs, such as heart, liver and muscle, which makes it difficult to detect small lesions in the chest and abdominal regions. Radiolabeled lipophilic organic cations, such as 4-(18F-benzyl)triphenylphosphonium (18F-BzTPP), have also been proposed as radiotracers for tumor imaging by PET (34-38). Their high uptake in the heart and liver may also impose a significant challenge for early detection of small tumors in the chest and abdominal regions. Thus, there is an unmet need for the radiotracers that have high tumor-selectivity (high tumor uptake with little accumulation in non-cancerous organs) and are sensitive to the mitochondrial potential changes at the early stage of tumor growth.</p><p>In our previous studies, we have demonstrated that the 64Cu-labeled TPP cations, such as 64Cu(DO3A-xy-TPP), are able to localize in the mitochondria of U87MG glioma cells. MicroPET imaging data also show that the tumor could be visualized clearly as early as 30 min p.i. (14). Considering both the tumor uptake selectivity, 64Cu(DO3A-xy-TPP) has a significant advantage over 99mTc-Sestamibi, the radiopharmaceutical currently available for both myocardial perfusion and tumor imaging (24-33, 39-45). However, the high liver uptake of 64Cu(DO3A-xy-TPP) remains a significant challenge for its clinical applications as a new PET radiotracer for imaging tumors.</p><p>In this study, we used TPEP as the mitochondrion-targeting molecule. We are interested TPEP cation because of the phosphoryl group that may improve radiotracer excretion kinetics from the liver. As expected, the liver uptake of 64Cu(DO3A-xy-TPEP) was significantly lower (p < 0.01) than that of 64Cu(DO3A-xy-TPP) (Figure 3), even though 64Cu(DO3A-xy-TPEP) (log P = -1.69 ± 0.11) is 10x more lipophilic than 64Cu(DO3A-xy-TPP) (log P = -2.67 ± 0.21). The tumor uptake of 64Cu(DO3A-xy-TPEP) is comparable to that of 64Cu(DO3A-xy-TPP) at >30 min p.i. (Figure 2). As a result, tumor/liver ratios of 64Cu(DO3A-xy-TPEP) are >3x better than those of 64Cu(DO3A-xy-TPP). On the basis of these results, we believe that 64Cu(DO3A-xy-TPEP) is a better PET radiotracer than 64Cu(DO3A-xy-TPP) for imaging the MDR-negative tumors. 64Cu(DO3A-xy-TPEP) also has significant advantages over 99mTc-Sestamibi with respect to the tumor uptake and T/B (tumor/heart, tumor/liver, tumor/lung and tumor/muscle) ratios.</p><p>The exact localization mechanism remains unknown even though the results from in vitro assays show that 64Cu(DO3A-xy-TPP) is able to localize in mitochondria of glioma cells (14). Many factors can influence biological properties of the 64Cu-labeled TPEP cations. Previously, we found that replacing DO3A in 64Cu(DO3A-xy-TPP) with DO2A leads to formation of 64Cu(DO2A-xy-TPP)+, and results in a dramatic reduction in liver uptake without altering its tumor uptake. In this study, a similar reduction in liver uptake was seen for 64Cu(DO2A-xy-TPEP)+ (Figure 3); but 64Cu(DO2A-xy-TPEP)+ had a rapid tumor washout over the 2 h study period. As a result, its tumor/liver ratio is significantly lower (p < 0.01) than that of 64Cu(DO3A-xy-TPEP).</p><p>64Cu(NOTA-Bn-xy-TPEP) has the same molecular charge as 64Cu(DO3A-xy-TPEP). They also share the almost identical lipophilicity with log P values being -1.69 ± 0.11 and -1.75 ± 0.03, respectively. However, the tumor uptake and T/B ratios of 64Cu(NOTA-Bn-xy-TPEP) are significantly lower (p < 0.01) than those of 64Cu(DO3A-xy-TPEP) (Figure 3). Similar adverse effect was also seen for the 64Cu-labeled TPP analogs (16). Among six 64Cu radiotracers evaluated in this study, 64Cu(NOTA-Bn-xy-TPEP) has the lowest tumor uptake and poorest T/B ratios in non-cancerous organs, such as the heart, liver, lungs and muscle. Thus, it is reasonable to believe that the coordination geometry of the 64Cu-DO3A chelates might play a significant role in tumor uptake and tumor retention of the 64Cu-labeled TPP and TPEP cations.</p><p>The lipophilicity (log p = -1.92 ± 0.03) of 64Cu(DOTA-Bn-xy-TPEP)- is also close to that of 64Cu(DO3A-xy-TPEP) (log p = -1.69 ± 0.11) despite of their difference in molecular charge. Their tumor uptake (Figure 3) is comparable within the experimental error. It seems that the negative charge in64Cu(DOTA-Bn-xy-TPEP)- does not affect its capability to localize in tumor cells. However, the uptake of 64Cu(DOTA-Bn-xy-TPEP)- in the heart, liver and muscle was significantly higher than that of 64Cu(DO3A-xy-TPEP) at >30 min p.i. As a result, its T/B ratios was significantly lower than that 64Cu(DO3A-xy-TPEP).</p><p>Linkers have a significant impact on tumor uptake and tumor/heart ratios of 64Cu-labeled TPEP cations. For example, the extra acetamido group in 64Cu(DOTA-xy-TPEP) results in a significantly lower liver uptake than that of 64Cu(DO3A-xy-TPEP). However, the tumor/liver ratios of 64Cu(DOTA-xy-TPEP) and 64Cu(DO3A-xy-TPEP) compare well within the experimental error (Figure 4) due to a faster tumor washout of 64Cu(DOTA-xy-TPEP). Substitution of xylene with PEG2 leads to a significant reduction of the tumor and liver uptake. As a result, the tumor/liver ratios of 64Cu(DO3A-PEG2-TPEP) and 64Cu(DO3A-xy-TPEP) are also comparable.</p><p>In addition, linkers also affect the metabolic stability of 64Cu radiotracers. For example, 64Cu(DO3A-xy-TPEP), 64Cu(DOTA-Bn-xy-TPEP)- and 64Cu(NOTA-Bn-xy-TPEP) have no metabolism during their excretion via renal and hepatobiliary routes as evidenced by the lack of metabolite(s) in urine and feces samples. 64Cu(DOTA-xy-TPEP) remains intact during excretion via the renal excretion; but only ∼15% of it remains intact in the feces sample (Figure 6). Since the only difference between 64Cu(DO3A-xy-TPEP) and 64Cu(DOTA-xy-TPEP) is the extra acetamido group, the metabolic instability of 64Cu(DOTA-xy-TPEP) is most likely caused by the cleavage of CO-NH bond during hepatobiliary excretion.</p><p>The radioactivity detected in urine and feces samples represents only the portion excreted from the renal and hepatobiliary routes. The remaining activity is still "trapped" in organ tissues. We examined the stability of 64Cu(DO3A-xy-TPEP) in the liver, and found that there was no intact 64Cu(DO3A-xy-TPEP) in the liver homogenate (Figure SIII: top). While the identity of these four major radiometric peaks (Figure SIII: bottom) remains unknown, the size-exclusion HPLC chromatographic pattern of 64Cu(DO3A-xy-TPEP) is very similar to that of the 64Cu-labeled TETA-Octreotide in the rat liver homogenate (46). Based on the results from stability studies of 64Cu complexes with tetraazamacrocycles (46-50), we believe that these radiometric peaks are likely caused by transchelation of 64Cu to the proteins, such as superoxide dismutase (SOD) abundant in the liver and kidneys (51).</p><p>The metabolism of the 64Cu-labeled biomolecules (antibodies and peptides) and 64Cu complexes of tetraazamacrocycles has been investigated extensively (46-59). These studies clearly show that kinetic inertness of the 64Cu chelate is particularly important for the in vivo stability of 64Cu radiotracers, and their instability is often caused by transchelation of 64Cu from the 64Cu-BFC chelate to proteins (46, 51). For target-specific 64Cu radiotracers, the tumor uptake is predominantly determined by receptor binding of targeting biomolecules (antibodies or peptides). The BFCs for 64Cu chelation should be those which form the 64Cu-BFC chelates with high kinetic inertness in order to minimize the liver radioactivity. For the 64Cu-labeled TPEP cations, however, the BFC contributes greatly to the radiotracer tumor uptake and excretion kinetics from non-cancerous organs, such as liver and lungs. The optimal BFC should be those which result in the 64Cu radiotracer with high tumor uptake and the best T/B, particularly tumor/heart and tumor/liver, ratios. From this point view, DO3A and DOTA are suitable BFCs for 64Cu-labeling of TPEP cations. It must be noted that the radioactivity "trapped" inside the liver due to transchelation of 64Cu is only a small portion of the injected radioactivity. Whenever possible, one has to consider the radioactivity excreted from renal and hepatobiliary routes (urine and feces samples), as well as the radioactivity "trapped" in various organ tissues.</p><!><p>In summary, we evaluated six new 64Cu-labeled TPEP cations for their biodistribution properties and excretion kinetics using the athymic nude mice bearing U87MG human glioma xenografts. It was found that most the 64Cu radiotracers described in this study have significant advantages over 99mTc-Sestamibi with respect to their tumor/heart and tumor/muscle ratios. TPEP seems to be a better mitochondrion-targeting molecule than TPP because of the lower liver uptake and better tumor/liver ratios of 64Cu(DO3A-xy-TPEP) than that of 64Cu(DO3A-xy-TPP). BFCs and molecular charge also have significant impact on biological properties of 64Cu-labeled TPEP cations. For example, replacing DO3A with DO2A results in 64Cu(DO2A-xy-TPEP)+, which has a lower tumor uptake and T/B ratios than 64Cu(DO3A-xy-TPEP). Substitution of DO3A with NOTA-Bn has a significant adverse effect on tumor uptake of the 64Cu radiotracer. However, the use of DOTA-Bn to replace DO3A has minimal impact on the radiotracer tumor uptake; but the tumor/liver ratio of 64Cu(DOTA-Bn-xy-TPEP)- is not as good as that of 64Cu(DO3A-xy-TPEP), probably due to the aromatic benzene ring in DOTA-Bn. Addition of an extra acetamido group in 64Cu(DOTA-xy-TPEP) results in a lower liver uptake; but the tumor/liver ratios of 64Cu(DOTA-xy-TPEP) and 64Cu(DO3A-xy-TPEP) are well comparable, probably due to a faster tumor washout of 64Cu(DOTA-xy-TPEP). Substitution of xylene with PEG2 also leads to a significant reduction in both tumor and liver uptake. As a result, 64Cu(DO3A-PEG2-TPEP) and 64Cu(DO3A-xy-TPEP) share very similar tumor/liver ratios. On the basis of both tumor uptake and T/B ratios of the 64Cu radiotracers, we believe that both DO3A and DOTA are suitable BFCs for the 64Cu-labeling of TPP cations. Among the six 64Cu radiotracers evaluated in this tumor-bearing animal model, 64Cu(DO3A-xy-TPEP) is of particularly interest due to its higher tumor uptake and better T/B ratios (particularly tumor/heart, tumor/liver and tumor/muscle). 64Cu(DO3A-xy-TPEP) will be further evaluated for its potential as a new PET radiotracer to monitor the MDR transport function in tumors of different origin.</p>

PubMed Author Manuscript

Aspergiloid I, an unprecedented spirolactone norditerpenoid from the plant-derived endophytic fungus <i>Aspergillus</i> sp. YXf3

An unusual C 18 norditerpenoid, aspergiloid I (1), was isolated from the culture broth of Aspergillus sp. YXf3, an endophytic fungus derived from Ginkgo biloba. Its structure was unambiguously established by analysis of HRMS-ESI and spectroscopic data, and the absolute configuration was determined by low-temperature (100 K) single crystal X-ray diffraction with Cu Kα radiation. This compound is structurally characterized by a new carbon skeleton with an unprecedented 6/5/6 tricyclic ring system bearing an α,βunsaturated spirolactone moiety in ring B, and represents a new subclass of norditerpenoid, the skeleton of which is named aspergilane. The hypothetical biosynthetic pathway for 1 was also proposed. The cytotoxic, antimicrobial, anti-oxidant and enzyme inhibitory activities of 1 were evaluated.

aspergiloid_i,_an_unprecedented_spirolactone_norditerpenoid_from_the_plant-derived_endophytic_fungus

Introduction<!>NMR data (<!>Experimental General experimental procedures<!>Fungal material, cultivation, extraction and isolation<!>X-ray crystallographic analysis of 1

<p>Plant-derived fungi, which have drawn considerable attention from natural product chemists, have been proved to be a rich source of bioactive natural compounds [1,2]. Recently, a wide variety of biologically active and structurally unique metabolites were isolated from these types of microorganisms [3][4][5][6], demonstrating their promise as a source of novel and/or bioac-tive natural products. Our previous chemical investigation of the bioactive secondary metabolites produced by the endophytic Aspergillus sp. YXf3 associated with Ginkgo biloba led to the isolation of new p-terphenyls and novel types of diterpenoids including pimarane-type diterpenoids (sphaeropsidins A and B, aspergiloids D and E), a cleistanthane-type diterpenoid (aspergiloid C), and norcleistanthane-type diterpenoids (asergiloids A, B, and F-H), many of which were reported from this microorganism for the first time [7][8][9]. Interestingly, sphaeropsidins A and B were also discovered from both Aspergillus chevalieri and phytopathogenic fungus Sphaeropsis sapinea, displaying anti-gram-positive bacterial, antiviral, antiprotozoal and phytotoxic activity [10][11][12]. We further focused on the fractionation containing the minor terpenoid constituents with characteristic signals for terminal vinyl group detected by 1 H NMR from the liquid fermentation broth of Aspergillus sp. YXf3 and isolated a novel norditerpenoid, namely, aspergiloid I (1) (Figure 1). Herein, we report the production, isolation, structure characterization, and biological activity of 1, a rare spirolactone metabolite with a novel carbon skeleton. ). These data show that 1 has two double bonds and one carbonyl which require three degrees of unsaturation, thus, 1 must also contain three rings.</p><!><p>The gross structure of 1 was initially deduced by comprehensive analysis of its 1D and 2D NMR data. The 13 C NMR and HSQC spectra of 1 allowed all protons to be assigned to their respective carbons. The 1 H, 1 H three-bond couplings from H-1 to H-3 observed in the COSY experiment established a spin system from C-1 to C-3 (Figure 2). The COSY correlation between H-8 and H-9 revealed C-8 to C-9 connectivity. A terminal vinyl moiety H-14/H 2 -15 was also confirmed by 1 The structure of 1 was further confirmed by a low-temperature (100 K) single-crystal X-ray diffraction experiment, which is shown in Figure 3. As compound 1 has a relatively high percentage of oxygen, it shows enough anomalous dispersion of Cu Kα radiation and allows to determinate the absolute stereochemistry with the Hooft parameter 0.17 (15) for 992 Bijvoet pairs by single-crystal X-ray diffraction experiment [13]. Therefore, the absolute configurations of the chiral centers in</p><!><p>The melting point was measured on a Beijing Taike X-5 stage apparatus and reported without correction. The optical rotation was recorded using a Rudolph Autopol III polarimeter. The UV spectrum was obtained on a Hitachi U-3000 spectrophotometer.</p><p>The CD spectrum was measured on a JASCO J-810 spectrometer, and the IR spectrum (KBr) was obtained on a Nexus 870 FTIR spectrometer. NMR data were acquired using a Bruker AVANCE III-500 NMR spectrometer at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR. The chemical shifts were given in δ (ppm) and referenced to the solvent signal (DMSO-d 6 , δ H 2.50, δ C 39.5; CDCl 3 , δ H 7.26, δ C 77.1) as the internal standard, and coupling constants (J) are reported in Hz. The high resolution mass measurement was conducted on an Agilent 6210 TOF LC-MS spectrometer. Silica gel (200-300 mesh; Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 gel (Pharmacia Biotech, Sweden) were used for column chromatography (CC). Semipreparative HPLC was conducted on a Waters ODS (250 × 4.6 mm, 5 μm) on a Hitachi HPLC system consisting of a L-7110 pump (Hitachi) and a L-7400 UV-vis detector (Hitachi). All other chemicals used in this study were of analytical grade.</p><!><p>The fungal strain Aspergillus sp. YXf3 was isolated by one of the authors (Z.K.G.) from a healthy leaf of Ginkgo biloba collected in the campus of Nanjing University (Nanjing, P. R. China), in October 2008 [7]. The strain was cultured on MEA (consisting of 20 g/L malt extract, 20 g/L sucrose, 1 g/L peptone, 20 g/L agar and deionized water) at 28 °C for 5 days.</p><!><p>Colorless crystal of 1 was obtained by crystallizing from a solution of 2 mL methanol with two drops of distilled water. The single crystal X-ray diffraction data were collected at 100 K with Cu Kα radiation (λ = 1.54178 Å) on a Bruker APEX DUO CCD diffractometer, equipped with an Oxford Cryostream 700+ cooler. Structures were solved using the program SHELXS-97 [15], and refined anisotropically by full-matrix least-squares on F 2 using SHELXL-97. The absolute configurations were determined by computation of the Hooft parameter [13], in all cases yielding a probability of 1.000 that the reported configuration is correct. Crystal data: C</p>

Beilstein

Quantum and quasi-classical calculations for the S+ +\nH2(v, j)\n\xe2\x86\x92SH+(v\xe2\x80\xb2,\nj\xe2\x80\xb2)+H reactive collisions

State-to-state cross sections for the S+ + H2(v, j) \xe2\x86\x92 SH+ (v\xe2\x80\xb2, j\xe2\x80\xb2) + H endothermic reaction are obtained with quantum wave packet(WP) and quasi-classical (QCT) methods for different initial rovibrational H2(v, j) over a wide range of translation energies. Final state distribution as a function of the initial quantum number is obtained and discussed. Additionally, the effect of the internal excitation of H2 on the reactivity is carefully studied. It appears that energy transfer among modes is very inefficient, that vibrational energy is the most favorable for reaction and rotational excitation significantly enhance reactivity when vibrational energy is sufficient to reach the product. Special attention is also paid on an unusual discrepancy between classical and quantum dynamics for low rotational levels while agreement improves with rotational excitation of H2, An interesting resonant behaviour found in WP calculations is also discussed and is associated to the existence of roaming classical trajectories that enhance the reactivity of the title reaction. Finally, a comparison with the experimental results of Stowe et al.[1] for S+ + HD and S+ +D2 reactions, finding a reasonably good agreement with those results.

quantum_and_quasi-classical_calculations_for_the_s+_+\nh2(v,_j)\n\xe2\x86\x92sh+(v\xe2\x80\xb2,\nj\x

Introduction<!>Quasi-classical calculations<!><!>Quantum calculations<!>Potential energy surface<!>Effect of the initial states of reagents<!>Resonances and roaming trajectories<!>State-to-state results<!>Comparison with experimental results<!>Conclusions

<p>Sulfur is one of the most abundant elements in space, after H, He, O, C and N. Its relative abundance with respect to H is 10−5. However, the relative abundance of all the sulfur containing molecules detected so far in space is several order of magnitudes lower. Sulfur has not being detected in ices in space, and it is therefore concluded that it must be present in highly refractive grains, not yet determined[2, 3].</p><p>The first step in the sulfur chemistry is the formation of its hydride, in neutral or cationic form. SH+ can be formed in collisions of atomic S with H3+, but H3+ ion is only abundant in cold molecular clouds. The recent detection of SH+ in hot regions, such as star formation regions[4], diffuse clouds[5, 6], and dense PDRs[7], indicates that there must by other routes to form this hydride cation. This low rotational N=1 → 0 transition of SH+(3∑−) was recently measured in the laboratory by Halfen and Ziurys[8].</p><p>One possible pathway is the (1)S++H2→SH++H reaction. This reaction is endothermic for H2(v = 0) by ≈ 0.86 eV[1, 9], but it may be the source of SH+ when considering vibrationally excited states of H2(v ≥ 2), as proposed by Agundez et al.[10].</p><p>Very recently, a potential energy surface (PES) for the ground quartet electronic state of this system has been calculated, and reaction rate constants were obtained using a quasi-classical trajectory (QCT) method[11]. The rates obtained increase substantially with increasing the initial vibrational state of H2, and were used to model SH+ fractional abundance as a function of the visual extinction, Av. For Av < 3, there is a notorious increase of the abundance of SH+ because at these high UV fluxes there is a relatively high abundance of vibrational excited H2. At Av > 4, however, the reaction of S with H3+ is the dominant route for the formation of SH+. The reaction of Eq. (1) is therefore expected to play an important role in the illuminated regions of photodissociation regions (PDR).</p><p>The aim of this work is to understand the effect of initial internal energy of H2, and to compare QCT with quantum calculations to check their validity. In addition, we study the state-to-state dynamics, to provide state-to-state cross sections to be used to determine the emission from excited rotational states, as recently done for CH+[12] and OH+[13].</p><p>The paper is organized as follows. Section II briefly describes the details of the QCT methods and of the quantum wave packet (WP) used in this work. Section III describes the results obtained and discuss the differences between quantum and classical results. Finally, section IV is devoted to extract some conclusions.</p><!><p>The QCT total reaction cross section is calculated as (2)σvj(E)=πbmax⁡2Pr(E)withPr(E)=NrNtot, where Nt is the maximum number of trajectories with initial impact parameter lower than bmax, the maximum impact parameter for which reaction takes place, and Nr is the number of trajectories leading to SH+ products. The QCT calculations have been performed using the code miQCT described previously[14]. The Hamilton equations are integrated using a step adaptive Adams-Bashforth-Moulton Predictor-corrector method[15], using the two Jacobi vectors r and R in cartesian coordinates and their generalized momenta, where r is the vector connecting the two hydrogens and R is the vector connecting the center of mass of H2 and S+. To calculate the total integral cross section of Eq. (2), a Monte Carlo sampling of initial conditions is done following the method of Karplus and co-workers[16]. 5 × 105 trajectories are run for each energy and initial rovibrational state. To select the initial conditions, the exact diatomic states of the reactants are obtained by solving the mono-dimensional Schrödinger equation to get the Evj eigen-values and consider high rovibrational excitations. The classical turning points are then obtained for each individual initial vibrational state.</p><!><p>First Jz = jz is set choosing a random number in the interval [−min(j, J), min(j, J)]. Jx is set as a random number in the interval [−(J−|Jz|), J−|Jz|]. The absolute value of Jy is then fixed and its sign is determined randomly.</p><p>The velocity vector is set along the z axis is set as Ṙ =(0,0 vR), with vR=2μE.</p><p>r = |r| is set as one of the classical turning points of the initial state, selected randomly. Its polar angles θr, ɸr are set by random numbers in the [0, π] and [0, 2π] intervals, respectively.</p><p>The velocity vector ṙ is set as</p><p>(ṙ)x = vr (cos ɸr cos θr cos χ − sin ɸr sin χ)</p><p>(ṙ)y = vr (sin ɸr cos θr cos χ + cos ɸr sin χ)</p><p>(ṙ)z = vr sin θr cos χ,</p><p>with vr = j/(μBCr), sin χ = jz/(j sin θr), j = μBC r × ṙ and μBC = mBmC/(mBj + mC)</p><p>The end-over-end angular momentum then becomes, l= J − j, and the impact parameter is b = |l|/(μvR).</p><p>Finally the R vector is set as: (R)x=bsin⁡α(R)y=bcos⁡α(R)z=R02−b2+ξ, with α = arctan(−ly/lx) and ξ a random number in the interval {0, TvR}. The period T is the rovibrational period, which have to be determined precisely for a good sampling of the phase space. As the distribution of r is not isotropic in this case, the period is determined in two steps. We first estimate the vibrational and rotational periods as Tv = 4π/(Ev+1j − Ev−1j) and Tj = π/(jBυ), respectively, where Bv=〈Φv|ℏ22μBCr2|Φv〉 is the rotational constant of the initial vibrational level v. Then, the Hamilton equations of motion of the diatomic molecule are integrated until tmax = Tv +Tj, and the rovibrational period T is set as the time t when the lowest |r(t) − r(t = 0)| distance is found. We should remark here that which such procedure, the obtained period T may differ significantly from the initial guess, specially for high (v,j) states.</p><!><p>The quantum H2 (v, j) + S+ → HS+(v′, j′)+ H state-to-state cross sections are calculated as (3)σvj,v′j′(E)=12j+1∑Ω,Ω′σvjΩ,v′j′Ω′(E), where Ω and Ω′ are the projections of the total angular momentum vector, J, on the body-fixed z-axis of reactants and products Jacobi coordinates. The helicity dependent cross-section are defined as[17, 18] (4)σvjΩ,v′j′Ω′=πkvj2∑Jϵ(2J+1)|SvjΩ,v′j′Ω′Jϵ|2 with kvj2=2μE/ℏ2 and μ=mSmH2/(mS+mH2). J is the absolute value of the total angular momentum and ϵ=±1 is the parity under inversion of coordinates. The reaction probability for each total angular momentum, J, and initial state, v, j, is defined as (5)PυjJ(E)=22j+1∑ΩΩ′∑υ′j′ϵ|SυjΩ,υ′j′Ω′Jϵ(E)|2.</p><p>The SvjΩ,v′j′Ω′Jϵ matrix elements are calculated with a quantum WP method for each value of J, ϵ and initial state quantum numbers v, j, Ω. The MAD-WAVE3 program [19–21] has been used, using reactant Jacobi coordinates, and transforming to product Jacobi coordinates at each iteration as explained in Ref. [19]. The parameters used in the WP calculations are listed in Table I.</p><p>For J = 0 we also performed time-independent (TI) calculations with hyperspherical coordinates using the ABC code[22], with the parameters listed in Table II.</p><p>The helicity dependent cross section in Eq. (4) requires the summation over all the partial wave expansion up to J = 70 in this case, to get convergence up to 2 eV of translation energy. For this purpose we performed WP calculations for all J in the interval Ω, 30 for all the initial states (v, j, Ω) considered here. To save computational time, for J > 30, complete WP calculations were performed only for some J values, those multiple of 5. For intermediate J,|SvjΩ,v′j′Ω′Jϵ|2 matrix elements were calculated using the J-shifting interpolation method[12, 18].</p><!><p>The PES of Ref. [11] has been used in all the calculations, and some minimum energy paths for several HHS angles are shown in Fig. 1. The reaction is endothermic by ≈ 0.95 eV from H2(v = 0), with no barrier for a collinear configuration. As the HHS angle varies, a barrier for the reaction appears giving rise to a rather narrow cone of acceptance for the reaction to occur. For vibrationally excited H2, r is elongated and the internal energy increases, so that for v=2 the total energy is high enough to access SH+ products. This was already investigated previously for this system using a QCT method[11], finding a considerable enhancement of the reactivity when increasing initial vibration.</p><!><p>The total integral cross section (ICS) for the S++H2(v, j) → SH+ + H reaction and different initial v, j states of H2 are shown in Fig. 2, obtained with QCT and WP calculations. The ICS increases considerably with v, as it was reported prevously with QCT calculations[11]. Here, the effect of the initial rotational excitation is also reported and compared with quantum WP results. It is found that the reaction ICS increases also when increasing the rotation excitation, specially from j=0 to 1. This effect is found in QCT and WP results, but for j=0 the QCT ones understimate considerably the ICS, for both v = 2 and v = 3</p><p>The increase of the ICS with the initial vibrational excitation, v, is well understood. The vibrational excitation brings energy in the mode associated to the reaction coordinate that have to be broken in order to form the SH+ product. Additionally, the energy for v ≥ 2 is sufficient for the reaction to become exothermic. The effect of rotation is more surprising, specially because a significant difference is observed between QCT and WP calculations for j=0, in the cases of v = 2 and 3.</p><p>To understand the effect of the rotation, calculations for zero total angular momentum, J = 0, have been performed for several initial rotational states j and v = 2, shown in Fig. 3. In order to assess the convergence of quantum results, TI calculations were performed giving results in very good agreement with the WP ones. The QCT results for j=0 and j=1 are considerably lower than the quantum ones. However, for higher j′s the agreement improve.</p><p>The effect of the total angular momentum, J, on quantum and classical results can be seen in the opacity functions shown in Fig. 4 for several initial states of H2. For v = 2, j = 0, the QCT probabilities are always lower than the WP results. For v = 2, j = 1 the QCT results are only lower for J < 30 while for v = 2, j = 2, QCT and WP results are in good agreement. Thus, high end-over-end angular momentum, ℓ, seems to increase the QCT reactivity, providing results in better aggreement with the WP ones. For v = 3, j = 0, the reactivity for j = 0 is already relatively high, so that QCT and WP agree for J > 30.</p><p>These results indicates that in a QCT treatment, the system needs more rotational excitation to react, and that it is more effective when the H2 rotation is excited.</p><p>In order to go deeper inside the dynamics, the stereodynamics is analyzed and in Fig. 5 the helicity dependent cross section is shown for v = 2, j = 1 and 2. Ω is the projection of j on the z-axis, parallel to R, and Ω = 0 for j > 0 corresponds to an angular distribution with a maximum at collinear configurations, while Ω = ±j is a T-shaped configuration. Assuming that the orientation of j do not change significantly in the entrance channel, σΩ=0 > σΩ=j indicates that the reaction is favoured for collinear approaches as discussed previously[18, 23, 24]. For j = 1, this is the case for low energies, E < 0.8 eV . For higher energies, the angular cone of acceptance increases, and the dependence of the reactivity on the relative orientation decreases. For j = 2 this is also the case, but the dependence is less marked.</p><p>Additionally, QCT calculations were performed fixing the initial orientation to collinear or T-shaped configurations for several energies and v = 2, j = 0. It is found that the cross section is considerably larger for the trajectories starting at collinear configurations as compared to those starting at T-shaped configurations. Moreover, when setting the impact parameter to zero, b = 0 ≡ J = 0, the trajectories starting at T-shaped configurations showed a very small reaction probability, nearly negligible.</p><p>All these results lead us to make a simple model for the reaction dynamics in this system. The PES in the entrance channel is rather isotropic so that for j = 0 and J = 0 the orientation between the two reagents do not vary significantly in the entrance channel. Since a higher barrier is present at bent geometries, see Fig. 1, the reactivity is strongly enhanced for those trajectories starting at collinear configurations. As the angular momenta increase, the system rotate during the approach of the two reactants, leading them the opportunity of finding the channel towards products.</p><p>This explains not only the important enhancement of the reactivity with increasing initial rotation but also the differences appearing between QCT and WP calculations. In the QCT method, each trajectory samples its own path without exploring other regions of the configuration space. However, the wave packet is non local, and is delocalized in the angular coordinates, thus exploring collinear as well as T-hsaped configurations at once. This makes that for j = 0, J = 0 the wave packet leads to a higher reactivity than the classical trajectories.</p><p>Since rovibrational excitation has a significant effect, it is interesting to quantify this effect when vibrational and rotational quantum numbers v and j increase. In order to analyse this effect, QCT calculations were performed for several initial rovibrational states of H2(v=0,1,2; j=1,5), shown in Fig. 6. As it can be seen, both vibrational and rotational excitations provide a considerable enhancement of the reactivity for a given total energy, while collision energy is found to be much less efficient. It is also interesting to see that the enhancement of reactivity due to rotation increases when the vibrational excitation is bigger. For v = 0, the augmentation of the cross section is rather moderate when the j varies from 1 to 5, and the cross section starts to increase at energies far beyond the opening of the SH+ channel. For v = 1 however, the augmentation observed between j = 1 and j = 5 is considerable and for v = 2, the effect seems to grow even stronger.</p><p>It is interesting to point out that for this system, the distribution of the energy in the different modes can change significantly the reactivity. This indicates that energy is not effectively transferred from a mode to another. In order to quantify the influence of vibrational and rotational energy contribution on reactivity, and have a better insight on energy transfers, collisions of S+ with H2 in several rovibrational states with approximately the same internal energy have been performed. The benchmark was set to H2(v = 3, j = 1), for which reaction exothermicity is 0.5 eV. To obtain the approximately the same internal energy for the comparison, the appropriate rotational levels have been choosen for lower vibrational numbers, which are (v = 2, j = 8), (v = 1, j = 12) and (v = 0, j = 15). The results for collisions energies up to 2 eV are shown in Fig. 7.</p><p>As it can be seen, even with H2 having similar internal energy, the cross sections as a function of collision energy exhibits very different behaviour. The first observation is that vibrational energy is the more favorable to reactivity. As mentioned before, it is the mode that has to be activated to obtain reaction. The second observation is that there is a clear trend difference between vibrational states that are endothermic when j = 0 (v = 0, 1), for which cross section decrease to nearly zero at low collision energy while the exothermic ones (v = 2, 3) exhibit an important increase in the cross section at low collision energy behaving like typical barrierless exothermic reactions. Similar behaviour is also seen in Fig. 6. This difference is striking, as in all the cases, the reaction is exothermic and does not present any barriers. This confirms that the energy transfer between rotation and vibration is not effective, specially at low collision energies. As a consequence the reaction probability decreases significantly when collision energy become small for v=0 and 1, while there is always a probability to react when v>1. This leads to high cross sections for v>1 since at low collision energy the trajectories are deviated toward the well in the entrance channel leading to an increase of the impact parameter with decreasing energy. On the contrary, for v=0 and 1, cross section drops because the probability to react tends to zero.</p><p>The inefficiency of energy transfer in this system can be understood considering that the potential does not present a deep well allowing the formation of a stable complex. This implies that the important rotational effect on reactivity is not associated to the increase of energy, but rather to a major exploration of the PES, enhancing the probability to find the path towards products. This can also explain why the rotational excitation is more favorable to reactivity for higher vibrational levels. By providing a better exploration of the PES, the effect of rotation is thus magnified when the cone of acceptance is bigger, providing an interesting synergic effect of the rovibrational excitation for this reaction.</p><p>This result has important implications in astrochemistry. First of all, a significant difference of reactivity is expected between ortho and para hydrogen. Additionally, as the density of molecules is low in the ISM, molecules like H2 do not thermalise efficiently, and in particular in highly irradiated regions like PDR where rovibrational exctiation can be achieved by photon absorption. In this case, in order to modelize correctly the formation of SH+, it is necessary to consider state-to-state rate constants because of the important effects of rovibrational excitation.</p><!><p>The WP reaction probabilities, in Fig. 3, show resonant structures for collisional energies below 1 eV for all the j's studied. Since the PES only presents a very shallow well in the entrance channel, the apparition of this resonant behaviour is surprising, and needs to be analysized. The QCT reaction probabilities does not show such behaviour. However, when the collision time is analyzed, many long lived trajectories occurs at these relatively low collision energies. Typical examples of such long lived trajectories are depicted in Fig. 8. These trajectories clearly show that after a first impact, the H2 rotation gets excited (we remind that vibrational excitation is very inefficient in our system), and the remaining translation energy becomes to small to yield to dissociation. Thus, after reaching the outer classical turning point, the system flies back to collide again, and this process is repeated until the system gets enough translation energy to dissociate, either in the entrance or the product channels. These kind of trajectories can be classified as roaming trajectories[25–27]. Recently, such trajectories were found in triatomic systems, in the dynamics of the MgH+H reaction[28, 29].</p><p>In order to establish the link between these roaming trajectories and the quantum resonances we need to determine how important are these trajectories and what is their effect on reaction probabilities. The roaming probabilities, shown in top panel of Fig. 9 in the case of H2 (v=2,j=1) for J=0 and 10, clearly increases at low collision energy. This roaming probability is defined as the ratio between the number of roaming trajectories and the total number of trajectories, roaming trajectories being defined in a first approximation as those having a residence lifetime superior twice the average value at the same total energy. In the bottom panel of Fig. 9, reaction probabilities of direct and roaming trajectories are ploted (J=0). As expected, it appears clearly that roaming trajectories are more reactive than direct ones as the rotation of H2 is excited at the first impact, favouring reactivity. Since the probability to obtain roaming as function of energy match the range where quantum resonances appear and considering the enhancement of reactivity of these trajectories, we can conclude that the quantum resonances can be associated to a quantum manifestation of roaming trajectories.</p><p>According to Mauguière et al., roaming occure only in well defined roaming regions of the phase space clearly separated from non roaming regions, in which both reactive and inelastic trajectories are trapped for an arbitrarily long time[30]. In a quantum approach, all regions of the phase space are sampled at once, including regions associated with roaming trajectories, giving rise to a resolved resonant structure. However, in the global QCT probabilities, these resonances cannot be observed because the Monte Carlo procedure provide an average value over all the phase space. This is not always the case. If the roaming regions are dense enough in the phase space, resonances may appear classicaly as reported in ref. [31], where classical resonances mimicking the quantum ones have been observed for J=0 at collision energy near the threshold.</p><!><p>The quantum vibrationally resolved state-to-state cross sections obtained for several initial states are shown in Fig. 10. For initial v = 2 and E < 0.8 eV, the final vibrational distributions of SH+(v′) states is reduced to only v′ = 0, with a minor contribution from more excited states. As for exothermic reactions, the cross section decreases with increasing the collision energy, and the cross section at low energy increases with v, as discussed below. At higher energies, E > 1.6 eV, the cross section for v′ = 0 is still the highest but comparable to those of v′= 1, 2 and 3, and all of them rather small, ≈ 2 Å2.</p><p>For initial v = 3, j = 0, the final vibrational distribution of SH+ products correspond to a nearly equal proportion of v′ = 0 and 1. These two final states present the highest cross sections for E < 0.4 eV, while for E > 0.8 eV all final vibrational states present nearly the same cross sections. As the potential does not present any barriers in the exit channel, once the system reaches this region, it can be considered that it will not come back. An analogy with the case of late barrier reactions can thus be established and the Polanyi rules can be applied [32, 33]. Vibrational excitation of reactants will favorise the reactivity leading to higher cross sections and the collision energy will be preferentially deposited as vibrational energy of the products.</p><p>The state-to-state cross section to each individual rotational state, j′, of SH+ products are shown in Figs. 11 and 12 for collisions starting in v = 2, j = 0 and v = 3, j = 0, respectively. For collisions starting in v=2, j=0, the highest rotationally resolved cross sections are found at low collision energy for relatively low j′ values, the maximum being obtain for j′ = 3. When increasing collision energy and j′ the cross section decreases smoothly. Similar behaviour are found for j =1, 2 and 3.</p><p>The rotationally resolved cross section for v=3, j=0 are rather different to those of v=2. For v = 3 there is more available energy making possible to form SH+ products in vibrational levels v′=0 and 1. The highest cross sections are also found at low energy, but the rotational levels with the highest cross sections are found to be j′= 15 and 7 for v′=0 and 1, respectively. These values are considerably higher than the one obtain for v = 2, j = 0, where the maximum was found for j′=3.</p><p>The quantum WP computational cost increases with j, since (2j +1) calculations have to be performed (one per initial Ω value). Thus, this kind of calculations becomes prohibitive for high j. For this purpose QCT calculations could be well adapted. Since the QCT cross sections obtained for j = 0 and 1 are in general too low as compared with the WP ones, it is worth trying to analyze how good QCT calculations are for determining state-to-state cross sections for v = 2, j = 2 a moderate j for which the total reaction cross section works fairly well. In Fig. 13 the WP and QCT results are compared at three collision energies. In the three cases and for all the final vibrational states, v′, considered the agreement is rather good. In the present case the discretization of the quantum numbers has been done with the most common histogram binning. These results can be taken as an indication that to consider higher rotational excitations of the reactants, the QCT method would yield reasonably good results.</p><!><p>In order to get a better idea of the quality of the potential, and to check the consequence of an adiabatic treatment discarding spin-orbit couplings, QCT calculations were performed for S++HD and S++D2 to reproduce the experimental results published by Stowe et al.[1]. It is interesting to point out that in the case of D2, a spin orbit contribution to the cross section have been estimated experimentally showing an important effect of the inter-system crossing between the 4A″ state and the 2A″, both leading to SH+(3Σ−)+H(2S) as can be seen in Fig. 4 of Ref. [1]. In the case of HD, no spin-orbit contributions were reported. As the experiment was done at 300K, the initial conditions were chosen for a thermalized rovibrational population, and calculation were done for collisions energies up to 15 eV. In order to compare our results, the experimental results were extracted graphically, and the spin-orbit contribution have been substracted to the experimental value. The experimental and calculated cross section for HD and D2 as a function of collision energy are shown in Fig. 14.</p><p>The global behaviour of calculated results and experimental results is very similar. In the case of D2, the calculated cross section lies between the absolute experimental cross section and the corrected one. Considering that the spin orbit contribution is an estimation, we can consider the agreement as satisfactory, at least for energies lower than the experimental maximum.</p><p>In the case of HD, both calculations and experiment show that the production of SD+ is dominant. Additionally, the agreement obtained is very good for the cross section associated to the formation of SD+. However the calculated QCT cross section for the SH+ channel is largely underestimated compared to the experimental one. As no spin-orbit effect have been reported in the experimental work and the spin-orbit contribution is clear for D2, it is legitimate to think that the formation of SH+ in the case of HD is dominated by the spin-orbit contribution while the formation of SD+ is dominated by the adiabatic reaction on the quartet state, as suggested by the good agreement between theory and experiment. If the interpretation is correct, this effect would be quite interesting, but calculations including the different spin-orbit states and their couplings will be necessary. Considering the experimental result with D2, taking in account spin-orbit contribution seems also important in order to study the reactivity to confirm and understand this hypothesis. As this contribution is stronger at lower collision energy, estimating this effect for very low collision energy and high vibrational level may be very interesting as an increase in the cross section at low energy will have an important effect on the calculated rate constants.</p><p>Another interesting point to note is that a maximum in the cross sections is observed experimentally for both HD and D2 at 6 eV and 8 eV respectively. This maximum is associated to the total dissociation (S++H+H) which becomes dominant at high collisions energy leading to a collapse of the probability to form SH+[34]. This maximum is also observed in the calculations, were the maximum in cross section found for HD is found at lower energy than the maximum obtained with D2. However, the calculated maxima are found at higher energies that the one found in the experiment. The discrepancy probably comes from the lack of precision of repulsive part of the potential at very high energy as it was not optimized to describe accurately this region in this work. The difference in the position of the maxima can be explained by the difference in the isotropy of the repulsive potential. Indeed, in Ref. [14], it was shown that a bigger anisotropy of the repulsive potential was favouring the total dissociation mechanism. By changing the position of the center of mass, the anisotropy of the potential increases in the case of HD, making the total dissociation mechanism more favorable than in the case of D2. As a consequence, the hydride formation cross sections drop faster for HD, and the maximum appears at lower collision energy.</p><!><p>In this work, a detailed study of the effect of rovibrational excitation on the reaction between S++H2 is done by performing WP and QCT dynamical calculations on the quartet state ab initio PES developed by Zanchet et al[11]. It is found that for j = 0, QCT method is unable to reproduce WP results, but a good agreement is found between the two methods for higher rotational levels. To get a better insight on the reaction, stereodynamics study is also performed. As a result of our calculations, it is found that vibrational energy is the more favorable to the formation of the SH+ product. Rotational excitation is also found to have an important effect on reactivity by providing a better exploration of the PES during the reaction and increasing the probability to find a path toward the products. Additionally, a synergy is observed between the vibrational and the rotational excitation, leading to a major effect of the rotation when the vibrational energy is higher. In this context, the reactivity strongly depends on the rovibrational state of H2. This effect of rotation have an additional indirect consequence leading to the presence of resonances in the WP calculations. They are explain by a roaming mechanism which implies a rotational excitation of H2 after a first impact, trapping the system for a while before a second impact with H2 rotationally excited. Roaming mechanism thus present an enhanced reaction probability compared to the direct one. This reaction being important from an astrochemical point of view, as it is one of the route to form SH+, it is necessary to provide state-to-state data to consider this reaction properly. In this scope, we present in this work the first state-to-state and state-to-all accurate WP reaction cross sections for S++H2(v, j) for (v = 2, j = 0,1,2) and (v = 3, j = 0). Finally, a comparison with the experimental data published by Stowe et al. [1] exhibits satifactory agreement for the contribution of the quartet state considered in this work, but demonstrate the necessity to take in account spin-orbit couplings to get a better description of this reaction.</p>

PubMed Author Manuscript

A rechargeable molecular solar thermal system below 0 °C

An optimal temperature is crucial for a broad range of applications, from chemical transformations, electronics, and human comfort, to energy production and our whole planet. Photochemical molecular thermal energy storage systems coupled with phase change behavior (MOST-PCMs) offer unique opportunities to capture energy and regulate temperature. Here, we demonstrate how a series of visiblelight-responsive azopyrazoles couple MOST and PCMs to provide energy capture and release below 0 C. The system is charged by blue light at À1 C, and discharges energy in the form of heat under green light irradiation. High energy density (0.25 MJ kg À1 ) is realized through co-harvesting visible-light energy and thermal energy from the environment through phase transitions. Coatings on glass with photo-controlled transparency are prepared as a demonstration of thermal regulation. The temperature difference between the coatings and the ice cold surroundings is up to 22.7 C during the discharging process. This study illustrates molecular design principles that pave the way for MOST-PCMs that can store natural sunlight energy and ambient heat over a wide temperature range.

a_rechargeable_molecular_solar_thermal_system_below_0_°c

Introduction<!>Synthesis and UV-Vis absorption spectra of bidirectional visible-light-driven photoswitches<!>Theoretical analysis of bidirectional visible-light-driven photoswitches<!>Design of low-temperature charging/discharging MOST-PCMs<!>Charging/discharging below 0 C<!>Energy storage time and energy density<!>Conclusions<!>Conflicts of interest

<p>Thermal management is crucial in our modern society, regardless of whether we are considering chemical transformations, electronics, human comfort, energy production or our whole planet. Thermal management materials based on specic heat capacity or phase change are seeing increased use in applications such as electronics, and domestic and industrial heat management. [1][2][3][4][5] Phase change materials (PCMs) are a broad class of materials whose latent heat during a phase transition from solidto-liquid can be used for energy storage applications. Latent heat storage offers a signicant advantage if the application involves temperature cycles close to the melting point since in those cases, the corresponding storage density of sensible thermal storage is small. In building applications, phase change materials made from paraffins, salt hydrates, fatty acids or ice can be used as central heat sinks 6 and also in oors, windows or walls. 7,8 Common to all "traditional" thermal energy storage materials is that they operate via heat transfer, both in energy input and energy output. 9 This leads to design challenges and scaling factors that restrict practical performance and implementation.</p><p>Molecular solar thermal (MOST) systems have been recognized as a promising avenue to harvest and store thermal energy. [10][11][12][13][14][15] In the charging process, a stable isomer of a photochromic molecule absorbs photon energy and is converted into a high-energy metastable isomer, thereby storing solar energy in chemical bonds. The MOST system is discharged when the metastable isomer switches back to the stable isomer by external stimuli, with the release of stored energy in the form of heat. While the MOST system shares some properties with PCMs, the process of energy storage and release in the MOST system is controlled by photons and molecular thermodynamics, 16,17 whereas in PCMs it is controlled by heat transfer. Recently, combining the functions of MOST and PCMs into a single component material (MOST-PCM) has been utilized to add storage capacity to the MOST system since the charging of the system is not only happening via solar irradiation but also by taking energy directly from the environment. [18][19][20][21] This dual input leads to an increased energy density by almost 100%. 19 Another attractive feature is added to PCMs; since the solidication of the cis liquid is not happening spontaneously, the phase change is locked by the photochemical system. This feature dramatically extends the functionality of the MOST-PCM combination since the phase change is controlled by external stimuli and no insulation is needed to hold the latent heat.</p><p>However, a severe limitation of MOST-PCMs based on azomolecules studied until now is their inability to be charged and discharged in the solid state in cold environments, especially below 0 C, because of the high melting point (T m ) of cisisomers. This is a critical condition since many applications such as thermo regulated fabrics, 22 or functional coatings will need to be able to function at that temperature. 23 Generally, the trans-cis photoisomerization of azo molecules requires a large free volume 24,25 and can only occur in the surface layers of transcrystals, thus preventing the charging process in the neat solid state. If the ambient temperature exceeds the cis-isomer T m , the generated cis-isomer melts into a liquid and exposes new transcrystal surfaces, and nally the trans-crystals are entirely transformed into cis-liquids. But most reported T m values of cisisomers are in the range of 20-200 C, 26 which means that their photoisomerization from trans-crystals to cis-liquid cannot occur at low ambient temperatures. On the other hand, although some cis-isomers can maintain liquid states below 0 C due to their supercooling behavior to achieve discharging at low temperature, the charging process is still at room temperature (27 C), limiting the versatility of the system. 19,20 Another challenge for the MOST-PCM is that the charging process generally needs UV light irradiation, since UV light causes damage to materials and the human body, and comprises a small fraction (4.5%) of the total solar spectrum, 27 resulting in the low utilization efficiency of solar energy. To date, only one study has reported the utilization of ortho-functionalized azobenzene derivatives to store both visible light energy and room temperature ambient heat. However, this system could not be charged below 0 C, and the energy density was in the 0.07-0.15 MJ kg À1 range. 28 ortho-Substitution can increase the energy of trans-isomers 29 or decrease the energy of cis-isomers, 30 so that the DH iso of this type of azo molecule decreases to only 6-25 kJ mol À1 (0.01-0.05 MJ kg À1 ). 28 Therefore, a reversibly charging/discharging and visible-light-energy storage MOST-PCM working at lowtemperature remains to be explored.</p><p>Here, we report new arylazopyrazoles as MOST-PCMs, which are rechargeable below 0 C by visible light, as illustrated in Fig. 1. In addition, by co-harvesting the visible light energy and low-temperature ambient heat, an energy density of 0.25 MJ kg À1 is achieved, which is an increase of 67% over previous comparable systems. 28 Furthermore, the cis-isomer has a halflife of 22 days at 0 C, demonstrating its stable energy storage capacity. The combination of high energy density, storage time, and the fact that the system can be charged at low temperatures provides the opportunity to explore the function of the material in a new type of optically regulated MOST-PCM window as a proof-of-concept study, designed to illustrate the function of the MOST-PCM system in a coating. Glass coated with arylazopyrazoles is prepared as a miniature energy storage window. One point to highlight is that the novel windows can be charged and discharged at À1 C by 400 nm blue light and 532 nm green light, respectively. During the discharging process, the surface temperature of the window can reach from À1 C up to 21.7 C (a temperature increase of 22.7 C), corresponding to a thermal power output of 256.2 W m À2 during a continuous period of 60 s. Charging and discharging energy at low temperatures has potential implications for functional clothing, 31 advanced sunglasses, deicing 23 and home heating 32 under ice-cold conditions, thereby increasing thermal comfort and reducing the energy consumption of conventional heating.</p><!><p>The fundamental principle to realizing low-temperature working and visible-light energy storing MOST-PCM systems is to design photochromic molecules that drive phase transition with visible light at low temperature. The introduction of a 4thiomethyl group and changing the bridging positions between the azo group and pyrazole ring was expected to achieve bidirectional visible light switching through extending the pconjugation. Accordingly, three 4-methylthioarylazopyrazoles (S3, S4, and S5) were designed and their synthetic routes are shown in Scheme 1.</p><p>In previous studies, 33 arylazopyrazoles were usually prepared by the Mills reaction of nitrosobenzene analogs and aminopyrazoles, but in this method it was difficult to synthesize arylazopyrazoles with electron-donating groups because the electron-donating groups would destabilize nitrosobenzene analogs. 34 Hence, we rst prepared 3(5)-nitroso-1H-pyrazole, which was then coupled with aniline analogs to give S3(5)-H. S3 and S5 were subsequently produced in one pot by N-methylation at two selectable positions, benetting from the proton transfer and tautomerism of 1H-pyrazole. S4 was formed via diazo-coupling and cyclization reactions.</p><p>Their photoisomerization behaviors were studied using UV-Vis absorption spectra in an acetonitrile solution. As shown in Fig. 2b-d, all trans-isomers exhibited single and intense absorption bands in the 350-400 nm region (3 max ¼ 25-32 Â 10 3 M À1 cm À1 ) due to p-p* transition. Compared to the reported 4methoxyarylazopyrazole (O4, 342 nm), 35 the p-p* l max of S3 and S4 were red-shied to about 360 nm, and further to 385 nm for S5 (Table S1 †). The trans/cis relative absorption of S3, S4 and S5 at 400 nm was strong (Table S1 †), and hence it was possible to realize trans-cis isomerization using visible light. The photostationary states (PSSs) at different wavelengths (from 365 to 532 nm) were studied (Fig. S1 †), and the isomeric compositions are presented in Table S2. † As a result, 400 nm blue light induced a near-quantitative yield (>95%) of trans-cis isomerization for S5, while only $85% for S3 and S4. The higher transcis photoconversion of S5 was attributed to its p-p* l max closer to 400 nm and stronger trans/cis relative absorption at l ¼ 400 nm. Exciting the tail of n-p* bands of three cis-isomers using green light (532 nm) resulted in cis-trans isomerization. The high overlap between the n-p* band of cis-S4 and the longwavelength absorption band of trans-S4 led to a relatively low cis-trans conversion (85%). In contrast to cis-S4, cis-S3 and cis-S5 exhibited n-p* transitions red-shied to 25 nm and 36 nm (Table S1 †), respectively, which led to partial separation of the n-p* bands of the cis and trans isomers, thereby inducing high (91% S3) to near-quantitative (>95% S5) isomerization.</p><!><p>The geometries of O4, S3, S4, and S5 and their electronic transition characteristics were calculated using density functional theory (DFT) modelling 36 to evaluate the relationship between molecular structures and photophysical properties (see Section 3 in the ESI †). All trans-isomers exhibited a planar structure with a C-N-N-C dihedral angle of 180.0 (Fig. 2e and S5 †), which resulted in symmetry-forbidden n-p* transitions (S 0 / S 1 ) with negligible oscillator strength (f ¼ 0.00, Table S4 †). Compared to trans-O4, the 4-methylthioarylazopyrazole series showed more effective extension of the p-conjugated system due to the increase of p-p conjugation by introducing the 4-SMe group, [37][38][39] as indicated by their frontier molecular orbitals (Fig. S7-S10 †). Consequently, the energy gap between the HOMO (p orbital) and LUMO (p* orbital) was smaller for the 4methylthioarylazopyrazole series (3.714, 3.776, and 3.542 eV for S3, S4, and S5, respectively, as depicted in Fig. 2f), leading to a bathochromic shi of their p-p* transition (S 0 / S 2 ). The increased red-shi of S5 was caused by a "complete" conjugation pathway between 5-pyrazole and the azo group that further expands the p-conjugation of the system. 40,41 cis-S4 showed a nearly T-shaped conformation with a C-C-N-N dihedral angle of 82.8 (Fig. 2e and S5 †), resulting in a weak n-p* transition with f of only 0.0048. In contrast, cis-S3 and cis-S5 were found to disfavor the T-shaped conformation owing to the presence of the ortho nitrogen atom (Fig. 2e and S5 †). Therefore, their n-p* absorbance bands were remarkably enhanced (f ¼ 0.1068 and 0.1096 for cis-S3 and cis-S5, respectively, Table S4 †), and nearly quantitative conversions from cis to trans isomers were achieved.</p><!><p>Thus, all the above data indicate that S5 can be used as a bidirectional visible-light-driven photoswitch, which provided nearquantitative trans-cis and cis-trans photoconversions in acetonitrile solution. However, pristine S5 was not able to store both visible-light energy and phase transition latent heat at low temperature since the photoisomerization from trans-crystals to cis-liquid was inhibited even at room temperature. To charge and discharge energy below 0 C, two additional principles are considered: (i) the T m of the cis-isomer should be below 0 C, thus forming amorphous cis-liquids to store photon energy and ambient heat; (ii) the trans-isomer should have a T m and crystallization point (T cry ) much higher than 0 C, thus forming trans-crystals to release energy. In order to adjust the T m of the trans and cis isomers, we varied the length of linear alkyl chains with or without a vinylic end group on the thioalkyl group of S5, denoted as An-S5 and Bn-S5 (Fig. 3a and Scheme S1 †). Studying the phase behaviour of the systems using differential scanning calorimetry (DSC), we nd that the trans-isomers with longer and intermediate alkyl chain lengths (n ¼ 6-12 for An-S5, and n ¼ 7, 9, and 11 for Bn-S5) showed T m (40-60 C) and T cry above 0 C (10-45 C, Tables S5 and S6 †). The cis-isomers with intermediate alkyl chain lengths had the lowest T m (5 C for A6-S5 and À1 C for B7-S5), as shown in Fig. 3b, c and S13, S14. † Hence, B7-S5 could undergo a reversible visible-light-triggered trans-crystal 4 cis-liquid transition in cold environments thanks to its low cis-isomer T m (below 0 C) and high transisomer T cry (34 C). This property illustrates, to the best of our knowledge, the rst example of a functional low-temperature visible-light controlled energy storage MOST-PCM system.</p><!><p>The charging and discharging processes of B7-S5 at À1 C are discussed as follows. As shown in Fig. 3d and Video S1, † aer 400 nm light (40 mW cm À2 ) irradiation, trans-B7-S5 in the orange crystal state lost birefringence and melted into red liquids, which then switched back to a crystal state by 532 nm light (110 mW cm À2 ) irradiation. UV-Vis spectroscopy was used to record the photoisomerization yields during irradiation of a neat sample of B7-S5 with a mass of 2 mg and a thickness of about 50 mm. During the charging process (Fig. 3e), the trans-cis isomerization of B7-S5 in a neat state proceeded easily and produced a high yield of photoisomerization (93%) at 40 min, slightly lower than that in dilute solutions (>95%). During the discharging process (Fig. 3f), the trans-isomer content of the sample increased exponentially, and a near-quantitative (95%) cis-trans isomerization was achieved within 2 min of irradiation. X-ray diffraction (XRD) analyses were carried out to further understand and verify the reversible trans-crystal 4 cis-liquid behavior of B7-S5 (Fig. 3g). The sharp peaks at 2q of 5-35 before irradiation corresponded to the regular stack of azo molecules in trans-crystals. Aer exposure to 400 nm light (40 mW cm À2 ), the peaks disappeared, indicating that cis-B7-S5 had an amorphous structure. Subsequently, XRD patterns were recovered when the sample was irradiated with 532 nm light (110 mW cm À2 ). The X-ray crystal structure of trans-B7-S5 reveals an antiparallel packing, and several weak contacts such as alkylalkyl, alkyl-phenyl, and alkyl-pyrazolyl dominating the intermolecular interactions (Fig. 3e). Presumably, the absence of strong intermolecular interactions in trans-B7-S5 offered enough exibility to the system, which was benecial for the charging process. 42,43</p><!><p>The cis-isomers of azo molecules can thermally relax into transisomers in the dark spontaneously, which determines the energy storage stability of cis-B7-S5. The change of absorbance value at p-p* l max (385 nm) as a function of time was measured in acetonitrile solution between 25 and 40 C (Fig. 4a and S15 †). The thermal isomerization rate constants, k cis/trans (Table S8 †), were calculated based on the rst-order reaction kinetics at 25 C, 30 C, 35 C, and 40 C, respectively. Based on the Arrhenius equation, cis-B7-S5 was found to have a half-life t 1/2 of 22.4 days at 0 C, indicating its stable thermal energy storage capacity at low temperatures. Thermal cis / trans kinetics was also studied in neat states (Fig. S16 and Table S9 †), and cis-B7-S5 liquid still had a t 1/2 as long as 6.3 days at 0 C. It is ubiquitous to observe a lower t 1/2 of photoswitches in condensed states than in solution. Cis-isomers tend to adopt fully relaxed geometries in solution, whereas in condensed states different geometries may be preferred due to intermolecular interactions, characterized by lower isomerization barriers. 44 Additionally, B7-S5 demonstrated excellent photon-harvesting ability with a quantum yield F trans/cis of 0.39 AE 0.01 for photoisomerization in acetonitrile solution (Fig. 4b and S17 †), similar to other reported azopyrazoles compounds. 19,41,45 To evaluate the energy density of the MOST-PCM, the isomerization enthalpy DH iso of the thermally induced cis-liquid to trans-liquid reversion reaction and crystallization enthalpy DH cry of the trans-liquid to trans-crystal transition were measured by DSC. As shown in Fig. 4c, the cis-B7-S5 liquid revealed a broad exothermic peak over 60-120 C during the thermally activated cis-trans isomerization, and the integrated area under the peak represented a DH iso of 0.14 MJ kg À1 (44 kJ mol À1 ). This result was consistent with our calculations (49 kJ mol À1 , Table S7 †) based on DFT and similar to the pristine azobenzene (<50 kJ mol À1 ). Furthermore, the DSC cooling curve displayed a sharp exothermic peak at around 33 C with a DH cry of 0.11 MJ kg À1 (35 kJ mol À1 ), which was due to the trans-liquid to trans-crystal transition. Therefore, the total thermal energy density of the MOST-PCM was 0.25 MJ kg À1 (79 kJ mol À1 ). According to F trans/cis and DH iso , the solar efficiency h was estimated to be up to 1.3% (see Section 6 in the ESI †), which was one of the highest values reported for azo-based MOST systems (0.2-1.3%). 19,28 Rechargeable energy storage coatings To illustrate the practical applications of B7-S5, trans-B7-S5 and diethoxydimethylsilane modied chain-like silica were co-dissolved in ethanol and isopropanol (1 : 1, v/v) solution, and then the mixture was drop-cast onto a glass substrate to form rechargeable coatings, as shown in Fig. 5a. The modied chainlike silica formed a transparent porous network structure 46 on glass substrates to prevent the leakage of cis-liquid (Fig. S18 †). Various patterns were created on the same rechargeable glass substrate through selectively writing/erasing processes (Fig. 5b).</p><p>First, a mask was placed on the glass and irradiated with 400 nm blue light. The exposed area of the glass was transformed from the initial opacity to semi-transparency, and the color changed from orange to red. As a result, the target pattern was written on the glass substrate. Subsequently, the erasing process could happen when the substrate without a mask was exposed to 400 nm blue light irradiation, resulting in a globally semi-transparent glass. And then, this glass substrate could be further patterned via selectively irradiating with 532 nm green light. Finally, the glass recovered to its opaque state aer exposure to 532 nm green light without a mask. The transmittance spectra of the rechargeable glass under different light irradiations were recorded (Fig. 5c). The glass sheet in a discharged state had a low transmittance of less than 10% in the wavelength range of 500-800 nm, and aer exposure to 400 nm blue light, the glass sheet is charged with the transmittance up to $70% in the wavelength range of 650-800 nm. In addition, the rechargeable glass showed good durability under alternating blue light and green light irradiations (Fig. 5d).</p><p>Thanks to the high transmittance of the charged state glass sheet and the low-temperature phase transition of B7-S5, the rechargeable glass had the potential for use as a photochromic solar thermal energy storage window in daily life, especially in cold winters. As a proof-of-principle study, the rechargeable glass sheets were installed on a miniature house model as windows (size 10 by 12 mm and coating thickness $400 mm). The house model was placed in À5 C surroundings to ensure that the surface temperature of the windows was around À1 C. As shown in Fig. 5e, upon exposure to 400 nm blue light (40 mW cm À2 ), the window stored visible light energy and low-temperature ambient heat while transforming from opacity to semitransparency. Then, by triggering it with 532 nm green light (110 mW cm À2 ), the stored energy was rapidly released on demand as high-temperature heat.</p><p>A high-resolution infrared thermal imaging camera was used to track the temperature changes of the window when exposed to 400 nm (40 mW cm À2 ) and 532 nm light (110 mW cm À2 ) (Fig. 5f, S19 and Videos S2-S5 †). During the 400 nm light irradiation (charging process), the window exhibited a temperature difference of about 3 C above the ambient temperature, indicating a weak photothermal effect. The charged window reached 21.7 C at 60 s during the 532 nm light irradiation (discharging process), about 22.7 C higher than the cold surroundings. A control experiment of irradiating the discharged window with 532 nm light showed a much lower temperature change (9.3 C), which means that the temperature change between the charged window and environment was mainly due to cis-trans isomerization. Assuming that the cis-trans isomerization was fully completed at approximately 60 s, the corresponding thermal power output was estimated to be 256.2 W m À2 . Such high-temperature heat release also means that the B7-S5 molecules on the surface of the window can act as a photon-driven molecular heat pump, upgrading thermal energy from low to high temperature. Furthermore, the optically controlled heat release makes it possible to reach about an order of magnitude higher temperature gradients than is possible with traditional MOST window coating concepts. 32 These solar thermal energy storage coatings show unprecedented performances, including visible-light trigger/storage, high energy density, and recyclable ice-cold charging/discharging, thus holding great promise for future energy management systems.</p><!><p>In conclusion, we have successfully designed a series of bidirectional visible-light switching azo molecules and applied them as MOST-PCMs for storing and releasing solar energy below 0 C. The molecular design strategies are summarized as follows: (i) the 4-thioalkyl substituent on azo molecules shis the p-p* absorption bands to long-wavelength, enabling bidirectional visible light photoisomerization; (ii) replacing one phenyl ring on the azo molecules with a pyrazole ring increases the half-life of the metastable cis-isomer; (iii) varying the length of thioalkyl chains changes the intermolecular forces, which could adjust the T m of both trans and cis isomers. Eventually, reversible visiblelight-triggered trans-crystal 4 cis-liquid transitions are achieved below 0 C. Accordingly, the azo molecules can simultaneously store visible-light energy and low-temperature ambient heat to achieve a high energy density (0.25 MJ kg À1 ).</p><p>Moreover, a rechargeable coating is prepared by drop-coating a solution containing azo photoswitches and modied chain-like silica on the glass surface. The coating shows potential as energy storage windows due to optical transmittance in the charged state, but it is also clear that more work is needed to increase the optical transmittance of the material. Future studies could focus on red-shiing l max of azo molecules to the near-infrared region to fabricate efficient semitransparent energy storage windows. Other possible application areas are functional coatings and fabrics with controllable heat release functions. We note that the structure-property relations derived from the chemical design provide a blue-print for how to design future MOST-PCM systems with tailored temperature functions and optimised optical properties. We envision that this work can open an avenue for the design of advanced MOST-PCM systems that store natural sunlight and ambient heat over a wide temperature range.</p><!><p>The authors declare no competing interests.</p>

Royal Society of Chemistry (RSC)

THE EFFECTS OF THE 5-HT3 RECEPTOR ANTAGONIST TROPISETRON ON COCAINE-INDUCED CONDITIONED TASTE AVERSIONS

Although cocaine readily induces taste aversions, little is known about the mechanisms underlying this effect. Recent work has shown that cocaine\xe2\x80\x99s actions on serotonin (5-HT) may be involved. To address this possibility, the present experiments examined a role of the specific 5-HT receptor, 5-HT3, in this effect given that it is implicated in a variety of behavioral effects of cocaine. This series of investigations first assessed the aversive effects of the 5-HT3 receptor antagonist tropisetron alone (Experiment 1). Specifically, in Experiment 1 male Sprague-Dawley rats were exposed to tropisetron (0, 0.056, 0.18 and 0.56 mg/kg) prior to the pairing of a novel saccharin solution. Following this, a non-aversion-inducing dose of tropisetron (0.18 mg/kg) was assessed for its ability to block aversions induced by a range of doses of cocaine (Experiment 2). Specifically, in Experiment 2 animals were given access to a novel saccharin solution and then injected with tropisetron (0 or 0.18 mg/kg) followed by an injection of various doses of cocaine (0, 10, 18, and 32 mg/kg). Cocaine induced dose-dependent taste aversions that were not blocked by tropisetron, suggesting that cocaine\xe2\x80\x99s aversive effects are not mediated by 5-HT, at least at this specific receptor subtype. At the intermediate dose of cocaine, aversions appeared to be potentiated, suggesting 5-HT3 may play a limiting role in cocaine\xe2\x80\x99s aversive effects. These data are discussed in the context of previous examinations of the role of serotonin, dopamine, and norepinephrine in cocaine-induced aversions.

the_effects_of_the_5-ht3_receptor_antagonist_tropisetron_on_cocaine-induced_conditioned_taste_aversi

1. INTRODUCTION<!>2.1 Apparatus<!>2.2 Subjects<!>2.3 Drugs and Solutions<!>2.4.1 Habituation<!>2.4.2 Conditioning<!>3. EXPERIMENT 1<!>4.1 Conditioning<!>4.2.1 Free feeding access<!>4.2.3 Restricted access<!>4.2.4 Tropisetron feeding assessment<!>4.3 Statistical Analysis<!>5.1 Dose Response Assessment<!>5.2 Feeding Assessment<!>6. EXPERIMENT 2<!>7.1 Conditioning<!>7.2 Statistical Analysis<!>8.1 Antagonism Assessment<!>9. DISCUSSION

<p>Drugs of abuse, including cocaine, have been shown to have both rewarding (Kosten et al., 1997, Nomikos and Spyraki, 1988, Wise et al., 1992) and aversive (Ettenberg, 2004, Ferrari et al., 1991, Goudie et al., 1978) effects, and the balance of these effects is thought to contribute to their overall abuse potential (Hunt and Amit, 1987, Kohut and Riley, 2010, Riley et al., 2009). Understanding the neurochemical basis for these affective properties may be important in understanding the differences in individual vulnerability to drug abuse (Cunningham et al., 2009, Freeman et al., 2008, for a review see Riley, 2011).</p><p>In relation to cocaine, its action as a dopamine transporter (DAT) inhibitor appears to be largely involved in its rewarding effects (Chen et al., 2006, Ritz et al., 1987; for a review and discussion of other monoamine involvement, see Uhl et al., 2002). However, the mechanism underlying its aversive effects is less understood. Although relatively fewer reports have investigated cocaine's aversive effects, several have implicated its actions on DA (Freeman et al., 2005, Serafine et al., 2012a, Serafine et al., 2012b). For example, Freeman and colleagues demonstrated that cocaine and the selective DAT inhibitor GBR 12909 induce similar dose-dependent acquisition of conditioned taste aversions (CTA; see Freeman et al., 2005). Specifically, they reported that aversions induced by both compounds at the highest dose tested (50 mg/kg) were comparable in both the rate of acquisition and degree of the aversion. Recently, our laboratory has demonstrated an involvement of DA via the use of the cross-drug preexposure preparation (Serafine et al., 2012a). Specifically, animals exposed to GBR 12909 prior to aversion conditioning with cocaine displayed attenuated cocaine-induced aversions suggestive of adaptation or tolerance to some common stimulus properties, presumably DA related, during preexposure (De Beun et al., 1996, Gommans et al., 1998, Serafine and Riley, 2009). Further, Serafine and colleagues reported that the nonselective DA receptor antagonist haloperidol blocked the acquisition of cocaine-induced CTAs (Serafine et al., 2012a), directly implicating a role of DA in cocaine's aversive effects (see also Hunt et al., 1985).</p><p>Although DA is highly implicated in the aversive effects of cocaine, recent work has also shown that cocaine's actions on serotonin (5-HT) may also be involved. For example, Sora and colleagues (Sora et al., 1998, Sora et al. 2001) found that transgenic knock-out (KO) mice without the 5-HT transporter (SERT) displayed an increase in cocaine-induced conditioned place preferences. They interpreted this increase in SERT KO mice to be a function of the removal of the aversive effects of cocaine (presumably mediated via its action on 5-HT), allowing for a greater overall rewarding effect of cocaine (presumably mediated via its actions on DA; see also Uhl et al., 2002). According to this position, cocaine's actions on SERT limit the rewarding effects of reward and the removal of the transporter in the KO mice eliminates this aversive effect. In support of this suggestion, our laboratory has recently reported that preexposure to cocaine significantly attenuated aversions induced by fluoxetine, a selective 5-HT reuptake inhibitor (see Serafine and Riley, 2010). Given that such an attenuation is typically interpreted as a function of adaptation to some aversive stimulus property shared between both drugs, this attenuation suggests a role for 5-HT in aversions induced by the two compounds (since 5-HT levels are enhanced by SERT inhibition by both cocaine and fluoxetine). Interestingly, preexposure to fluoxetine had no effect on cocaine-induced aversions, suggesting that although the two drugs share stimulus properties, they are not identical (see Serafine and Riley, 2010)</p><p>Although 5-HT may mediate in part the aversive effects of cocaine, it is not known what specific receptor system is involved in this effect. Of the range of 5-HT receptor subtypes, a few (specifically, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C and 5-HT3) have been implicated in the behavioral effects of cocaine, including its affective (rewarding) properties (Harrison and Markou, 2001, Parsons et al., 1998, Kankaanpaa et al., 2002 respectively; see, Hayes and Greenshaw, 2011, Muller and Huston, 2006 for reviews). For example, the 5-HT3 receptor antagonist Y-25130 has been reported to attenuate cocaine's ability to lower intracranial self stimulation thresholds in rats (Kelley and Hodge, 2001; for similar attenuating effects of the 5-HT3 receptor antagonist MDL722 on cocaine-induced place preferences, see Kankaanpa et al., 2002; Suzuki et al., 1992). Interestingly, the 5HT3 receptor antagonist tropisetron has been reported to block place aversions induced by 1-phenylbiguanide (PBG, a 5HT3 receptor agonist; Higgins et al., 1993), suggesting that this receptor subtype may mediate the aversive effects of drugs acting on 5-HT receptors. Accordingly, Experiment 2 examined the ability of tropisetron to affect cocaine-induced taste aversions. If cocaine induces aversions that are mediated in part by its ability to increase 5-HT activity at 5-HT3 receptor subtypes, tropisetron should attenuate such effects. Prior to this assessment, the ability of tropisetron to induce aversions on its own was first examined (Experiment 1).</p><!><p>All subjects were individually housed in stainless-steel, hanging wire-mesh cages on the front of which graduated Nalgene tubes could be placed for fluid presentation. Subjects were maintained on a 12:12 light-dark cycle (lights on at 0800h) and at an ambient temperature of 23 °C. Except where noted, food and water were available ad libitum</p><!><p>The subjects were 96 experimentally naïve, male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, Indiana) approximately 75 days old and between 250 and 350 g at the start of the experiment. Procedures recommended by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996, 2003) and the Institutional Animal Care and Use Committee at American University were followed at all times. Animals were handled daily approximately 2 weeks prior to the initiation of the study to limit the effects of handling stress during conditioning and testing.</p><!><p>Tropisetron hydrochloride (synthesized at the Chemical Biology Research Branch of the National Institute on Drug Abuse) was prepared in saline at a concentration of 0.5 mg/ml and was subsequently filtered through a 0.2 μm filter to remove any contaminants and administered intraperitoneally (IP). Cocaine hydrochloride (generously provided by the National Institute on Drug Abuse) was dissolved in distilled water at a concentration of 10 mg/ml and was subsequently filtered through a 0.2 μm filter to remove any contaminants and administered subcutaneously (SC). Cocaine doses are expressed as the salt. Saccharin (sodium saccharin, Sigma) was prepared as a 1 g/l (0.1%) solution in tap water.</p><!><p>Following 24-h water deprivation, subjects were given 20-min access to tap water daily. This daily access was repeated until consumption stabilized, i.e., subjects approached and drank from the tube within 2 S of its presentation and water consumption was within 2 ml of the previous day for a minimum of 4 consecutive days with no consistent increase or decrease. Throughout the study, fluid was presented in graduated 50-ml Nalgene tubes and measured to the nearest 0.5 ml by subtracting the difference between the pre- and post-consumption volumes.</p><!><p>On Day 1 of conditioning, all subjects were given 20-min access to the novel saccharin solution. Immediately following this presentation, animals in each experiment were rank ordered based on saccharin consumption and assigned to treatment groups (n = 7 - 8 per group) such that overall consumption was comparable among groups. Immediately after rank ordering animals were injected with tropisetron or vehicle (Experiment 1) or tropisetron or vehicle followed by an injection of cocaine (0, 10, 18, 32 mg/kg) 30 min later (Experiment 2).</p><!><p>Although administration of tropisetron and other 5-HT3 receptor antagonists alone typically does not result in any observable effects (Hendrie, 1990), when using pharmacological antagonists to assess mechanism in the CTA design, it is important to consider the possibility that administration of the antagonist prior to saccharin and cocaine could impact aversion learning independent of its effects of cocaine, e.g., by affecting taste sensitivity or drinking in general. One way to circumvent this issue is to administer the antagonist after saccharin consumption but prior to cocaine (Bienkowski et al., 1997, Freeman et al., 2008, Serafine et al., 2012b). Since many compounds when administered immediately after saccharin can (at least at some doses) induce CTAs on their own (for a discussion of this issue, see Freeman et al., 2008, Serafine et al., 2012b), it is important to determine a dose of the antagonist that does not induce a CTA alone prior to assessing its effect on cocaine-induced aversions. Accordingly, in Experiment 1 animals were given access to a novel saccharin solution and injected with one of a number of doses of tropisetron to assess its ability to induce aversions. Following conditioning, the effects of tropisetron on food consumption were monitored as a collateral assessment of any potential behavioral suppression induced by the antagonist.</p><!><p>During conditioning, 31 subjects were given access to saccharin and injected immediately thereafter with 0, 0.056, 0.18 or 0.56 mg/kg tropisetron, yielding Groups 0, 0.056, 0.18 and 0.56; specifically, Group 0 (n = 7), Group 0.056 (n = 8), Group 0.18 (n = 8), and Group 0.56 (n = 8). The vehicle group (Group 0) was matched in volume to the group receiving the high dose of tropisetron (Group 0.56). The specific doses (0.056 mg/kg, 0.18 mg/kg, and 0.56 mg/kg) used in this initial assessment were based on the doses of tropisetron used by Higgins and colleagues in their assessment of the effects of tropisetron on PBG-induced place aversions (see above, Higgins et al., 1993). The 3 days following this initial conditioning trial were water-recovery days during which animals were given 20-min access to tap water (no injections followed this access). This alternating procedure of conditioning/water recovery was repeated for a total of four complete cycles. Following the last water-recovery session after the fourth conditioning trial, animals were given 20-min access to both saccharin and tap water in a final two-bottle aversion test. Specifically, both the saccharin- and water-filled Nalgene tubes were placed on the cages simultaneously with the placement of the tubes (left or right side) counterbalanced across subjects to prevent positioning effects. No injections were administered after this test.</p><!><p>Following the two-bottle test, subjects were returned to ad libitum water access for 11 days during which no saccharin or injection was given. Food access was also ad libitum and was measured over the last 3 days of this period. The absolute amount of food consumed was determined by subtracting the amount of food remaining in the cage or fallen through the cage floor from the total food initially made available.</p><!><p>On the next day, 50% of the average daily amount of food consumed during free access was made available for 23 h (the calculation and preparation of the food took approximately 1 h during which time subjects had no access to food or water). At the conclusion of this 23-h period, any food which had fallen through the cage floor was measured and subtracted from the total food initially made available (on 23-h 50% restricted access sessions animals always consumed the entirety of the food and as such only the fallen food was measured). Subjects were then given 23 h of ad libitum access to food. Consumption was measured after 2 h and then again at the end of the end of the 23-hr ad libitum access period. This alternating schedule of 23-h 50% restricted access followed by 23-h ad libitum access period (with consumption measured at 2 and 23-h after food presentation) was repeated a total of three times.</p><!><p>Following the last cycle of the above phase, subjects again were given 23-h of restricted access followed by 23-h access to free feeding. Immediately prior to free-food access subjects were given a Baseline Session in which they were given an IP injection of vehicle (matched in volume to the highest dose of tropisetron, 0.56 mg/kg). They were given the 23-h ad libitum access period (with consumption measured at 2 and 23-h, as previously described). Following the 5th 50% restricted access day, subjects were given a Test Session where they were given an IP injection of either vehicle or tropisetron 30-min prior to the ad libitum access to food. The dose administered matched the dose given during conditioning (0, 0.056, 0.18, and 0.56 mg/kg) for individual subjects. Consumption was measured 2 h following food presentation.</p><!><p>The differences in mean saccharin consumption during conditioning were analyzed using a 4 x 4 mixed model ANOVA with the between-subjects factor of Group (0, 0.056, 0.18 and 0.56 mg/kg) and the within-subjects factor of Trial (1–4). Trial differences were analyzed using a paired samples t-test with a Bonferroni correction setting the significance to p ≤0.0083. The differences in percent saccharin consumed and total fluid consumed during the two-bottle test were analyzed using a one-way ANOVA with the between-subjects variable of Group (0, 0.056, 0.18 and 0.56 mg/kg). A 4 x 2 mixed model ANOVA with a between-subjects factor of Group (0, 0.056, 0.18 and 0.56 mg/kg) and a within-subjects factor of Session (Baseline or Test) was used to analyze the 2-h access food consumption following the vehicle or drug injection. Where appropriate, Tukey post hoc analysis was used to determine specific group differences. All significance levels were set at p ≤0.05 unless otherwise noted.</p><!><p>The 4 x 4 mixed model ANOVA on saccharin consumption during conditioning revealed a significant effect of Trial [F (3, 81) = 41.949, p < 0.001]. Regarding the Trial effect, paired samples t-tests revealed Trial 1 consumption was significantly less than all other trials (Trial 2, Trial 3, Trial 4, all ps < 0.001). There was no effect of Group and no significant Trial X Group interaction (see Figure 1). Specifically, all groups consumed saccharin at high levels and significantly increased consumption over repeated trials.</p><p>Analysis of the percent saccharin consumed on the final two-bottle aversion test revealed that subjects injected with the high dose of tropisetron (Group 0.56) consumed significantly less percent saccharin than all other subjects (Groups 0, 0.18, and 0.056; all ps < 0.05), indicating that the high dose (0.056 mg/kg) induced a significant CTA relative to vehicle and the two lower doses of tropisetron. There were no significant differences in total fluid consumed among groups (data not shown).</p><!><p>The 4 x 2 mixed model ANOVA revealed a significant effect of Session [F (1, 27) = 7.719, p = 0.010]. In relation to the Session effect, overall food intake increased from Baseline to Test. Specifically, average food intake was approximately 6.8 and 7.6 g on the Baseline and Test, respectively. There was no effect of Group and no significant Session X Group interaction.</p><!><p>In Experiment 1, various doses of tropisetron (0, 0.056, 0.18, and 0.56 mg/kg) were examined to establish a dose of the antagonist that could be administered following saccharin consumption (and prior to cocaine) without the likelihood of inducing an aversion on its own which might confound any interpretation of its antagonist effects on cocaine. As noted, only the highest dose (0.56 mg/kg) tested induced a CTA and even here this was evident only after repeated conditioning trials and in the relatively sensitive two-bottle test (for an overview of the relative sensitivy of the one- vs two-bottle aversion test, see Batsell and Best, 1993, Spector et al., 1981). This general lack of an effect of tropisetron is supported by its failure to affect food consumption at any dose and consistent findings that 5-HT3 receptor antagonists rarely produce effects on their own and that their activity appears inert except at toxic doses (Hendrie, 1990). Given that there was no difference among doses during the feeding assessment and that previous research has shown tropisetron's ability (at 0.1 mg/kg) to block place aversions induced by the 5-HT3 receptor agonist PBG (Higgins et al., 1993), 0.18 mg/kg was used in the following assessment of the effects of tropisetron on cocaine-induced CTAs.</p><!><p>Subjects in Experiment 2 were run in two replicates (n = 32: Replicate 1; n = 32: Replicate 2). During conditioning, subjects were given a novel saccharin solution to drink followed by an injection of 0.18 mg/kg tropisetron (based on the results from Experiment 1). Thirty min following this injection, different groups of subjects were given a SC injection of cocaine (10, 18 or 32 mg/kg) or vehicle (matched in volume to 32 mg/kg cocaine), yielding eight experimental groups, specifically, vehicle-vehicle (V0; n = 8), vehicle-10 mg/kg cocaine (V10; n = 8), vehicle-18 mg/kg cocaine (V18; n = 8), vehicle-32 mg/kg cocaine (V32; n = 8), tropisetron-vehicle (T0; n = 8), tropisetron-10 mg/kg cocaine (T10; n = 8), tropisetron-18 mg/kg cocaine (T18; n = 8) and tropisetron-32 mg/kg cocaine (T32; n = 8). The first letter in each group designation refers to the pretreatment drug (Tropisetron, T, or Vehicle, V); the number refers to the dose of cocaine given during conditioning. The specific doses of cocaine used in this assessment were based on previous work reporting the ability of these doses to produce graded aversions that ranged from little to intermediate to near complete suppression (see Ferrari et al., 1991, Freeman et al., 2005). Such a dose range provides behavioral effects that are subject to modulation and allow for attenuation or potentiation of aversions to be seen. The 3 days following this initial saccharin presentation were water-recovery days, during which animals were given 20-min access to tap water (no injections followed this access). This alternating procedure of conditioning and water recovery was repeated for a total of four complete cycles. Following the last water-recovery session after the fourth conditioning trial, animals were given 20-min access to saccharin and water in a final two-bottle aversion test (as described in Experiment 1).</p><!><p>The differences in mean saccharin consumption during conditioning were analyzed using a 2 x 2 x 4 x 4 mixed model ANOVA with the between subjects factors of Pretreatment Drug (tropisetron or vehicle), Replicate (1 or 2) and Conditioning Dose (0, 10, 18 and 32 mg/kg cocaine) and the within subjects factor of Trial (1–4). The differences in percent saccharin consumed and total fluid consumed during the two-bottle test were analyzed using a 2 x 4 univariate ANOVA with the between-subjects variables of Pretreatment Drug (Vehicle or Tropisetron) and Conditioning Dose (0, 10, 18 and 32 mg/kg cocaine). Where appropriate, Tukey post hoc analysis was used to determine specific group differences. All significance levels were set at p ≤0.05 unless otherwise noted.</p><!><p>The 2 x 2 x 4 x 4 mixed model ANOVA revealed no significant effect of Replicate. Consequently, the data across the two replicates were pooled for presentation. The mixed model ANOVA did reveal a significant effect of Trial [F (3, 144) = 4.180, p = 0.007] and Conditioning Dose [F (3, 48) = 16.453, p < 0.001] but no significant effect of Pretreatment Drug [F (1, 48) = 1.097, p = 0.3]. There was a significant a significant Trial X Conditioning Dose interaction [F (9, 144) = 25.527, p < 0.001]. In relation to the significant Trial X Conditioning Dose interaction, Tukey post hoc tests, collapsed across Pretreatment Drug, revealed no significant differences between Conditioning Dose on Trial 1. On Trial 2, subjects, injected with 32 mg/kg cocaine differed from all other groups (all ps < 0.024). On Trials 3 and 4, subjects injected with 32 mg/kg cocaine again differed from all other groups (all ps < 0.01) and subjects injected with 18 mg/kg and 10 mg/kg differed from vehicle (both ps < 0.011) (see Figure 3).</p><p>Analysis of the percent saccharin consumed on the final two-bottle aversion test revealed no significant effect of Pretreatment Drug [F (1, 56) = 0.198, p = 0.658] or Pretreatment Drug X Conditioning Dose [F (3, 56) = 2.505, p = 0.068] interaction. There was a significant effect of Conditioning Dose [F (3, 56) = 32.517, p < 0.001]. In relation to the significant effects of Conditioning Dose, Tukey post hoc tests, collapsed across Pretreatment Drug, revealed that all drug-injected subjects drank significantly less than subjects injected with vehicle (0 mg/kg) (all ps < 0.001). Subjects injected with 32 mg/kg drank significantly less than subjects injected with 10 mg/kg (p = 0.030) (see Figure 4). There were no significant differences in total fluid consumed among groups (data not shown).</p><!><p>In Experiment 2, the role of 5-HT3 receptor antagonism on cocaine's aversive effects was examined. Specifically, animals were treated with the 5HT3 receptor antagonist tropisetron immediately prior to aversion conditioning with cocaine. As described, cocaine induced dose-dependent aversions that were not significantly affected by pretreatment with tropisetron. The failure of tropisetron to impact aversions induced by cocaine may be a function of a number of factors including the dose administered during pretreatment as well as the pretreatment interval. However, as noted above, Higgins and his colleagues have reported that tropisetron did attenuate place aversions induced by the 5HT3 agonist PBG at a dose (0.1 mg/kg) and a pretreatment time comparable (30 min) to those used in the present assessment (see Higgins et al., 1993). Its failure to affect cocaine in the CTA preparation is unlikely a function of these specific parametric conditions.</p><p>That tropisetron did not attenuate cocaine-induced aversions suggests instead that the 5HT3 receptor subtype does not mediate the aversive effects of cocaine. Although the 5-HT3 receptor subtype may play no role in cocaine-induced aversions, several other reports support a role for 5-HT in general in cocaine's aversive effects. As previously described, Sora and colleagues reported KO mice with a SERT deletion displayed an increase in cocaine-induced conditioned place preferences (Sora et al., 1998), an effect interpreted as being a function of the reduction in cocaine's aversive effects normally mediated by 5-HT (see Uhl et al., 2002 for a summary of the relative roles and interactions of DA, 5-HT and NE in the rewarding and aversive effects of cocaine). This interpretation is supported in part by the fact that KO mice with the SERT deletion display attenuated cocaine-induced CTAs relative to wild-type (Jones et al., 2010). Consistent with these findings, Serafine and Riley (2010) have recently reported that preexposure to cocaine significantly attenuated aversions induced by fluoxetine, suggesting some adaptation or tolerance to their common aversive effects. Interestingly, preexposure to fluoxetine had no effect on cocaine-induced aversions, suggesting that although the two drugs share aversive stimulus properties (enhanced 5-HT levels by SERT inhibition through both cocaine and fluoxetine administration), these effects are not identical (Serafine and Riley, 2010). The fact that 5-HT in general appears to be involved in cocaine's aversive effects, whereas 5-HT3 receptor antagonism was without effect may simply argue that other 5-HT receptor subtypes may be involved.</p><p>Although tropisetron had no antagonist effect on cocaine-induced aversions, aversions induced by the low dose of cocaine (10 mg/kg) appeared potentiated (both during the acquisition of the aversion as well as on the two-bottle test). For example, on the two-bottle test five of the eight subjects in the tropisetron-pretreated group drank 0 ml of saccharin, whereas none of the vehicle pretreated animals displayed this level of near complete suppression of consumption (see Figure 5). In fact, seven of the eight tropisetron pretreated subjects drank less than all but two of the animals pretreated with vehicle. The ability of tropisetron to impact aversions induced by cocaine is unlikely a function of the combined aversive effects of tropisetron and cocaine. While in Experiment 1, the high dose of tropisetron (0.56 mg/kg) was aversive as assessed in the two-bottle aversion test, the dose of tropisetron chosen for Experiment 2 (0.18 mg/kg) did not induce an aversion on its own after multiple conditioning trials or in the two-bottle assessment. These results suggest that activity at 5-HT3 receptors might actually limit cocaine's overall aversive effects and antagonism at these receptors potentiate the aversive effects of cocaine. It is interesting in this context that Higgins and colleagues reported tropisetron attenuated morphine-induced place preference and argued that 5-HT3 antagonism limits morphine reward by potentiating its aversive effects (Higgins et al., 1993), an effect supported by Bienkowski and colleagues who reported that tropisetron (0.1 mg/kg, SC) potentiates morphine-induced taste aversions (Bienkowski et al., 1997).</p><p>The finding that 5-HT3 receptor antagonism may potentiate cocaine's aversive effects parallels recent work with NE and its possible role in cocaine's aversive effects. For example, while NET KO mice display attenuated aversions (Jones et al., 2010) and preexposure to desipramine weakens (Serafine and Riley, 2009) aversions induced by cocaine, the NE antagonists prazosin and propranolol potentiate cocaine-induced aversions (Freeman et al., 2008). Further, preexposure to cocaine potentiates the aversions induced by the NET inhibitor desipramine (Serafine and Riley, 2009). Both of these findings suggest that under specific conditions NE activity may limit cocaine's aversive effects (for a review, see Serafine and Riley, In Press).</p><p>Although the current experiment provides no evidence that 5-HT3 receptor activation is involved in cocaine's aversive effects, the role of 5-HT remains unknown. Prior work with cocaine indicates that its aversive effects are complex and multifaceted, mediated primarily by DA and modulated in part by NE and 5-HT activity. Further examination of the brain amines and their receptors will allow for a more comprehensive understanding of the mechanism underlying cocaine's aversive effects. Given that drug use is thought to be a function of the balance of the rewarding and aversive effects of a drug, understanding these aversive effects and the factors that modulate them may lead to better prevention and treatment of cocaine abuse.</p>

PubMed Author Manuscript

Tandem Catalysis: Transforming Alcohols to Alkenes by Oxidative Dehydroxymethylation

We report a Rh-catalyst for accessing olefins from primary alcohols by a C\xe2\x80\x93C bond cleavage that results in dehomologation. This functional group interconversion proceeds by an oxidation-dehydroformylation enabled by N,N-dimethylacrylamide as a sacrificial acceptor of hydrogen gas. Alcohols with diverse functionality and structure undergo oxidative dehydroxymethylation to access the corresponding olefins. Our catalyst protocol enables a two-step semisynthesis of (+)-yohimbenone and dehomologation of feedstock olefins.

tandem_catalysis:_transforming_alcohols_to_alkenes_by_oxidative_dehydroxymethylation

<p>Enzymes perform one-carbon dehomologations of alcohols via the intermediacy of an aldehyde. For example, DNA demethylases oxidize alcohols to aldehyde intermediates that are decarbonylated to generate alkanes and arenes.1 Lanosterol demethylase performs a tandem oxidation and dehydroformylation to generate alkenes (Figure 1a). In contrast, while dehomologation of alcohols to generate alkanes has been achieved with various homogeneous catalysts,2 initial efforts to convert alcohols into olefins used heterogeneous catalysis and resulted in side reactions, including dehydration, olefin isomerization, and cracking, due to high reaction temperatures (>380 °C).3c Inventing ways to access olefins remains a primary focus due to their versatility as building blocks for materials and medicines.4 To achieve a mild, selective, and more general alcohol to alkene transformation, we thus focused on developing a bioinspired cascade.</p><p>Our laboratory reported a dehomologation that transforms aldehydes into olefins via transfer of the formyl group and hydride onto a strained olefin acceptor, such as norbornadiene.5a Morandi coined such processes shuttle catalysis.6 Nozaki and Sorensen reported complementary dehydroformylations, through Ir-catalysis or photocatalysis, respectively (Figure 1b).5b, 3d In Sorensen's study, he illustrated one oxidative dehydroxymethylation of a neopentylic alcohol, although a mixture of products was observed.3d Given precedence for both transfer hydrogenation7,8 and transfer dehydroformylation,5a we focused on the use of tandem Rh-catalysis to achieve a more general alcohol dehomologation to alkenes (Figure 1c).9</p><p>We set out to identify one catalyst capable of both transfer hydrogenation and transfer hydroformylation.10 Using 1-dodecanol 1a as a model substrate, we began our studies with a catalyst known to activate aldehyde C–H bonds ([Rh(cod)OMe]2, 3-OMeBzOH, and Xantphos, Table 1).5a Upon successful oxidation of alcohol 1a, we imagined the resulting aldehyde could undergo dehydroformylation to the alkene 2a or decarbonylation to the alkane 3a. From an initial survey, we discovered that selectivity for alkene vs alkane was influenced by the acceptor. In the absence of an acceptor, we observed undecane 3a as the only product (10% yield). In stark contrast, by using strained olefin acceptors A1 and A2, we observed 1-undecene (2a, 32% and 18% respectively), along with undecene isomers (iso-2a, 16:1 and 2.3:1, 2a:iso-2a). Using ketones as acceptors (A3–4) resulted in decarbonylation to undecane 3a. While using electron-deficient olefin acceptors, such as enone A5 or acrylonitrile A6, a mixture of 1-undecene 2a and undecane 3a was observed (1.4:1 and 1:3, 2a:3a). Using unsaturated ester or amide acceptors provided a major breakthrough in selectivity for the desired alkene 2a.</p><p>Unsaturated ester and amide acceptors (A7–A8) enabled selective formation of 1-undecene (2a, 33–35%, >20–17.5:1, 2a:3a). Use of N,N-dimethylacrylamide (DMAA) as an acceptor gave 1-undecene 2a in 95% yield and >20:1 selectivity.11 We reason DMAA affords improved reactivity because it can bind more effectively to the Rh-catalyst in comparison to other Michael acceptors (A5–A8, A10). We found that the byproduct was N,N-dimethylpropionamide, which arises from the hydrogenation of DMAA. The use of N-vinylpyrrolidone (A9) or the α-methyl substituted acrylamide A10 resulted in diminished reactivity (3–5%). Previously, we found that both CO and H2 were transferred to our strained olefin acceptor, norbornadiene A1.5a In contrast, we do not observe transfer hydroformylation, yet catalyst turnover still proceeds in the presence of CO generation, as quantified by GC-thermal conductivity detection (see SI).12</p><p>With this catalyst-acceptor combination, we performed the dehomologation of primary alcohols (Table 2a). Allylbenzene 2b was obtained (93% yield) from 4-phenyl-1-butanol, without isomerization to a conjugated olefin. 3-Phenyl-1-propanol and derivatives with electron-donating and electron-withdrawing groups gave styrenes (2c–e) in 85–93% yields. Heterocyclic alcohols, such as those with pyridine and indole, were tolerated (2f, 85%; 2g, 77%). A primary diol gave diene 2h in 88% yield, in the presence of double the amount of DMAA (6 equivalents). A β,β-disubstituted alcohol transformed to internal olefin 2i in 91% yield. Alcohols bearing alkenes and tertiary alcohols underwent dehomologation (2j, 82%; 2k, 87%). Next, we explored 1,3- or 1,4-diols and 2-, 3- or 4-amino derived alcohols (2l–s). Allylic ether 2l and amine 2o were obtained in 81% and 92% yields respectively, without allylic C–O or C–N bond cleavage or debenzylation. Enol and enamine derivatives (2m, 2n, 2p–s) can be accessed (75–83% yields). Enamine formation occurred preferentially over allyl amine formation to afford 2q (80% yield). We obtained tri-substituted enamide 2r in 75% yield from alcohol 1r. With most alcohols, excellent chemoselectivities (>20:1) were observed. In contrast, use of 3-phthalimido-1-propanol gave a 4:1 mixture of oxidation-dehydroformylation (2s) and oxidation-decarbonylation (3s). When cis- or trans-1t was used, β-hydride elimination occurred preferentially at the less substituted position to give 2t1. In addition, we found that allylic alcohols (4a-c) underwent oxidative dehydroxymethylation (75–95% yields), with only 1.5 equivalents of DMAA needed (Table 2b).13</p><p>Next, we explored applications (Scheme 1). By combining hydroboration-oxidation with oxidative dehydroxymethylation, a one-carbon dehomologation of 1-dodecene 6 was achieved on gram scale to give 1-undecene 2a (82% yield, Scheme 1a). This two-step process provides valuable odd-numbered carbon olefins from readily available deven-numbered carbon olefins.14c A two-carbon dehomologation of olefins can be achieved by combining olefin dihydroxylation and oxidative dehydroxymethylation. For example, we found that 1-dodecene 6 could be transformed to 1-decene 2v (Scheme 1a).15 The transformation occurs efficiently with molecules that are more structurally complex (Scheme 1b). Benzyl protected deoxycholic acid derivative 8a gave olefin 9a (81% yield), with no debenzylation. We probed chemoselectivity by using triol 8b, with alcohols bearing different steric bulk. We observed oxidation-dehydroformylation of the primary alcohol and selective oxidation of the less hindered secondary alcohol to afford 9b (66% yield). Diol 8c underwent oxidative dehydroxymethylation and secondary alcohol oxidation to access (+)-yohimbenone 9c. Based on this result, we improved our previous synthesis of (+)-yohimbenone 9c by shortening the sequence to two steps.5a, 16</p><p>While further studies are warranted, on the basis of literature reports2,5,6,11,17 and our own observations, we propose the following pathway (Scheme 3). Exchange between the benzoate counterion in Rh-complex A and an alcohol affords B. Intermediate B undergoes β-hydride elimination to give Rh-hydride C. Coordination of DMAA to C generates intermediate D. Hydrometallation of DMAA followed by protodemetalation provides the aldehyde and regenerates complex A. Oxidative addition into the aldehyde C–H bond by A generates acyl-Rh-hydride F. Reductive elimination of 3-methoxybenzoic acid generates acyl-Rh G. CO deinsertion to H, followed by β-hydride elimination, yields Rh-hydrido-carbonyl I. Olefin exchange with DMAA generates Rh-hydride J. Hydrometallation of DMAA gives complex K, and CO is extruded to make L. Finally, protodemetalation regenerates complex A.</p><p>To support the proposed mechanism, control experiments and deuterium-labeling experiments were carried out. Under standard conditions, neopentylic alcohol 1w oxidizes to aldehyde 10 in 90% yield (Scheme 4a). Incorporation of a quaternary carbon alpha to the carbonyl suppressed dehydroformylation. These results support the intermediacy of an aldehyde in the catalytic cycle. Of note, aldehyde 11 undergoes dehydroformylation under standard conditions (Scheme 4b), showing similar reactivity to our previous report,5a but with a more economical acceptor (i.e., norbornadiene vs DMAA). Replacing the benzoate counterion with chloride suppressed both oxidation and dehydroformylation (Scheme 4a and b). In the absence of DMAA, dehydrogenation of alcohol 1w was not observed (Scheme 4a). In contrast, decarbonylation of aldehyde 11 gave ethyl benzene 3c (78% yield, 5.5:1 3c:2c, Scheme 4b). These observations highlight the importance of both the benzoate counterion and DMAA. In support of the protonation of intermediate E (Scheme 3), we observed deuterium incorporation at the β-position of DMAA when using deuterated isopropanol D-12 (Scheme 4c). Hydrogen-deuterium exchange is possible during dehydroformylation via the benzoate counterion acting as a proton shuttle (Scheme 4c and d).5a</p><p>Using competition experiments, we studied the chemoselectivity of this cascade (Scheme 2, see SI for details). Aldehydes undergo dehydroformylation in preference to primary alcohols undergoing oxidative dehydroxymethylation, with 60:1 selectivity. Primary alcohols oxidize faster than secondary and benzylic alcohols faster than aliphatic. These observations support that alcohol oxidation is the turnover limiting cycle in this novel cascade.</p><p>Established strategies for constructing olefins, including the Wittig olefination,18 the Heck reaction,19 and olefin metathesis,20 generate carbon-carbon bonds. In contrast, our strategy contributes to emerging routes to olefins that involve C–C bond cleavage.21 These methods represent examples of a one-carbon dehomologation of carbon frameworks and thus hold promise for various applications, including the conversion of biomass into feedstocks.22 Moreover, such transformations increase retrosynthetic flexibility by allowing the interconversion of two common functional groups.14</p>

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Ascorbate oxidation by iron, copper and reactive oxygen species: review, model development, and derivation of key rate constants

Ascorbic acid is among the most abundant antioxidants in the lung, where it likely plays a key role in the mechanism by which particulate air pollution initiates a biological response. Because ascorbic acid is a highly redox active species, it engages in a far more complex web of reactions than a typical organic molecule, reacting with oxidants such as the hydroxyl radical as well as redox-active transition metals such as iron and copper. The literature provides a solid outline for this chemistry, but there are large disagreements about mechanisms, stoichiometries and reaction rates, particularly for the transition metal reactions. Here we synthesize the literature, develop a chemical kinetics model, and use seven sets of laboratory measurements to constrain mechanisms for the iron and copper reactions and derive key rate constants. We find that micromolar concentrations of iron(III) and copper(II) are more important sinks for ascorbic acid (both AH 2 and AH − ) than reactive oxygen species. The iron and copper reactions are catalytic rather than redox reactions, and have unit stoichiometries: Fe(III)/ Cu(II) + AH 2 /AH − + O 2 → Fe(III)/Cu(II) + H 2 O 2 + products. Rate constants are 5.7 × 10 4 and 4.7 × 10 4 M −2 s −1 for Fe(III) + AH 2 /AH − and 7.7 × 10 4 and 2.8 × 10 6 M −2 s −1 for Cu(II) + AH 2 /AH − , respectively.

ascorbate_oxidation_by_iron,_copper_and_reactive_oxygen_species:_review,_model_development,_and_deri

<!>Ascorbic acid chemistry review<!>.−<!>⋅<!>ROS reactions<!>9<!>Transition metal reactions. Catalytic or redox?<!>Reactions of the radical anion.<!>Other model uncertainties.<!>Methods<!>Online measurements of Fe(II), Fe(III) and Cu(II).<!>Results and discussion<!>Ascorbate oxidation via the catalytic, redox or OH<!>⋅−<!>Fe(II) + O 2 .<!>Implications and conclusions

<p>Ascorbic acid is of great interest in food, where it is both an essential vitamin and a natural preservative. Ascorbic acid is also vital for plants. It not only plays a role in photosynthesis, cell growth and signal transduction, but also helps defend from oxidative stress as the most abundant water-soluble antioxidant in plants [1][2][3][4] . Because of its importance for food and in plants, food chemists and botanists have performed the vast majority of studies of ascorbic acid oxidation chemistry [1][2][3][4][5][6][7] .</p><p>In mammalian systems, ascorbic acid is a common and important molecule with roles in metabolic function, oxidative stress responses and immune system maintenance taking place in epithelial lung lining fluid and other areas in the body 8,9 .</p><p>In an air pollution context, inhaled particulate matter, a highly complex and variable mixture of inorganic and organic compounds, encounters the lung lining fluid containing substantial concentrations of ascorbic acid. Growing evidence indicates that transition metals in inhaled particles are particularly active components capable of inducing a wide range of negative health effects including myocardial infarction, adverse birth outcomes and respiratory illnesses [10][11][12] . A leading hypothesis for how airborne particles induce health effects is via oxidative stress, and redox-active transition metals such as iron and copper have been heavily implicated in the ability of particles to generate reactive oxygen species and therefore potentially contribute to aerosol toxicity 13,14 . For example, soluble iron and copper in synthetic lung fluid correlate with the formation of reactive oxygen species OH ⋅ and H 2 O 2 15, 16 . Because ascorbic acid is a key antioxidant in lung lining fluid, ascorbic acid consumption is one of the assays used by atmospheric chemists to quantify aerosol oxidative potential 17 ; aerosol oxidative potential is proving to be better at predicting adverse health outcomes than particle mass 18 . Ascorbate/ ascorbic acid consumption has been observed to be positively correlated with total iron and copper concentrations in ambient aerosol 14 .</p><p>Ascorbic acid can have both pro-and anti-oxidant roles, and it reacts with reactive oxygen species (ROS) and transition metals. Ascorbic acid can be readily oxidized by undergoing a one-or two-electron transfer, terminating the free radical-mediated chain reactions in foods and tissue, reducing lipid peroxidation and deterioration of foods 6 . The autoxidation of ascorbic acid by oxygen in the presence of transition metals, especially cupric (Cu(II)) and ferric (Fe(III)) ions accounts for the majority of loss of this ascorbic acid activity in food. Despite its role as an efficient antioxidant, ascorbic acid can also accelerate oxidative deterioration of flavor and color in food through Fenton-type radical reactions 6,19 . This pro-oxidant effect occurs when transition metal ions are present, and the level of available ascorbic acid is relatively low and not sufficient to scavenge the radicals formed by Fenton-type reactions. In both food and physiological conditions, the key loss pathways for ascorbic acid are via ROS and transition metals, especially Fe(III) and Cu(II). However, for the transition metal reactions, the stoichiometries, mechanisms and rate constants are all very uncertain. Further, while the ROS ascorbic acid reactions are reasonably well understood from a mechanistic standpoint, the range of rate constants in the literature for these reactions spans about a factor of 15 [20][21][22][23][24][25] .</p><p>Here we develop a model in the Kinetics Preprocessor (KPP) 26 environment based on available ascorbic acid chemistry with ROS and free iron and copper chemistry from the literature. The model is validated against measurements of the formation of dehydroascorbic acid (DHA), the main oxidation product of ascorbic acid in the presence of micromolar concentrations of Fe(II), Fe(III) and Cu(II) at pH 2.8 and 7.0. The measurements at pH 2.8 were made to develop an online measurement of ascorbic acid consumption by ambient particulate matter 17 , and allow us to probe the chemistry of ascorbic acid, AH 2 . Additional measurements were made at pH 7.0 to probe the reactions of the deprotonated form, AH − . We also use measurements of ascorbate loss and/or OH ⋅ formation from Lin and Yu 27 and Charrier and Anastasio 15 at ~ pH 7 to further constrain the model. We then use the model to constrain the mechanisms and derive rate constants for the catalytic reactions of Fe(III) and Cu(II) with both AH 2 and AH − in the presence of oxygen.</p><!><p>Here we use 'ascorbic acid' to mean the sum of the protonated form, AH 2 and the deprotonated form AH − , and the chemical formulas to indicate the individual species. pH dependence. Ascorbic acid reacts with several species of ROS, as well as the oxidized forms of several transition metals (Fig. 1). As ascorbic acid (AH 2 ) can readily lose a proton to form the ascorbate anion (AH − ), (pK a,1 = 4.1; pK a,2 = 11.8) both AH 2 and AH − play roles in chemistry at low and neutral pHs. Typically, AH 2 and Reactions with hydroxyl and hydroperoxyl radicals and superoxide. OH ⋅ reactions with both AH 2 and AH − (Table 1, R4 and R7) appear to proceed at close to diffusion-controlled collision rates. The rate constants for these reactions fall in the ranges (4.5-7.9) × 10 9 M −1 s −1 at pH 1-1.5 and (1-11) × 10 9 M −1 s −1 at pH 7-11 respectively (Supplementary Table S1). We adopt the rate constant of 7.9 × 10 9 M −1 s −1 from Redpath and Willson 21 for the oxidation of AH 2 by OH ⋅ and 1.1 × 10 10 M −1 s −1 from Buettner and Schafer 22 for AH − in our study, because the deprotonated ascorbic acid tends to react more rapidly than the protonated form.</p><p>Both AH 2 and AH − readily undergo one-electron oxidation by superoxide (O 2</p><!><p>), hydroperoxyl radical (HO 2</p><!><p>) and hydroxyl radical (OH ⋅ ) to form the ascorbate radical (A .− ) (R4-9, Table 1). The pK a of AH . is sufficiently low that the protonated radical (AH . ) can be ignored. The unpaired electron of A .− residing in the π-system makes A .− relatively unreactive 22 , however A .− can form DHA via disproportionation (R12,13, Table 25 and Cabelli and Bielski 24 . The experimental data disagree by a factor of 1.5-15, although they have the same shape, with a maximum in the observed rate at about pH 4.5. Because both AH 2 and the hydroperoxyl radical have similar pK a s (4.1 and 4.8 respectively), in the pH range 2-7, the contributions of R6 and R8 are difficult to separate, while at low and high pH R5 and R9 dominate, respectively. Because Nadezhdin and Dunford 25 neglected the AH 2 reactions (R5 and R6) in their discussion and their data span a smaller pH range, we use values based on the data and analysis by Cabelli and Bielski 24 . Cabelli and Bielski 24 conclude that it is not possible to deconvolute k 6 and k 8 , but the sum (0.356 k 6 + k 8 ) can be said to be equal to 1.22 × 10 7 M −1 s −1 . Because we use updated pK a s for ascorbic acid and HO 2 ⋅ (Tables 1 and 2) we adjust 24 , d Van der Zee and Van den Broek 36 , e Bielski et al. 37 , f Vislisel et al. 57 , g Dewhirst and Fry 47 , h Parsons et al. 45 , z Estimate numbers.</p><p>12d,e 2A 1). The rate constants for reactions of A ⋅− and HO 2 ⋅ and O 2 ⋅− were measured by Cabelli and Bielski 24 using radiolysis; using pH to select HO 2 ⋅ (pH = 1-3) or O 2 ⋅− (pH = 7.8-8), and were determined to be 5.0 × 10 9 M −1 s −1 and 2.6 × 10 8 M −1 s −1 , respectively.</p><!><p>Autooxidation. Ascorbic acid is an excellent electron-donor antioxidant. The relatively low reduction potential of ascorbate (0.19 V for DHA/AH − at pH 3.5) should allow it to be readily oxidized by molecular oxygen 5 . However, while this redox reaction is thermodynamically favorable, it is spin forbidden; molecular oxygen is a triplet with two unpaired electrons, while ascorbate is in the ground state 5 . The only ascorbate species that is capable of true autoxidation, determined after treating the solutions with Chelex resin to remove trace metals appears to be the ascorbate dianion (A 2− ) + O 2</p><!><p>. Because there is little A 2− at pHs below ~ 10 (Table 1 R2), the autoxidation rate for ascorbate (all forms) is slow, ~ 6 × 10 -7 s −1 at pH 7 5 . We verified this as part of our measurements (not shown).</p><!><p>Ascorbate reactions with iron and copper are central to metal-mediated antioxidant chemistry, and it is clear that ascorbate reacts overwhelmingly with the oxidized forms of the metals (Fe(III) and Cu(II)). The early studies of this reaction uniformly interpreted their data with catalytic mechanisms [28][29][30][31] such as:</p><p>) for Cu 2+ , CuOH + and CuSO 4 70 (q) for CuCl + and CuCl 2 78p</p><p>Other reactions specific to a subset of experiments , CuCl + and CuCl 2 . These forward and back reactions are written separately in the KPP input file. ∆ An upper limit is used for these reactions. i Gonzalez et al. 40 , j Miller et al. 58 , k Pham and Waite 51 , l De Laat and Le 38 , m Herrmann et al. 43 , n Stumm and Morgan 50 , o Powell et al. 42 , p Deguillaume et al. 41 , q Wang et al. 59 , r Lee et al. 60 , s Pham et al. 61 , t Pham et al. 62 , u Goldberg et al. 63 , v Wu et al. 64 , w Skogareva et al. 65 www.nature.com/scientificreports/ Following this, Buettner 32 reported rate constants for the bimolecular reactions of Fe(III) and Fe(III)/EDTA and Cu(II) with AH − at pH 7 in oxygenated solution, and suggested it was a catalytic reaction, although the rate constants they reported did not include an oxygen dependence. Later, Buettner and Jurkiewicz 19 instead described it as a redox reaction: and suggested a somewhat higher rate constant for the Fe(III)/EDTA complex. Subsequent modeling studies adopted the redox reaction 33,34 .</p><p>Overall, however, the ascorbate mechanistic literature does not support a significant role for the redox reaction. Most or all studies point to the catalytic reaction instead; this includes the original source of the rate constant used for the redox reaction in Buettner 32 , and the mechanistic studies described below. We also test the redox and catalytic mechanisms with our model ("Ascorbate oxidation via the catalytic, redox or OH ⋅ /HO 2 ⋅ /O 2 ⋅− Pathways").</p><p>Transition metal ascorbic acid reaction mechanism. Three detailed mechanisms have been proposed to describe the oxidation process of ascorbic acid by iron and copper [28][29][30][31] . All of them begin with the oxidized form (Fe(III) or Cu(II)), consume oxygen, and produce A .− or DHA plus a reduced form of oxygen (HO 2 . , O 2 .− or H 2 O 2 ). In the proposed mechanisms, metal, ascorbic acid and oxygen molecules form a complex, with the metal ion serving as a bridge that transfers one or two electrons from ascorbic acid to oxygen and maintains its valence. In Scheme A, proposed by Khan and Martell 31 , a ternary metal-ascorbate-oxygen complex forms in which one electron is transferred from AH 2 or AH − through metal ion to oxygen (Scheme 1).</p><p>Scheme A Khan and Martell 31 .</p><p>Subsequently, Jameson and Blackburn 28 and Jameson and Blackburn 29 suggested a mechanism that involves an initial two-electron transfer to oxygen and the formation of Cu(III) intermediates (Supplementary Scheme B, Supplementary Eqs. ( 7)-( 14)). Shtamm et al. 30 then proposed a two-electron transfer mechanism involving Cu(I)-Cu(II) redox couple (Supplementary Scheme C). Although there is no agreement on the step by step oxidation state of metal ions in the catalytic cycle, there is some evidence showing that the reducibility of metal ion was necessary for it to be an active catalyst. Khan and Martell 31 tested VO 2+ , Mn 2+ , Co 2+ , Ni 2+ and Zn 2+ , and of these only VO 2+ was able to catalyze the oxidation of ascorbic acid. Further it is not clear if A .− is an intermediate of the oxidation of ascorbic acid [28][29][30] or if ascorbic acid is directly oxidized to DHA 30 . The disproportionation reaction for A .− is now well established (R12 and 13, Table 1) and is sufficiently rapid to not be rate limiting in this mechanism. This difference can have a moderate effect on the fitted rate constants; for iron of more consequence is the amount of OH . that is produced.</p><p>The stoichiometry for the metal ion-catalysed oxidation reactions of ascorbic acid by oxygen is also debated. Khan and Martell 31 measured iron and copper-catalyzed oxidation of ascorbic acid by oxygen at pH 2-5.5 and reported the reaction was first-order in ascorbic acid, metal and oxygen. Jameson and Blackburn 28 investigated the copper-catalyzed oxidation of ascorbic acid by oxygen in 0.1 M potassium nitrate at pH 2-3.5 and found a first-order dependence on copper and ascorbate (AH − ) and half-order on oxygen. The same rate law (for AH − ) was investigated by Shtamm et al. 30 at pH 2.7-4. Consistent with the rate law derived by Jameson and Blackburn 28 the rate observed by Shtamm et al. 30 was inversely related to the pH, indicating the rate law only applies to ascorbate. Moreover, Jameson and Blackburn 29 suggest that the stoichiometry can change depending on the nature and concentration of electrolytes, finding evidence that high concentrations of chloride ions (0.1 M) shifted the dependence from 1st order on AH − to half order on total ascorbic acid (AH 2 + AH − ).</p><p>Transition metal rate constants. Measured and estimated reaction rate constants for AH 2 and AH − with Fe(III) and Cu(II) in the literature are summarized in Table 3. The literature is divided into values for catalytic reactions, including cases for which it is possible to re-calculate a value given for the redox reaction as a catalytic reaction, and values for the redox reactions. The various catalytic reaction stoichiometries as well as pH dependencies and other caveats are also shown in Table 3.</p><!><p>The main product of the AH 2 and AH − reactions is the radical anion, A .− (Fig. 1), which disproportionates to form AH − and DHA or AH 2 and DHA at low pH: 2A •− H + ⇔ AH 2 /AH − + DHA (R12 and R13, Table 1). There is a wide range of values for the equilibrium constant K 12 in the literature; Foerster et al. 35 found pH dependent values of 1.6-7.9 × 10 14 M -1 at pH 4-6.4 (recalculated for the form of the equilibrium constant above) and Buettner and Schafer 22 reported 5 × 10 14 M -1 at pH 7.4. More recently Van der Zee and Van den Broek 36 found a value of 1.7 × 10 16 M -1 at pH 7.4 using ESR to monitor A .− and improved calibration techniques. We adopt the value from Van der Zee and Van den Broek 36 for K 12 and calculate the equilibrium www.nature.com/scientificreports/ constant for R13 using K −1 13 = K −1 12 K AH 2 , where K AH 2 is the first dissociation constant of ascorbic acid. We include both reactions in the model due to the highly pH-dependent A .− decay rates. For the forward reactions 2A •− + H + → AH − + DHA (R12) and 2A •− + 2H + → AH 2 + DHA (R13), we use 7 × 10 4 and 8 × 10 7 M −1 s −1 based on the radiolysis study by Bielski et al. 37 . The rate constants are chosen from the two plateaus in the pH dependent rate constants they derived 37 .</p><!><p>For the model presented here, some of the chemistry is well established, including much of the ROS chemistry, acid-base equilibria, inorganic iron chemistry, and probe and buffer chemistry. There are several general sources of error and uncertainty for the set of reactions in Tables 1 and 2, in addition to the specific uncertainties described above. These include errors in the rate constants, which range from a few percent to a factor of ten or more. In some cases, reaction stoichiometries and product distributions are also uncertain. Measurement data was often collected in solutions containing other solutes that may impact reaction rates and/or reaction mechanisms, but how and at what concentrations other solutes effect the rates is not known. Temperature differences also introduce uncertainties when data are collected at different temperatures. Further, usually only a small number of the uncertain reactions are important for a given set of experimental conditions. Some of our validation data was collected at 37 °C to mimic physiological conditions. Unfortunately, almost all literature data available for the set of reactions used here were reported for room temperature, and temperature dependencies were not available. Because temperature dependence is reaction specific, and can even have different signs, we have only adjusted the small number of rate coefficients for which there is temperature dependence. Gas solubility is also temperature dependent; we use a dissolved oxygen content corresponding to the temperature of each experiment.</p><!><p>Model description and extraction of rate coefficients. The model (Tables 1 and 2) includes reactions that describe the chemistry of reactive oxygen species, iron, ascorbate, sulfate and chloride, and a few reactions specific to the detection of DHA or OH ⋅ corresponding to the experimental datasets used to validate the model. The model builds on previous models describing aqueous OH ⋅ production kinetics in the presence of iron and sulfate [38][39][40] . Additional reactions describing the copper 41,42 and chlorine 43 chemistry have also been updated. The ascorbic acid mechanism (Fig. 1) is built into the model based on the detailed review of the available literature (described above). The final form of the ascorbic acid-metal reaction is similar to Scheme A; however, we have also explored many other forms and stoichiometries of the reactants (below).</p><p>The chemical kinetics mechanisms were solved using the Kinetics Pre-Processor (KPP) 2.2.3 26 with the gfortran compiler and the Rosenbrock solver. For measurements using two reaction coils in series with different conditions (below), the model was run separately for each set of conditions, and the output of the first segment was used as an input for the second.</p><p>To solve for the rate constants of two unknown reactions, such as the two reactions needed for copper (R16 and R17) we employ a two-dimensional binary search algorithm, specifically, we vary the rate constants for the two key reactions on an 11 × 11 grid field. For each grid (a combination of two rate constants), we run the model for each Fe(III) or Cu(II) concentration for which we have a measurement and calculate a mean squared error. The MSE of each unit square is obtained by averaging the MSEs of the nearest four grid points. After one cycle, we arrive at a unit square centered by a minimum MSE and we then divide this square into a new 11 × 11 grid field. The range containing the minimum MSE is narrowed as this process is repeated, and after 4 times the rate constant combination with minimum MSE is determined to be the best fit.</p><p>For cases where we need to fit more than two rate constants, the grid search method is not efficient enough, thus, we use a coordinate search method instead. We start from randomly chosen initial rate constants for these reactions, along with an initial search range. Each time we vary one rate constant within the search range while keeping the other rate constants fixed, calculate corresponding MSEs, and update the rate constant with the one that produces minimum MSE. This process was applied to each reaction in turn and when this sees no improvement, we reduce the search range in order to continuously decrease the MSE. Finally, when the search range exceeds our required precision, the optimization stops.</p><p>Validation data. We measured the oxidation of ascorbic acid by Fe(II), (III) and Cu(II) by quantifying the oxidation product dehydroascorbic acid (DHA), as described in detail in Campbell et al. 17 . Briefly, DHA is reacted with o-phenylenediamine (oPDA) to produce a highly fluorescent product 3-(1,2-dihydroxyethyl)fluoro [3,4-b]quinoxaline-1-one (DFQ) with unit yield. DFQ is then quantified via fluorescence spectroscopy.</p><p>OH ⋅ formation from ascorbate reactions with Fe(II) and Cu(II) at around pH 7 were reported by Charrier and Anastasio 15 (2.8 µM OH ⋅ from 1 µM Fe(II) and 14 µM from 1 µM Cu at 24 h) and Lin and Yu 27 (0.3 µM OH ⋅ from 1 µM Fe(II) at 3.8 h and 8.8 µM from 0.3 µM Cu at 6.3 h); Lin and Yu 27 also reported ascorbate consumption. While the measurements are difficult to compare due to measurement differences and potential non-linear dependencies on both concentration and reaction time, the results appear to be in good agreement for Fe(II) and weak agreement for Cu(II).</p><p>Reagents and chemical preparation. All chemicals were obtained from Sigma-Aldrich. Ascorbic acid (≥ 99.0%), Chelex 100 sodium form, 0.1 M HCl solution, 0.1 M NaOH solution, CuSO 4 (≥ 99.0%), FeSO 4 (≥ 99%), Fe 2 (SO 4 ) 3 (≥ 98%), o-phenylenediamine (≥ 99.5%), DHA (≥ 96%), HEPES (≥ 99.5%) were used as received.</p><p>A 200 µM solution of ascorbic acid was prepared in Chelex-resin treated MilliQ water (resistivity ≥ 18.2 MΩ cm −1 ), to ensure as low as possible trace metal concentrations and minimize background DHA formation. The Vol:.( 1234567890</p><!><p>Measurements of ascorbic acid oxidation by iron and copper were conducted in an online instrument described in detail in Campbell et al. 17 . Briefly, a flow of 1.1 mL/min of 200 µM ascorbic acid is added to an equivalent 1.1 mL/min flow of either Cu(II)SO 4 , Fe(II)SO 4 or Fe(III) 2 (SO 4 ) 3 .</p><p>The reaction mixture was then incubated in reaction coil-1 (Supplementary Table S2) housed in ethylene glycol for 20 min at 37 °C. After passing through the reaction coil, a solution containing 46 mM oPDA in 0.1 M HCl was added at 1.1 mL/min and mixed with the ascorbic acid/metal reaction mixture for 10 min (at pH 2.8) at room temperature in reaction coil-2 (Supplementary Table S2). DHA formed by the oxidation of ascorbic acid/ ascorbate reacted rapidly with oPDA to form the highly fluorescent compound DFQ. The reaction mixture containing DFQ then passes through a fluorescence detection cell (details in Campbell et al. 17 ). The extent of ascorbic acid oxidation is then expressed in terms of µM DHA using a DHA calibration curve 17 . A summary of reaction conditions and dilution ratios are presented in Supplementary Table S2.</p><p>While water typically contains low levels of H 2 O 2 (not measured here but generally below 10 nM 44 ) in the absence of transition metals, there are no pathways to form radicals either from O 2 or H 2 O 2 . Consistent with this, in the absence of added metals, DHA formation in the reaction coils was below detection limits.</p><!><p>DHA loss pathways. Observations of the stability of DHA indicate it decreases with increasing pH; in pH 2-4, aqueous DHA solutions are stable for days, while at neutral pH, the half-life of DHA is around 20 min 45,46 . Our model includes three degradation pathways of DHA: reactions with the hydroxyl radical, H 2 O 2 and hydrolysis to produce 2,3-diketogulonic acid. For the reaction with hydroxyl radical, we estimate a rate constant of 1 × 10 10 M −1 s −1 , the diffusion limit. The hydrolysis rate constant is thought to be negligible at low pH but reaches (5.3-5.8) × 10 -4 s −1 at neutral pH 46,47 . DHA also reacts with H 2 O 2 with an estimated rate constant of 4.2 × 10 -2 M −1 s −1 and oxalyl l-threonate, cyclic oxalyl L-threonate and oxalate as the main products 45 . Calculated DHA degradation from these three pathways is negligible at low pH. At neutral pH, DHA degradation reaches 29-39% of total DHA formation in the first reaction coil for copper and 28% for iron, with hydrolysis as the main pathway. Although the reaction of DHA and oPDA is thought to be fast, with a reaction time of about 14 s, in the first (20 min) reaction coil where oPDA is absent, DHA degradation is quite significant. The evidence in favor of the catalytic reaction for iron is as follows. First, the redox pathway fails to produce enough DHA especially at pH 2.8. This is both because the redox reaction converts Fe(III) to Fe(II), and the system can only slowly reoxidize the Fe(II), so Fe(III) can consume only a limited amount of ascorbic acid. Also, the redox reaction seems to produce A ⋅− , which only produces DHA with 50% efficiency (R12, 13); for the catalytic reaction production of A ⋅− vs. direct formation of DHA is less of a settled question. Second, both the catalytic and redox reactions make more cumulative OH ⋅ from the Fe(III)-ascorbic acid reactions than observed by Lin and Yu 27 and Charrier and Anastasio 15 (discussed more in "Comparison with OH • formation data"), but the catalytic reaction is reasonably close to the observations ("Comparison with OH • formation data") while the redox reaction vastly overshoots. The catalytic reaction directly generates the OH ⋅ precursor H 2 O 2 , but it does not produce the Fe(II) needed for the Fenton reaction to convert H 2 O 2 to OH ⋅ as does the redox reaction.</p><!><p>The evidence in favor of the catalytic reaction for copper is as follows. In experiments where the extent of the reaction is high, as for Cu(II) at pH 7 (Fig. 3), DHA formation eventually stops increasing. The catalytic reaction is able to reproduce the general asymptotic shape of the DHA formation dependence on Cu(II) (Fig. 3) while the redox mechanism predicts a linear relationship (this is also observed for Fe(II), but the iron phenomenon likely depends more on autoxidation of Fe(II) to Fe(III) rather than the Fe(III) reacting with ascorbate). The reason the catalytic reaction produces an asymptotic behavior is that oxygen is consumed in the closed reaction tubes, limiting reactions 14-17 (Table 1). Additionally, when we include both the redox and catalytic pathways in the model using the coordinate search algorithm for pH 2.8 Fe(III) and pH 7 Cu(II), the optimization steps in the direction where rate constants for the redox reactions of AH 2 + Fe(III) and AH − + Cu(II) continuously decrease; for the other DHA data the model cannot differentiate between the catalytic and redox reactions. OH ⋅ production from the copper via both pathways falls between the two divergent observational results 15,27 . Further support for the catalytic mechanism comes from Jameson and Blackburn 28 , who reported that although some degree of charge transfer occurred within the copper-ascorbate complex, the complete one-electron redox reaction did not happen when there was no oxygen.</p><p>ROS reactions are only important in the experimental systems if OH ⋅ , HO 2 . and/or O 2 ⋅− concentrations are high, such as for conditions associated with experiments with added H 2 O 2 , or when Fe(II) is the dominant form of iron, for example. Were more DHA formation to be attributed to ROS chemistry, much higher concentrations of OH ⋅ would be needed, a situation clearly not supported by the OH ⋅ measurements by both Charrier and www.nature.com/scientificreports/ Anastasio 15 and Lin and Yu 27 . Our result indicates that for both Fe(III) and Cu(II), the catalytic pathway is always dominant compared to the ROS pathway, by 4-6 and 1-3 orders of magnitude for Fe(III) and Cu(II) respectively. However, in the Fe(II) case, the contribution of the catalytic pathway and ROS pathways are more comparable; the ratio of catalytic to ROS pathways decreases from 11 at 2.5 µM Fe(II) to 1.4 at 200 µM Fe(II). This is because Fe(II) produces ROS via reduction of molecular oxygen and the Fenton reaction, pathways not available to Fe(III). While the oxidized form of the Fe and Cu might be expected to dominate ascorbate consumption in many situations, the contributions of OH ⋅ , HO 2 ⋅ and/or O 2 ⋅− may be significant under some conditions. Given the rate constants (Table 1), OH ⋅ , HO 2 ⋅ /O 2 ⋅− need to be ~ 10 -9 , 10 -4 × [Fe(III)] or 10 -8 , 10 -3 × [Cu(II)], respectively to account for around half of ascorbate loss. OH ⋅ , HO 2 ⋅ in liquid phases in equilibrium with gas phase concentrations of 10 6 and 10 7 molec/cm 3 result in liquid phase concentrations of ~ 10 -3 and 10 -2 nM, respectively 48 . However, 1 and 2. Because of the additional uncertainties and lack of relevance of the high Fe(II) concentrations, only experimental data for Fe(II) concentration within 200 µM at pH 2.8 is shown in the figure . Table 3. Summary of rate constants for reactions of iron and copper with ascorbic acid. *The reaction appears to be catalytic, but these papers assumed a redox reaction; the rate has been recalculated here assuming an oxygen dependence. www.nature.com/scientificreports/ even with modest concentrations of organics, the radicals will be rapidly depleted away from the interface 48 .</p><p>Consistent with this, model calculations for lung lining fluid estimated concentrations of 10 -10 -10 -7 nM for OH ⋅ , 10 -6 -10 -4 nM for HO 2 ⋅ and 10 -3 -10 -1 nM for O 2</p><!><p>, with the highest concentrations associated with extremely high PM concentrations 33 . In comparison a typical concentration for iron and copper is around several micromolar in the bronchoalveolar lavage and can be even higher when exposed to highly polluted environments, and thus should usually be the dominant sink for ascorbic acid and ascorbate 49 .</p><p>Catalytic reaction rate constants. Fe(III). In the Fe(III)/AH 2 system, the model is very sensitive to the catalytic reactions AH 2 (R14) or AH − (R15) or both, depending on pH. We minimize the sum of MSEs for the two iron data sets (Fe(III) at pH 2.8 and 7) to derive rate constants for the catalytic reactions R14 and R15. Bestfit third-order rate constants [Fe(III)] [AH 2 /AH − ][O 2 ] for R14, R15 are 5.7 × 10 4 and 4.7 × 10 4 M −2 s −1 , respectively; these values produce good agreement with the DHA formation data for both Fe(III) and the Fe(II) over the concentration range as shown in Fig. 2. The AH − result is in good agreement (within 35%) with 3.5 × 10 4 M −2 s −1 from Buettner 32 , assuming a first order oxygen dependence in their study (Table 3). Our values for AH 2 and AH − are significantly lower than 4.0 × 10 5 and 2.4 × 10 7 M −2 s −1 reported by Khan and Martell 31 and the AHvalue after recalculation for the catalytic pathway from Lakey et al. 33 of 2.3 × 10 5 M −2 s −1 , Table 3.</p><!><p>Because the asymptotic behavior observed for very high Fe(II) at pH 2.8 is far outside the relevant range for environmental samples, we include only Fe(II) data up to 200 µM. The model is reasonably successful at reproducing DHA formation from Fe(II) at pH 2.8 at lower Fe(II) concentrations (Fig. 2), although it overpredicts DHA formation at high Fe(II) (> 100 µM). Ascorbate is only oxidized by OH ⋅ , HO 2 ⋅ , O 2 − and Fe(III), so in the presence of Fe(II) ascorbate oxidation should be controlled by the production of Fe(III) or ROS. Both of these pathways are initiated by the reduction of O 2 via R45 and enhanced by H 2 O 2 production from Fe(III) + ascorbic acid (R14, 15). The rate constant for R45, Fe(II) + O 2 is pH sensitive. Stumm and Morgan 50 suggest that when pH > 5 the rate increases with pH, with a second-order dependence on OH − concentration. At low pH (1-4), this rate constant is ~ 10 -5 M −1 s −1 and independent of pH. This rate constant has been found to increase by a factor of 10 for a 15 °C temperature increase 50 . Therefore, for pH 2.8 we use a rate constant of 10 -5 M −1 s −1 at room temperature and 10 -4 M −1 s −1 at 37 °C. For pH 7.0, a k 45 of 0.39 M −1 s −1 was suggested by Pham and Waite 51 which we adjusted upward to 3.9 M −1 s −1 at 37 °C.</p><p>The Fe(II) measurements (Fig. 2) show that initial AH 2 consumption/DHA production by Fe(II) is around one sixth of that of Fe(III), however, this likely results partly from conversion of Fe(II) to Fe(III) in the stock solution. Although the Fe(II) stock solution was made fresh daily, in the few hours required for the measurements Fe(II) slowly oxidizes to Fe(III). Rate constants for R45 at room temperature are ~ 0.11 M −1 s −1 for pH 6.0 and 0.17 M −1 s −1 for pH 6.5 51 . The pH of our 1 mM FeSO 4 stock solution is ~ 6.3, so about 13%/h of Fe(II) is oxidized to Fe(III). Because the degree of oxidation of the stock solution was not the same for each experiment, this may explain the larger error bars for Fe(II) (Fig. 2). The modelling results for Fe(II) shown here are based on the assumption that 10% of Fe(II) was oxidized to Fe(III) before each experiment.</p><p>Copper catalytic reaction rate constants. Compared to iron, there are more studies of the copper reactions with ascorbic acid, and more disagreements (Table 3). Here, the rate laws from Jameson and Blackburn 28 , Jameson and Blackburn 29 , Shtamm et al. 30 and Khan and Martell 31 are all tested. For simplicity, we use overall reactions for the catalytic pathway; the products of the catalytic reaction are H 2 O 2 and either DHA or A •− , depending on the charge balance of the equations.</p><p>Ascorbic acid oxidation by Cu(II) is more efficient than by Fe(III) (Figs. 2 and 3). For the 1:1:1 stoichiometry first proposed by Khan and Martell 31 , the third-order rate constants [Cu(II)][AH 2 /AH − ][O 2 ] for R16 and R17 that provide the best fit of the data are 7.7 × 10 4 and 2.8 × 10 6 M −2 s −1 , respectively (Fig. 3). The Cu(II) ion catalyzes the oxidation of ascorbic acid more efficiently at neutral pH than acidic pH; this is reflected in the much higher value for k 17 than k 16 . In the pH 7.0 measurements, the theoretical maximum DHA concentration in the second reaction coil is 100 μM, but similar to the observation for Fe(II), DHA formation reached a much lower maximum of about 45 μM at 2.5 µM Cu(II). The model matches this behavior well for Cu(II), due to a combination of the depletion of oxygen in the solution which suppresses DHA production at high Cu(II) concentrations, and the hydrolysis of DHA, which is significant at high pH.</p><p>The fitted rate constants for AH 2 and AH − are lower than 3.8 × 10 5 and 6.0 × 10 7 M −2 s −1 in Khan and Martell 31 . Using a first order oxygen dependence, (redox) rate constants for Cu(II) and AH − in Buettner 32 and Lakey et al. 33 can be converted into (catalytic) third-order rate constants k([Cu(II)][AH − ][O 2 ]) of 3.1 × 10 6 and 3.0 × 10 6 M −2 s −1 respectively, in good agreement with the rate constant we derived. Both the iron and copper data suggest our experimental data are in better agreement with measurement of Buettner 32 , while there might be an overestimation of the rate constants in Khan and Martell 31 .</p><p>If we instead use the rate law [Cu(II)][AH − ][O 2 ] 1/2 suggested by Jameson and Blackburn 28 and Shtamm et al. 30 with a 2.5 order rate constant we find a best-fit value for AH − of 1.2 × 10 5 M −3/2 s −1 . This value is about 1.5 orders of magnitude larger than 4.3 × 10 3 M −3/2 s −1 from Jameson and Blackburn 28 and 5.2 × 10 3 M −3/2 s −1 from Shtamm et al. 30 . Some or all of the difference could be due to the difference in temperature; as the earlier measurements were made at ~ 25 °C and ours were mostly at 37 °C or from the low concentrations of chloride ions 5 included in our experiments.</p><p>Nevertheless, this 1:1:0.5 rate law predicts higher DHA formation at high Cu(II) than observed in the pH 7.0 data. This is partly because the reaction's half-order oxygen dependence means O 2 is less depleted and is less able to limit DHA formation. The 1:1:1 stoichiometry of Cu(II), AH 2 /AH − and oxygen fits the shape of DHA formation curve better and arrives at a smaller MSE. However, the room temperature DHA hydrolysis rate used in the model may also underestimate DHA consumption.</p><p>Jameson and Blackburn 29 suggested a [Cu(II)][AH 2 + AH − ] 1/2 [O 2 ] 1/2 (1:0.5:0.5) rate law for solutions containing 0.1 M chloride ions. Our experiment includes chloride ions as follows: for pH 2.8 experiments the pH in the first reaction coil was adjusted with HCl resulting in 1.6 × 10 -3 M Cl − , and the oPDA mixed into the 2nd reaction coil is prepared in 0.1 M HCl increasing the Cl − concentration in the 2nd coil by 0.03 and 0.05 M for pH 2.8 and 7 experiments respectively, but DHA forms in the first reaction coil, so this is less important. This stoichiometry provides a much worse fit for our data than the other rate laws, implying our concentrations of Cl − are too low to significantly alter the Cu(II)-ascorbic acid reaction.</p><p>Comparison with OH ⋅ formation data. To further test the reactions we derived with data from the literature, we include benzoate (the OH ⋅ probe) and phosphate buffer reactions (R87-95) and calculate OH ⋅ production for the conditions used by Charrier and Anastasio 15 and Lin and Yu 27 . Although both of these studies only investigated Fe(II), our model includes reactions that oxidize this species to Fe(III), and thus we can use the data to constrain the products of the Fe(III) + AH 2 /AH − reaction. With A ⋅− + HO 2 ⋅ as products, the model produces OH ⋅ that exceeds the observations from both Charrier and Anastasio 15 and Lin and Yu 27 by 2 orders of magnitude, suggesting the products are H 2 O 2 + DHA. The H 2 O 2 + DHA combination of products produces 4 µM OH ⋅ formation from 1 µM Fe(II) after 24 h, in reasonable agreement with the Charrier and Anastasio 15 measurement of 2.8 µM. A similar calculation for the Lin and Yu 27 conditions overshoots the reported OH, resulting in a concentration that is about a factor of three higher, and somewhat overestimates consumption of ascorbate (the modeled concentration at 3.8 h is about 90% of the measured value). The discrepancy may be due to several factors, including the temperature difference, errors in various rate constants or the observational data, or incorrect assumptions about the products of the Fenton reaction; there is some evidence in the literature that the Fenton reaction can produce either OH ⋅ or Fe(IV), with a pH dependent product distribution. Fe(IV) production may be favored around neutral pH [52][53][54] . Our model only considers OH ⋅ as a product and could thus overestimate its formation.</p><p>For copper, the model results fall between the OH ⋅ formation measurements from Charrier and Anastasio 15 and Lin and Yu 27 , which are widely divergent from each other, at about 2.6 times the former and 27% of the latter. Although both the Lin and Yu 27 data and our model agree at nearly 100% ascorbic acid loss at ~ 6 h, ascorbic acid concentration predicted by the model decreases with time nonlinearly, different from the linear trend in the experiment. Several other aspects of Cu(I) and Cu(II)-ROS chemistry are uncertain; some additional discussion of the gaps can be found in the SI.</p><!><p>In recent years, there has been widespread application of acellular assays to measure particle-bound ROS and aerosol oxidative potential (OP), with measurements spanning large spatial, temporal and chemical spaces 11,18,55 . OP is proving to be better at predicting adverse health impacts than particle mass 11,18 . The complex interplay between OP assays, including the ascorbic acid assay, and redox-active PM components has widely been demonstrated 14,18,56 . Detailed understanding of assay responses is crucial to fully elucidate both the role of chemical composition on aerosol OP and to directly probe the role of aerosol OP in particle toxicity. Ultimately, this information should be translatable into policy that specifically targets the components in PM that drive OP (and</p>

Scientific Reports - Nature

Identification of a distinct desensitisation gate in the ATP-gated P2X2 receptor

P2X receptors are trimeric ATP-gated ion channels. In response to ATP binding, conformational changes lead to opening of the channel and ion flow. Current flow can decline during continued ATP binding in a process called desensitisation. The rate and extent of desensitisation is affected by multiple factors, for instance the T18A mutation in P2X2 makes the ion channel fast desensitising. We have used this mutation to investigate whether the gate restricting ion flow is different in the desensitised and the closed state, by combining molecular modelling and cysteine modification using MTSET (2-(Trimethylammonium)ethyl methanethiosulfonate). Homology modelling of the P2X2 receptor and negative space imaging of the channel suggested a movement of the restriction gate with residue T335 being solvent accessible in the desensitised, but not the closed state. This was confirmed experimentally by probing the accessibility of T335C in the P2X2 T18A/T335C (fast desensitisation) and T335C (slow desensitisation) mutants with MTSET which demonstrates that the barrier to ion flow is different in the closed and the desensitised states. To investigate the T18A induced switch in desensitisation we compared molecular dynamics simulations of the wild type and T18A P2X2 receptor which suggest that the differences in time course of desensitisation are due to structural destabilization of a hydrogen bond network of conserved residues in the proximity of T18.

identification_of_a_distinct_desensitisation_gate_in_the_atp-gated_p2x2_receptor

<!>Introduction<!>Molecular modelling<!>Molecular biology and electrophysiological recordings<!>Data analysis and statistics<!><!>Results<!><!>Results<!><!>Discussion<!>Supplementary data<!>Transparency document

<p>Ionic gate controls current flow in closed state, gate opens when ATP binds.</p><p>MTSET labelling suggests desensitisation gate in different place than closed state.</p><p>T18A affects desensitisation by disrupting a conserved hydrogen bond network.</p><!><p>The P2X family of ligand gated ion channels consists of seven paralogs (P2X1-7R) that form trimeric receptors which are activated by ATP. Structurally, P2XRs comprise a large extracellular region which contains the ATP binding sites, a transmembrane region formed by two transmembrane helices per subunit, and a typically small intracellular region formed by the N- and C-terminal regions. While P2XRs share their overall architecture there are differences in ATP sensitivity and time-course of the response to ATP [1]. The P2X1R and P2X3R are activated by low concentrations of ATP (EC50 < 1 μM) with currents that decay rapidly during the continued presence of agonist (within 1–2s) in a process called desensitisation. Conversely, the P2X7R has an EC50 of >300 μM and currents are sustained and can facilitate on repeated application [2]. The variation in agonist sensitivity is associated with differences in the extracellular ligand binding region of the receptor, for example recent work suggests that a P2X7R specific salt bridge contributes to the high concentrations of ATP required to activate the receptor [3].</p><p>The molecular basis of the differences in time-course of P2XR responses was first suggested from work showing that a splice variant in the intracellular C-terminus of the P2X2R could modify the rate of desensitisation and a study using chimeras showing the importance of the intracellular amino terminus of the receptor [4,5]. Subsequent work identified the importance of residues around the conserved YxTxK/R amino terminal sequence motif in determining the time-course of the response [6,7]. P2X2 receptors mutated in this region, such as T18A or T18N, switch the receptor from slowly to quickly desensitising (<1 s) [8].</p><p>The crystallisation of the zebrafish P2X4R provided a detailed structural model for ATP binding and opening of the transmembrane channel gate [9,10]. More recently stuctures of the human P2X3R in a range of states became available that provided the first structural information on the desensitised state of the receptor [11]. In the agonist-free, closed state, no structural information could be resolved on the intracellular regions. Two other forms of the hP2X3R resolved an ATP bound state with an open conformation of the ion channel with the intertwining of the N- and C-terminal regions to form an intracellular cap, and a desensitised ATP-bound form where the channel gate had moved and the intracellular cap had disassembled [11]. These studies provided the first structural models of the gating cycle of a P2XR and are consistent with the suggestion that there is a distinct desensitisation gate in the receptor [12].</p><p>We therefore devised a strategy to provide experimental evidence for the suggestion that the gate to ionic flow moves in the desensitised state. Pioneering cysteine scanning mutagenesis studies [[13], [14], [15], [16]] provided a good understanding of the location of the channel gate in the P2X2R that is consistent with structural work. One of the key findings in the location of the channel gate was the identification of a cysteine mutant (in hP2X2R; T335C) that blocks ionic flow when chemically modified by methanethiosulfonate compounds in the open, but not in the closed state of the receptor, and hence showed gated access. In this work we tested experimentally whether T335C is accessible in the desensitised state using a mutation in the intracellular amino terminus (T18A) that makes the hP2X2R rapidly desensitising, and we probed computationally how, at a mechanistic level, the T18A mutation might affect the time course of desensitisation.</p><!><p>Homology models for the human P2X2 receptor were based on the sequence entry GenBank AAD42947.1, which lacks the 12 N-terminal residues of the SwissProt entry P2RX2_HUMAN (Q9UBL9). For modelling the P2X2R open state, the P2X3R open state X-ray structure (PDB identifier: 5SVK), and accordingly, for the P2X2R closed and desensitised state models, PDB entries 5SVJ and 5SVL were used as templates. P2X2R and P2X3R sequences were aligned using MAFFT [17] (pairwise template-target sequence identity ∼ 50%) and manually inspected in Jalview (v2.10.5) [18] to adjust gaps and insertions based on the structure of the respective template [19]. Alignments and templates were used in Modeller v9.19 [20] to calculate 50 models for each state. The resulting models were ranked by their Discrete Optimized Protein Energy (DOPE) score. The best scoring models were used to generate a negative space image of the tunnel between the subunits using HOLE (v2.2.005 on Linux) [21] with a 8 Å pore radius parameter. Calculated channel descriptors were converted to a vmd_plot and visualised together with the receptor in VMD [22]. Pymol [23] was used for additional visualizations.</p><p>In total, six molecular dynamics simulations were performed for alternative P2X2R and P2X2R T18A models. Simulations were run in AMBER18 using the ff14SB and lipid17 force fields with the TIP3P water model [24,25] and ATP parameters as in previous work [26]. The set-up involved embedding the P2X2R in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer using PACKMOL-Memgen [27] and charge neutralisation with Cl- and Na+ at 0.15 M. At this stage, the T18A mutation was introduced into each replicate. The simulation protocol involved 5000 steps steepest descent and 5000 steps of conjugate gradient energy minimization, followed by two heating steps from 0 to 100 K at NVT, and 100K–303K at NPT conditions. A 20 ns holding step to equilibrate the system's periodic boundary condition dimensions was followed by 200 ns production runs which were analysed in cpptraj [28] for changes in the local environment of key residues in the cytoplasmic cap and visualised in R (Supplemental Figs. S1 and S2).</p><!><p>The constructs encoding wild type hP2X2R receptors were available from previous work [6]. Stage V Xenopus laevis oocytes were injected with cRNA synthesised with the T7 mMessage mMachine kit (Ambion). P2XR expressing oocytes were stored at 16 °C in ND96 buffer (in mM; NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, sodium pyruvate 5 and HEPES 5, at pH 7.6) with 50 μgml−1 of gentamycin and tetracycline for 3–7 days with the solution changed daily. For electrophysiological recordings the gentamycin and tetracycline were not present and the CaCl2 was replaced with 1.8 mM BaCl2.</p><p>Two electrode voltage clamp recordings were made using a Geneclamp 500B amplifier with a Digidata 1322A A-D convertor and pClamp 8.2 acquisition software (Molecular Devices) at a holding potential of −60 mV. ATP (magnesium salt, Sigma) was applied via a U-tube perfusion system. (2-(Trimethylammonium)ethyl methanethiosulfonate (MTSET) (1 mM) was applied in the bathing solution and/or co-applied with ATP.</p><!><p>Data are presented throughout as mean ± S.E.M. For peak currents the individual values were normalized to the mean peak current for the WT receptor in the batch of oocytes. Significant differences from WT were calculated by one-way analysis of variance followed by Dunnett's test using GraphPad Prism 6 (GraphPad software inc).</p><!><p>P2X2R Ionic gate in different conformation states. (A) hP2X2R homology model in the apo state (closed). The black box in (A) corresponds to the area displayed in other panels indicating the location of the channel gate. In (B), (C), (D) the voids are shown as a grey-dot tunnel. MTSET access through the transmembrane region is blocked by the activation gate near residue T335 (yellow, B). In the open state (C) and the desensitised state (D) residue T335 is accessible.</p><!><p>In the closed state the transmembrane channel is blocked by the activation gate near residues T335 and T338 (Fig. 1B). Upon ATP binding, the receptor transitions to the open state, (Fig. 1C), as the subunits move outward and the extracellular loop flexes. This flexing causes the TM helices to create a wider pore near the lateral fenestrations [29]. The T335 residues are pulled apart creating a wide opening between the subunits. In the desensitised state (Fig. 1D) T335 residues are even further apart, however at the same time, the V342 residues come closer together to form the desensitisation gate and stop/reduce ion flow.</p><p>Cα-Cα distances between T335 residues of different subunits vary between the closed, open and desensitised states ranging from 7.7 Å to 13.2 Å and 14.3 Å, respectively. This indicates changes in the accessibility of T335 to MTS compounds used in this study. Experimentally the accessibility of T335 and hence whether the activation gate is open can be tested by application of MTSET to the T335C mutant. MTSET, like other MTS compounds, can bind covalently to solvent accessible cysteines [30,31]. In case of T335C being accessible to MTSET and oriented towards the pore, covalent binding of MTSET blocks ion flow (see Fig. 1C). We determined how the T18A mutation [8] that makes the hP2X2 receptor rapidly desensitising affects the accessibility of T335C [32].</p><!><p>Effect of MTSET at different stages of ATP-response of P2X2 cysteine mutants. (A) MTSET (1 mM, 30s) in the absence of ATP had no effect on subsequent ATP currents at WT, T335C or T18A/T335C (fully recovered from desensitisation) showing inaccessibility of T335C in the closed state. Histogram of mean % inhibition of ATP response by MTSET. For all figures black bar indicates ATP application (100 μM, 10 s). (B) MTSET effects when co-applied with ATP showing gated access to residue T335C. There was no effect on the control WT even after the 3rd co-application. Co-application (30s) abolished responses in both the slowly desensitising mutant T335C and the fast desensitising T18A/T335 by more than 90% (C). To determine whether block at T18A/T335C was at the open or desensitised state, MTSET (1 mM, 30s) was applied to the desensitised channel (lower panel). Following washout and the normal period required for recovery from desensitisation (indicated by hashed line, full recovery in control upper panel) subsequent ATP responses were reduced by >90%. Data are shown as mean ± SEM, n = 3–6, ***p < 0.001. The corresponding states of the receptor are indicated on the right-hand side.</p><!><p>When MTSET was applied for 30 s to the desensitised receptor (T18A/T335C; either in the continued presence, or immediately after removal of ATP, Fig. 2C) the response to a subsequent application of ATP (after a 5 min wash/recovery period) was reduced by >90% demonstrating that T335C is accessible in the desensitised state. In summary our results show that T335C is not accessible to MTSET in the agonist free form, but is accessible in both the ATP-bound open and ATP-bound desensitised states indicating that the activation gate is still open in the desensitised state. This provides the first functional evidence for an additional barrier to ionic permeation in P2XRs, i.e. a desensitisation gate.</p><!><p>MD Simulation of P2X2R wildtype and T18A mutant (A) P2X2R TM and intracellular cap regions in cartoon representation with different subunits in light pink, grey and light blue – colour scheme as in Fig. 1. One instance of residues Y16, T18, D348 and K356 is shown as spheres. (B) Enlarged version of (A) with key residues shown as sticks and dotted lines indicating distance measurements shown in (C) and (D). (C) Representative example for Y16/D348 interaction monitored over a 200 ns molecular dynamics simulations for the P2X2R wildtype. Raw data shown in lightblue and rolling average over 10 data points in black. (D) As in (C) for the P2X2R T18A mutant with raw data shown in firebrick and rolling average over 10 data points in black. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</p><!><p>The restriction gate is different in desensitised and closed states. Residues lining the P2XR channel gate have been established by cysteine scanning mutagenesis studies well before X-ray structures became available. These were generally consistent with structural biology data (for review see Samways et al.(2014)). In this work we probed experimentally how far the P2X2R ion channel is still open in the desensitised state. This was achieved by using P2X2 wild type and P2X2 T18A receptors to test the accessibility of T335C in the open and desensitised state. MTSET is a water soluble, positively charged MTS-reagent that binds covalently to accessible cysteine residues. In our experimental set-up MTSET is added extracellularly and based on our P2X2R homology models can enter the transmembrane channel via the lower vestibule of the extracellular region of the P2X2R (Fig. 1A). The finding that T335C is not reacting with MTSET in the closed state, but is accessible in open and desensitised states is evidence for the activation gate still being open in the desensitised state, and implies a desensitisation gate as additional barrier to ionic permeation. Our work provides the first direct functional evidence that the channel gate moves in the desensitised state. This is in agreement with cadmium effects on cysteine mutants suggesting that the gate region within TM2 moves during desensitisation [33] and consistent with hP2X3R crystal structures [11].</p><p>Multiple factors contribute to desensitisation. Despite the availability of open and desensitised states P2X3R X-ray structures, the molecular mechanism of desensitisation and determinants of different desensitisation patterns in P2X receptor paralogs are not fully understood. It is however clear that different regions of P2X receptors can play a role in desensitisation. For instance, there is evidence from voltage clamp fluorometry that the kinetics of conformational changes in the extracellular region are associated with desensitisation [12]. A role of TM1 and TM2 in desensitisation has been established by TM helix swapping experiments [7], for instance hP2X1R can be made slowly desensitising when either hP2X1R-TM1 or -TM2 of are swappped with equivalent regions [34]. In the intracellular region, changes in intracellular calcium and ATP concentrations [35] and phosphorylation on T372 of the P2X2aR splice variant modulate P2X2R desensitisation [36]. The involvement of extracellular, transmembrane and intracellular regions of P2XRs in the modulation of desensitisation features indicates a multi-faceted process.</p><p>How does T18A affect desensitisation? A short stretch of N-terminal residues are conserved throughout the P2XR family (YXTXK/R) and point mutations in this region can have profound effects on time-course of the ATP response. For instance, in P2X2R the T18A mutation transforms the receptor from slowly desensitising to fast desensitising [8]. T18 was originally postulated to be part of a PKC phosphorylation site [8]), though this was later challenged [[37], [38], [39]]. Based on the P2X3R structure in the open state a structural role of the P2X2-T18 region as part of the cytoplasmic cap has emerged, where T18 with other highly conserved residues Y16, D348 and K355 forms an intersubunit hydrogen bond network. The side chains of T18 and Y16 lock the N-terminal beta-strand of the cytoplasmic cap to the C-terminal transmembrane helix via hydrogen bonds to D348, and via K355 to the adjacent short α-helix perpendicular to the membrane (Fig. 3). Our MD simulations suggest that the T18A modification destabilises the tethering between Y16 and D348, and affects structure and dynamics of the cytoplasmic cap. Based on the hP2X3 X-ray structures in different states Mansoor et al. proposed the disassembly of the cytoplasmic cap as key step in desensitisation [11]. In agreement with this proposal the effect of the T18A modification on the dynamics of the conserved H-bond network involving Y16, T18, D348 and K355 we detected in our MD simulations provides a plausible mechanistic explanation how this mutation may accelerate desensitisation.</p><!><p>The following are the Supplementary data to this article:Supplemental Table 1Supplemental Table 1Suppl Fig. 1.pdfSuppl Fig. 1.pdfSuppl Fig. 2.pdfSuppl Fig. 2.pdf</p><!><p>coi_disclosure _spl_AS_splcoi_disclosure _spl_AS_spl</p>

PubMed Open Access

Identification of drugs targeting multiple viral and human proteins using computational analysis for repurposing against COVID-19

The SARS-CoV2 is a highly contagious pathogen that causes a respiratory disease named COVID-19. The COVID-19 was declared a pandemic by the WHO on 11th March 2020. It has affected about 5.38 million people globally (identified cases as on 24th May 2020), with an average lethality of ~3%. Unfortunately, there is no standard cure for the disease, although some drugs are under clinical trial. Thus, there is an urgent need of drugs for the treatment of COVID-19. The molecularly targeted therapies have proven their utility in various diseases such as HIV, SARS, and HCV. Therefore, a lot of efforts are being directed towards the identification of molecules that can be helpful in the management of COVID-19.In the current studies, we have used state of the art bioinformatics techniques to screen the FDA approved drugs against thirteen SARS-CoV2 proteins in order to identify drugs for quick repurposing. The strategy was to identify potential drugs that can target multiple viral proteins simultaneously. Our strategy originates from the fact that individual viral proteins play specific role in multiple aspects of viral lifecycle such as attachment, entry, replication, morphogenesis and egress and targeting them simultaneously will have better inhibitory effect.Additionally, we analyzed if the identified molecules can also affect the host proteins whose expression is differentially modulated during SARS-CoV2 infection. The differentially expressed genes (DEGs) were identified using analysis of NCBI-GEO data (GEO-ID: GSE-147507). A pathway and protein-protein interaction network analysis of the identified DEGs led to the identification of network hubs that may play important roles in SARS-CoV2 infection. Therefore, targeting such genes may also be a beneficial strategy to curb disease manifestation. We have identified 29 molecules that can bind to various SARS-CoV2 and human host proteins. We hope that this study will help researchers in the identification and repurposing of multipotent drugs, simultaneously targeting the several viral and host proteins, for the treatment of COVID-19.

identification_of_drugs_targeting_multiple_viral_and_human_proteins_using_computational_analysis_for

Introduction:<!>Methods:<!>Molecular docking of FDA approved drugs in SARS-CoV2 proteins:<!>The differential gene expression (DEGs) analysis:<!>Protein-protein interaction network analysis:<!>Results and discussion:<!>Molecules docking to SARS-CoV2 Structural proteins:<!>Drugs targeting multiple SARS-CoV2 proteins<!>Conclusions

<p>Novel zoonotic viruses with potential for rapid spread and significant pathology pose a grave threat to humans. During the last few decades many epidemics of viral diseases have occurred such as Ebola, Zika, Nipah, Avian influenza (H7N9), HIN1, Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoV1), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV)(1) (2).</p><p>In the end of 2019, mysterious pneumonia cases begin to emerge in China's Wuhan city. A novel coronavirus, which was later renamed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) was found to be the causative organism and the disease was termed "coronavirus diseases-19" (COVID-19) (3). COVID-19 is the world's worst pandemic and has so far affected about 5 million people globally (identified cases as on 19th May 2020), with average lethality of ~3%. Infection with SARS-CoV2 results in acute respiratory distress syndrome (ARDS) leading to lung injury, respiratory distress and lethality. Elderly patients and those with comorbidities have been reported to be at risk of higher mortality.</p><p>The SARS-CoV2, SARS-CoV1 and MERS-CoV belongs to the family of Coronaviridae and β-coronavirus genus (4). While bats are considered to be the origin of SARS-CoV1 and SARS-CoV2, the intermediate host that led to human transmission of SARS-CoV2 is still unknown.</p><p>Sequence analysis reveals that SARS-CoV2 is similar to coronavirus identified in Malayan pangolins (Manis javanica) (5). The SARS-CoV2 genome is 29.8 -29.9kb positive-sense single stranded RNA with 5′-cap and 3′-poly-A tail. Its genome is organised into two segments that encode non-structural (Nsp) and structural proteins. The first segment is directly translated by ribosomal frameshifting into polyprotein 1a (486 kDa) or 1ab (790 kDa) (ORF1a, ORF1ab) which results in generation of non-structural proteins and formation of replication-transcription complex (RTC) (1,6). Discontinuous transcription of the viral genome results in formation of subgenomic RNAs (sgRNAs) containing common 5′-and 3′leader and terminal sequences which serve as the template for subgenomic mRNA production (6). The ORF1a/1ab covers the two-thirds of the whole genomic length and encodes for the 16 non-structural proteins (Nsp1-16), which play critical role in various viral processes. The second segment at the 3′-terminus of the genome encodes the four main structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins (6). The life cycle of SARS-CoV2 starts with its entry into the host cell through receptor mediated endocytosis initiated by the binding of its Spike protein to the ACE2 receptor. Subsequently, uncoating of the virus particle releases the genome, which is translated to generate replication-transcription complex proteins. The viral RTC complex then generates full length negative sense RNA which is subsequently transcribed into full length genome. The viral genome and structural proteins are assembled into virions near the ER and Golgi interface and are transported out of the cell through vesicles by the process of exocytosis (7).</p><p>The detailed understanding of the clinical manifestations and the underlying molecular mechanisms that drive disease pathogenesis are still unclear. There is no standard cure for the disease and currently the therapeutic regimen involves symptomatic treatment and previously approved drugs against other viral infections and diseases. Worldwide efforts to develop vaccines and drug against SARS-CoV2 are ongoing. Based on the similarity and information available from other coronaviruses, repurposing of approved drugs is among the best and rapid strategies to identify potential drug candidates (8). In this context, the computational techniques can quickly identify novel molecules that target viral proteins to suggest candidates for repurposing. Hence, during the COVID pandemic a lot of studies have been reported using a variety of such strategies (9).</p><p>The in-silico studies have identified many drugs that can target viral proteins viz. RNAdependent RNA polymerase (RdRp), Spike, Membrane, 3CL pro and human proteins such as angiotensin converting enzyme 2 (ACE2) which serves as receptor for SARS-CoV2. Among them zanamivir, indinavir, saquinavir, lopinavir, and remdesivir are notable (10,11). There are many drugs such as baricitinib (12), lopinavir (10), ritonavir (13), remdesivir (14,15), hydroxychloroquine (16,17), arbidol (18) etc., that are currently under trial to treat SARS-CoV-2 infection. However, only a few studies have reported targeting more than one viral protein with a single molecule or using combination therapy. In this study we attempted to identify molecules that can simultaneously bind to multiple proteins of the SARS-CoV2. The strategy to target multiple proteins originates from the fact that individual viral proteins play specific role in multiple aspects of viral lifecycle such as attachment, entry, replication, morphogenesis and egress.</p><p>Single molecules that can potentially target many viral proteins can perturb viral lifecycle at multiple points and thereby can be highly efficient in curbing SARS-CoV2 infection. In addition, the molecules that simultaneously target multiple viral proteins will have a higher barrier towards emergence of resistant mutants.</p><p>In this work, we have used the 3D-structures of the SARS-CoV2 proteins to identify FDA approved drugs that can bind to these proteins using bioinformatics methods. The FDA approved drugs were chosen so that they can be quickly repurposed for treating COVID19.</p><p>Additionally, we also analyzed if the identified molecules can affect the host proteins that get differentially expressed as a result of SARS-CoV2 infection. These molecules can be used as modulators of both the SARS-CoV2 and human proteins.</p><!><p>Protein structure modelling:</p><p>The SARS-CoV2 proteins for which there is no crystal structure reported were modelled using Modeller v9.22 (19) (homology modeling) or obtained from I-TASSER server (threading) (20).</p><p>The modelling template for each protein was identified by performing Delta-BLAST against the PDB database. Proteins were modelled using either single or multiple templates based on the query coverage. The final homology modeling was performed using the modeller9. 22.</p><p>Further, the model stereochemistry and other structural parameters were assessed using standalone PROCHECK tool. The proteins for which suitable templates were not found were obtained from I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/).</p><!><p>The structures of the FDA approved drugs were obtained from the DrugBank (https://www.drugbank.ca/) repository. Ligands were prepared by Schrödinger LigPrep wizard ligands using the default parameters.</p><p>The active site of the modelled proteins were identified using either of the two methods 1) the ligand binding pocket, if the co-crystal structures are available or 2) the active site was predicted using sitemap algorithm in Schrodinger v9.3 molecular modelling software (21). The proteins with active site pocket volume of <150 Å 3 were removed as smaller pockets may not be amenable to docking. Finally, 13 proteins were selected for docking. The molecular docking was performed using the Glide module of Schrodinger molecular modelling software (www.schrodinger.com/glide). The molecules showing a docking score of -8.5 (roughly corresponding to 1 µm) (22) or better were selected for further analysis.</p><!><p>The differentially expressed genes were identified by analysing the data from NCBI GEO (GEO ID: GSE-147507) that contains data on cell lines infected with various virus including SARS-CoV-2. The above dataset also included an RNA sequencing study done using lung tissue of two normal and one COVID-19 patients. The DEGs were identified using limmavoom with the criteria of |log2FC≥1| and p-value≤0.01. The DEGs were further studied for their involvement in various pathways, processes and diseases using Ingenuity Pathway Analysis (IPA).</p><!><p>The identified DEGs were mapped for their interactions with other human proteins using HIPPIE v2.2 which contains 14855 proteins and 411430 interactions. The reported proteinprotein interactions with a minimum score of 0.63 (medium confidence, 2 nd quartile) (23) were used for creation of the network using Cytoscape v3.7.2. The largest interconnected component was extracted and connectivity of individual nodes (degree) were calculated to assess their importance. The calculations were performed on the high performance Linux cluster. The flowchart of the methodology is presented in Fig. 1. The differentially expressed genes were identified using analysis of GEO data. Analysis was done to identify important host proteins, enriched pathways, and processes, etc. The DEGs were searched against databases to identify their modulators.</p><!><p>As stated earlier a total of 13 viral proteins (Table 1) were selected for molecular docking. The computational analysis of ligands binding to various proteins is a powerful method to quickly identify potential molecules for further analysis. These methods have been successfully used in various studies (24). In the first stage, the molecules were docked into the SARS-CoV2 proteins using Glide module of Schrodinger (www.schrodinger.com/glide) in standard precision (SP) mode. The redocking was done to ensure the appropriate selection of top hits.</p><p>The molecules were then ranked using Glide score as implied in Schrodinger.</p><p>We adapted the following notion for our drug repurposing analysis: 1. drugs that can inhibit viral entry into host cell by perturbing the function of surface glycoproteins like the spike, membrane and envelope protein. Preventing the function of other non-structural proteins that play accessory role in viral processes such as Nsp2, Nsp4 and Nsp10. 4. drugs that can also affect differentially expressed host proteins in COVID-19 along with the viral proteins.</p><!><p>The hallmark feature of coronaviruses is their transmembrane spike (S) glycoprotein as this protein is reason for its name "Corona" in Latin meaning, "Crown". The spike protein exists as homo-trimers. Each monomer is about 180kDa and has two distinct subunits S1 and S2. While octreotide, and lapatinib bind to spike protein with appreciable affinity (Fig. 2). Other groups have also predicted the binding of posaconazole to spike protein which further substantiates our analysis (27). Posaconazole is an antifungal agent used in the prevention of invasive fungal infections and is also shown to inhibit the entry of Chikungunya virus (28) and replication of Zika and Dengue viruses by binding to oxysterol-binding protein (sterol transporter) (29).</p><p>Octreotide is a long-acting somatostatin analogue used for treatment of gastrointestinal tract bleeding, hepatocellular carcinoma and hemorrhage associated with Cytomegalovirus induced colitis (30) (31). Mefloquine is an antimalarial drug used in chloroquine resistant malaria.</p><p>Nebivolol is an antihypertensive molecule with a very good safety profile in subjects with obstructive respiratory comorbidities (32) and can be an important drug to consider in SARS like diseases. The docking score of -8.5 indicates that nebivolol binds to spike protein with good affinity (Fig. 2). antibodies against N protein suggesting its role in eliciting humoral immune response (37,38).</p><p>Our study predicts that ribavirin, vasopressin, octreotide, and capreomycin bind to N protein (Fig. 4). Of these, capreomycin, a polypeptide (isolated from Streptomyces capreolus) is used in the treatment of multidrug resistant tuberculosis. Previous studies report α-ketoamides, lopinavir and ritonavir as inhibitor of 3CL pro (45,46).</p><p>Indinavir is shown to inhibit HIV protease by blocking its active site and leads to immature virus particle formation, however high doses have been linked to lipodystrophy syndrome (47).</p><p>Naldemedine, is a μ-opioid receptor antagonist used for the treatment of opioid-induced constipation (48). Our study also predicts that tenofovir, nebivolol, ribavirin, nilotinib, lanreotide, ibrutinib, mefloquine, lopinavir, desmopressin, pasireotide, and methotrexate are among top molecules binding to the protease PL pro . An interesting observation is the identification of folic acid as a high affinity ligand of PL pro (Fig. 7). its activity (49). Our analysis shows that lanreotide, methotrexate, octreotide, cangrelor, and pibrentasvir bind to the helicase with high affinity (Fig. 8). Pibrentasvir, is a HCV NS5A</p><p>inhibitor effective against all HCV genotypes (50). Methotrexate acts as an antimetabolite and thus used as an antineoplastic drug. It is also anti-inflammatory and used in treatment of inflammatory diseases like rheumatoid arthritis. It decreases the de novo synthesis of purines and pyrimidines and forms dimers with thymidylate synthase (TS), hence also used as antiparasitic drug. Methotrexate is also shown to effectively reduce replication of Zika and Dengue viruses (51). The most vital enzyme responsible for the replication/transcription of the viral genome is the RNA-dependent RNA polymerase (RdRp) also known as Nsp12. The primer for RdRp RNA synthesis is synthesized by Nsp8 (52). Our analysis shows that cobicistat, capreomycin, pibrentasvir, elbasvir, indinavir and remdesivir among others can bind with RdRp (Fig. 9).</p><p>Cobicistat is known to inhibit the cytochrome-mediated metabolism of HIV protease and was approved in 2012 by FDA as pharmacoenhancer for HIV treatment (53). Other groups have also predicted that cobicistat and capreomycin can inhibit SARS-CoV2 protease (54) (55).</p><p>Pibrentasvir and elbasvir are HCV NS5A inhibitors and indinavir is potent HIV protease inhibitor (56). The molecules we identified to bind to RdRp can serve as potential alternatives to remdesivir.</p><p>The Nsp15 is EndoRNase with endoribonuclease activity. It cleaves the 5′ and 3′ of uridylate residues in RNA by forming 2′-3′cyclic phosphodiester. Its mechanism is similar to that of RNase A, RNAse T1 and XendoU (57). Its NendoU activity can interfere with the host's innate immune response and masks the exposure of viral dsRNA to host dsRNA sensors (58). In our analysis, drugs such as octreotide, desmopressin, macimorelin, and simeprevir were found to target Nsp15 (Fig. 10). Nsp14 is the 3'-5'exonuclease that plays a role in proofreading mechanism (59). Nsp14 contains four conserved DE-D-D acidic and a zinc-finger (ZnF) domain (60). Our molecular docking predicted that cangrelor, venetoclax, pimozide, nilotinib, droperidol, nebivolol, indacaterol, ezetimibe, simeprevir, siponimod, lapatinib, elagolix bind to Nsp14 (Fig. 11).</p><p>Pimozide, a calmodulin inhibitor is shown to inhibit Chikungunya virus secretion (61).</p><p>Moreover, it binds to the envelope protein of HCV and inhibits infection with many HCV genotypes (62). Ezetimibe is shown to inhibit formation of capsid-associated relaxed circular DNA of Hepatitis B Virus (HBV) (63) and is also shown to inhibit Dengue infection by interfering in formation of replication complex (64). Indacaterol is the β2-adrenoceptor agonist and used in the treatment of chronic obstructive pulmonary disease (COPD) since it induces bronchodilation effect (65). It is a promising candidate for therapeutics against SARS-CoV2 due to its ability to regulate genes involved in suppressing proinflammatory cytokine production and attenuation of airway hyper-responsiveness (66). However, dose and treatment schedule needs to be evaluated due to its counter effect on the expression of RNase L which is vital for antiviral response. respectively (41,81). Lanreotide was the only molecule that showed appreciable binding affinity with Nsp10 (Fig. 14).</p><!><p>Cangrelor: is a P2Y12/13 inhibitor and is used as antiplatelet drug. The abnormal blood clotting is an increasingly recognized complication of COVID-19 and adverse prognosis.</p><p>Therefore, it has been suggested to use thrombolytics/antiplatelet agents in the early stages of infection (82,83). Importantly in our screening Cangrelor was found to bind to multiple SARS-CoV2 proteins. Among the important targets are the main protease, spike protein, exonuclease, and helicase. Interestingly the target of Cangrelor in humans is P2Y12/13 which is also found to be differentially expressed as a result of SARS-CoV2 infection (Supplementary table 2).</p><p>Additionally, there are no reported drug interactions between investigational COVID-19 therapies and Cangrelor (97). Therefore, this drug can be important for repurposing to treat the COVID-19. However, it was found to bind with many SARS-CoV2 proteins therefore it has to be seen whether it has a privileged scaffold or it is a promiscuous binder. The short plasma half-life upon intravenous administration may limit its efficacy against SARS-CoV2.</p><p>Nilotinib: is a potent tyrosine kinase inhibitor and is used as an anticancer drug. It is found to bind to multiple SARS-CoV2 proteins. It was intriguing to see it binding with two nonstructural proteins (Nsp2 and Nsp14) with high affinity. A literature survey showed that it has been reported to exhibit antiviral activity against Human Cytomegalovirus (99). The mechanism is not very clear; however, it has been surmised that it may disrupt the productive replication of the virus. The SARS-CoV2 coronavirus depends on Abl2 kinase activity to fuse and enter into the cells. The kinase inhibitor imatinib is already under clinical trials for COVID-19 (https://clinicaltrials.gov/ct2/show/NCT04357613). Thus, other kinase inhibitors that are binding to viral proteins can also be potential candidates for repurposing.</p><p>Lapatinib: is another potent kinase inhibitor, similar to nilotinib, that is showing good binding with the SARS-CoV2 proteins (S-protein, Nsp4 and Nsp13). Lapatinib is a HER2 (ligandindependent receptor tyrosine kinase) inhibitor used in the treatment of HER2-positive breast cancers (84). Interestingly, HER2 inhibition is shown to activate TBK1 through cGAS-STING pathway which plays a crucial role in anti-viral innate immune signaling (80). Hence, lapatinib can be effective in perturbing SARS-CoV2 replication as well as upregulating anti-viral signaling.</p><p>Lancreotide: a long-acting analog of the drug somatostatin widely used in the treatment of Graves' ophthalmopathy, Acromegaly and Endocrine tumors ( 85),( 86),( 87), (88), is found to have high binding affinity to multiple SARS-CoV2 proteins such as PL pro (Nsp3), Nsp10, 13 and 16.</p><p>Octreotide: another somatostatin analog similar to lancreotide. It is used in the treatment of diarrhoea, pancreatic neuroendocrine tumors and massive hemorrhage caused by cytomegalovirus colitis (89),( 90), (40). This drug also binds to the multiple proteins of SARS Cov2 virus which includes the structural proteins S and N and the helicase, endonuclease and methyl transferase. Study suggested, several anticancer drug have potential target of SARS-Cov2 as repurposing drugs (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239789/).</p><p>Capreomycin: a polypeptide isolated from Streptomyces capreolus used in the treatment of multidrug resistant tuberculosis and co-infected (HIV) is also found to bind with 2'OMT, nuclease and RdRp proteins. However, this compound has serious nephrotoxicity and ototoxicity, which has to be taken in into account (39). Pibrentasvir: the hepatitis C virus NS5A inhibitor was found to bind with helicase and RdRp proteins.</p><p>Indinavir: HIV protease inhibitor and major component of highly active antiretroviral therapy (HAART) for treatment of HIV/AIDS was found to bind RdRp and 3CL pro protease with good affinity.</p><p>Venetoclax: is a BCL-2 (antiapoptotic protein) inhibitor is used for the treatment of chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) (93). It is found to bind with Nsp16 and exonuclease.</p><p>Alatrofloxacin a fluroquinolone antibiotic that targets the DNA gyrase enzyme was found to bind with Nsp2 and Nsp4 proteins with good affinity.</p><p>A heatmap (Fig. 15) was generated using the docking scores to summarize the binding of important drugs to multiple proteins. The detailed list drugs and their docking scores is given in supplementary table 1. As stated earlier, the differentially expressed genes were identified by analyzing the data from NCBI GEO (GEO ID: GSE-147507) using limma-voom with the criteria of |log2FC≥1| and p-value≤0.01. The differentially expressed genes were then analyzed using IPA and Cytoscape.</p><p>Details are given in Supplementary Table 2. A protein-protein-interaction network analysis was done using Cytoscape to identify the network hubs based on their interactions with other proteins using Degree centrality. The giant component was extracted from the network with 1446 nodes containing 1770 interactions. Top 5% of the proteins (total 72) were selected as hubs based on the degree centrality for further analysis.</p><p>A search for the DEGs (136) at drug-gene interaction database (DGIdb) resulted in the identification of 352 drugs. (Supplementary table 3). Further, we wanted to know if some of them can also target viral proteins. An intersection with drugs identified early revealed 29 drugs that can target both viral and human proteins. It is worth to mention that among the human proteins many of them (e.g. IL2RG, DAPP1, CCL7, IFIT1, MMP8, FOS) are hubs i.e. very important proteins in the generated protein-protein-interaction network. Therefore, the drugs with multi-targeting ability against these proteins as well as SARS-CoV2 proteins can have a significant therapeutic utility for COVID-19 which is a novelty of this study (Supplementary Table 4).</p><p>The analysis indicated that some of proteins upregulated during SARS-CoV2 infection are also targeted by the drugs identified to bind to viral proteins in our analysis. TUBB48 (Tubulin Beta 4A Class IVa) is upregulated during SARS-CoV2 infection. Previous study suggests that druginduced microtubule depolymerization results in reduction of infectious virus particle release due to defect in spike protein incorporation into the virions. Paclitaxel, which targets the N protein, is a cytoskeletal drug which stabilizes the microtubule polymer formation and protects it from disassembly (94). The transcription complex Activator protein 1 (AP1) is composed of homo/hetero dimers of Fos, Jun, CREB and others ATFs. The studies on the SARS-CoV1 infection in the Vero and Huh7 cell shows that nucleocapsid protein is the potent activator of (AP-1) (95). Interestingly, asthmatic patients show higher expression of c-fos in their epithelial cells. It is also observed that TNF-α induced ROS and intracellular glutathione depletion in the airway epithelial cells induces the production of AP-1 and leads to the pulmonary fibrosis (96,97). Our analysis suggests that paclitaxel and bromocriptine which dock with nucleocapsid and Nsp4 proteins can also effectively bind to c-Fos and thereby would be beneficial in inhibiting the SARS-CoV2 as well as in alleviating lung injury observed in COVID19. The transcriptome analysis revealed that S100/calgranulin is upregulated during SARS-CoV2 infection.</p><p>Calgranulin is polypeptide released by the activated inflammatory cells such as leukocytes, PBMC phagocytes and lymphocytes and is accumulated at the sites of chronic inflammation.</p><p>It is the ligand for RAGE receptors and is the major initiator of cascading events in inflammation amplification (98). Interestingly, the differential gene expression analysis of PBMCs from SARS patients shows the higher expression of this calgranulin families (97) and this protein is found in higher quantity in the Bronchoalveloar Lavage Fluid (BALF) and sputum of patients with inflamed lungs, COPD, and ARDS (99). Our analysis suggests that the anti-inflammatory agent methotrexate which has high affinity to the Nsp13 protein of SARS-CoV2 also shows appreciable binding to calgranulin and can thereby be useful to curtail systemic inflammation in lungs observed during COVID19 in addition to its inhibitory effect on SARS-CoV2. Transcriptomic analysis also suggests increased expression of endogenous prolactin, which leads to prolactin induced STAT5 activation and its pathways. Prolactin has a dual role in human physiology functioning as a hormone (secreted from anterior pituitary gland) and cytokine (secreted by immune cells). It causes anti-apoptotic effect and induces proliferation in immune cells in response to antigens leading to increased production of immunoglobulin, cytokines, and autoantibodies (100). We envisage that prolactin may be one of the significant player in trigger of cytokine storm implicated in COVID19. Interestingly, our study suggests that zidovudine which target the O'-methyl transferase (Nsp16) can also bind to prolactin and can be of high significance in management of COVID19 due to dual ability to affect Nsp16 and prolactin.</p><!><p>The overall goal of this study was to identify molecules that can dock with multiple SARS-CoV2 proteins that play vital role(s) in the viral lifecycle. Our study predicted several promising drug candidates with high binding affinity towards many of SARS-CoV2 proteins.</p><p>These drugs will be very effective than drugs that target single viral proteins due to their ability which targets the exonuclease is also a promising agent due to its ability to regulate genes involved in suppressing pro-inflammatory cytokine production and attenuation of airway hyper-responsiveness (71). We also observed that the molecules that bind to Nsp4, 13, 16 and N proteins can also bind to human proteins that play a pivotal role in disease pathogenesis by promoting inflammatory signalling leading to cytokine storm thereby suggesting that the molecules such as a paclitaxel, methotrexate, and zidovudine can have a dual beneficial effect in the management of COVID19. Overall our study predicts promising agents with potential to inhibit crucial viral processes, upregulate anti-viral host response and alleviate severe lung disease condition thereby providing attractive avenues for design of potential and multipronged therapeutic strategies against COVID 19.</p>

ChemRxiv

Surprising Conformers of the Biologically Important A·T DNA Base Pairs: QM/QTAIM Proofs

For the first time novel high-energy conformers–A·T(wWC) (5.36), A·T(wrWC) (5.97), A·T(wH) (5.78), and A·T(wrH) (ΔG = 5.82 kcal·mol−1) (See Graphical Abstract) were revealed for each of the four biologically important A·T DNA base pairs – Watson-Crick A·T(WC), reverse Watson-Crick A·T(rWC), Hoogsteen A·T(H) and reverse Hoogsteen A·T(rH) at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of quantum-mechanical theory in the continuum with ε = 4 under normal conditions. Each of these conformers possesses substantially non-planar wobble (w) structure and is stabilized by the participation of the two anti-parallel N6H/N6H′…O4/O2 and N3H…N6 H-bonds, involving the pyramidalized amino group of the A DNA base as an acceptor and a donor of the H-bonding. The transition states – TSA·T(WC)↔A·T(wWC), TSA·T(rWC)↔A·T(wrWC), TSA·T(H)↔A·T(wH), and TSA·T(rH)↔A·T(wrH), controlling the dipole-active transformations of the conformers from the main plane-symmetric state into the high-energy, significantly non-planar state and vice versa, were localized. They also possess wobble structures similarly to the high-energy conformers and are stabilized by the participation of the N6H/N6H′…O4/O2 and N3H…N6 H-bonds. Discovered conformers of the A·T DNA base pairs are dynamically stable short-lived structures [lifetime τ = (1.4–3.9) ps]. Their possible biological significance and future perspectives have been briefly discussed.

surprising_conformers_of_the_biologically_important_a·t_dna_base_pairs:_qm/qtaim_proofs

Introduction<!><!>Introduction<!>Density functional theory calculations of the geometry and vibrational frequencies<!>Single point energy calculations<!>Evaluation of the interaction energies<!>Estimation of the kinetic parameters<!>QTAIM analysis<!>Calculation of the energies of the intermolecular H-bonds<!>Results and their discussion<!><!>Results and their discussion<!><!>Results and their discussion<!>Conclusions<!>Author contributions<!>Conflict of interest statement

<p>Investigation of the dynamics of the isolated DNA base pairs by both the experimental and especially theoretical methods is urgent biophysical task of exceptional importance (Keepers et al., 1982; Pechenaya and Volkov, 1984; Volkov, 1995; Auffinger and Westhof, 1999). At this, the researchers are convinced that exactly the intrinsic conformational dynamics of the DNA base pairs largely determines the functionally important dynamical behavior of DNA and this approach has no reasonable alternatives.</p><!><p>For the first time it was revealed novel high-energy conformers A·T(wWC) (5.36), A·T(wrWC) (5.97), A·T(wH) (5.78) and A·T(wrH) (ΔG = 4.98 kcal/mol under normal conditions) at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of quantum-mechanical theory in the continuum with ε = 4.</p><!><p>Spontaneous thermal fluctuations or breathing of DNA enables the opening of the DNA base pairs, making reactive their chemical groups, that are normally hidden inside the DNA double helix, available for hydrogen exchange involving imino and amino groups, chemical modification (e.g., by formaldehyde, that is a toxic, mutagenic and carcinogenic compound leading to fatal consequences or mutagenesis) and important protein-DNA interactions by the participation of the regulatory proteins (Lazurkin et al., 1970; Frank-Kamenetskii and Lazurkin, 1974; Lukashin et al., 1976; Chay, 1979; Frank-Kamenetskii, 1981, 1983, 1985; Guéron et al., 1987; Guéron and Leroy, 1995; von Hippel et al., 2013; Frank-Kamenetskii and Prakash, 2014). Notably, that reactions of the hydrogen exchange and formaldehyde interaction with DNA were the first documented cases evidencing the opening of the DNA base pairs (Lazurkin et al., 1970; Frank-Kamenetskii and Lazurkin, 1974; Lukashin et al., 1976; Chay, 1979; Guéron et al., 1987).</p><p>Moreover, it is believed that opening of the DNA base pairs with a defined probability ~10−5 (Lazurkin et al., 1970; Frank-Kamenetskii and Lazurkin, 1974; Lukashin et al., 1976; Frank-Kamenetskii, 1981, 1985; Guéron et al., 1987; Guéron and Leroy, 1995; von Hippel et al., 2013; Frank-Kamenetskii and Prakash, 2014) precedes the melting of DNA, that is represent the two-state model according to which each base pair is suggested to stay in the closed or open states (Frank-Kamenetskii and Lazurkin, 1974; Lukashin et al., 1976; Chay, 1979; Frank-Kamenetskii, 1981, 1983; Guéron et al., 1987; Singh and Singh, 2017). Exactly this model could quantitatively explain in details the melting of DNA processing in the multistate way due the different length and heterogeneous sequence (Vologodskii et al., 1984; Wartell and Benight, 1985; Wada and Suyama, 1986; SantaLucia, 1998). At this, predicted lifetimes of the open state of the base pairs lie in the sub microsecond range (~10−7 s) (Craig et al., 1971; Porschke and Eigen, 1971; Frank-Kamenetskii, 2011). However, according to the literature data it remains unknown, what the nature of the open state of the DNA base pairs is and whether there is a barrier on the potential energy surface for providing its existence (Lavery, 1994; Stofer et al., 1999; Yang et al., 2015).</p><p>It was also demonstrated by NMR experiment (Nikolova et al., 2011, 2013) a Hoogsteen breathing consisting in the flipping of the Watson-Crick DNA base pair from the usual anti-conformation to the less favorable syn-conformation with probability ~10−2, representing another pathway for the reaction of formaldehyde attack on DNA (Bohnuud et al., 2012).</p><p>Since the model of two states—H-bonded base pair and opened base pair—is not able to describe in details the dynamical behavior of DNA, which experimentally manifests itself in a number of its physico-chemical properties (Lazurkin et al., 1970; Craig et al., 1971; Porschke and Eigen, 1971; Frank-Kamenetskii and Lazurkin, 1974; Lukashin et al., 1976; Chay, 1979; Frank-Kamenetskii, 1981, 1983, 1985, 2011; Vologodskii et al., 1984; Wartell and Benight, 1985; Wada and Suyama, 1986; Guéron et al., 1987; Lavery, 1994; Guéron and Leroy, 1995; SantaLucia, 1998; Stofer et al., 1999; Giudice et al., 2001; Ababneh et al., 2003; Coman and Russu, 2005; Nikolova et al., 2011, 2013; Bohnuud et al., 2012; von Hippel et al., 2013; Frank-Kamenetskii and Prakash, 2014; Yang et al., 2015; Singh and Singh, 2017), the searching of new conformational states of the DNA base pairs near their Watson-Crick global minima has been intensified (Keepers et al., 1982; Pechenaya and Volkov, 1984; Volkov, 1995; Giudice et al., 2003; Pérez et al., 2007; Lindahl et al., 2017).</p><p>The modeling of the conformational heterogeneity of the Watson-Crick A·T DNA base pair allowing the existence of the semiopen states in DNA, which is associated with the presence of the weak C2H…O2 H-bond in it, and their support by the semi-empirical quantum-chemical MNDO/H (Hovorun, 1997) and PM3 (Kryachko and Volkov, 2001) methods presented in the papers (Hovorun, 1997; Kryachko and Volkov, 2001) seems attractive. Moreover, none of these interesting ideas has been confirmed by ab initio methods.</p><p>Nowadays in the literature it does not present the data confirming the presence of the stable conformational states in the isolated Watson-Crick DNA base pairs, except canonical ones (Lavery, 1994; Stofer et al., 1999). It is obviously connected with the lack of the new ideas according as the structural features of the complementary foundations, so the nature of the intermolecular interactions, first of all of the H-bonds responsible for the presence of the conformers, which differs from the classical ones.</p><p>In present work basing on our previous data (Brovarets' and Hovorun, 2014a,b,c, 2015a,b; Glushenkov and Hovorun, 2016), we adhere to the idea that the pyramidalized amino group of the adenine (A) DNA base can simultaneously form two antiparallel N6H/N6H′…O4/O2 and N3H…N6 H-bonds with thymine (T) DNA base, thus supporting high-energy non-planar conformers of the biologically important A·T DNA base pairs. We succeeded to localize the transition states (TSs) connecting the main plane-symmetrical conformers of the A·T base pairs (global minimum) with the established significantly non-planar high-energy conformers. On the basis of the obtained data, we expressed the assumption according the possible biological importance of the discovered conformers of the canonical A·T DNA base pairs.</p><p>We chose as the object of the investigation of the biologically-important A·T DNA base pairs, in particular–Watson-Crick A·T(WC), reverse Watson-Crick A·T(rWC), Hoogsteen A·T(H) and reverse Hoogsteen A·T(rH) base pairs (Donohue and Trueblood, 1960; Haschemeyer and Sobell, 1963; Hoogsteen, 1963; Tchurikov et al., 1989; Liu et al., 1993; Parvathy et al., 2002; Sühnel, 2002; Zagryadskaya et al., 2003; Brovarets', 2013a,b; Alvey et al., 2014; Nikolova et al., 2014; Yang et al., 2015; Poltev et al., 2016; Zhou, 2016; Sathyamoorthy et al., 2017; Szabat and Kierzek, 2017; Ye et al., 2017).</p><p>Thus, the reverse A·T(rWC) Watson-Crick or so-called Donohue DNA base pair (Donohue and Trueblood, 1960), which is formed by the rotation of one of the bases according to the other by 180° around the N1–N3 axis of the Watson-Crick A·T(WC) DNA base pair, has been registered in the bioactive parallel-stranded DNA (Tchurikov et al., 1989; Parvathy et al., 2002; Brovarets', 2013a,b; Poltev et al., 2016; Szabat and Kierzek, 2017; Ye et al., 2017).</p><p>The A·T(H) Hoogsteen base pair (Hoogsteen, 1963) is formed due to the rotation on 180° of the A DNA base relative to the T DNA base around the C9-N9 axis from the anti (WC) to syn (H) conformation, representing itself alternative DNA conformation that is involved into a number of biologically important processes such as recognition, damage induction, replication and has been actively investigated in the literature (Hoogsteen, 1963; Brovarets', 2013a,b; Alvey et al., 2014; Nikolova et al., 2014; Yang et al., 2015; Zhou, 2016; Sathyamoorthy et al., 2017). In particular, in the canonical DNA double helix Watson–Crick base pairs exist in a dynamic equilibrium with sparsely populated (~0.02–0.4%) and short-lived (lifetimes ~0.2–2.5 ms) Hoogsteen base pairs (Zhou, 2016).</p><p>At this, the reverse A·T(rH) Hoogsteen or so-called Haschemeyer–Sobell base pair (Haschemeyer and Sobell, 1963), that is formed by the rotation of one of the bases by 180° around the N7–N3 axis of the base pair according the other base (Brovarets', 2013a,b), also plays important biological role (Liu et al., 1993; Sühnel, 2002; Zagryadskaya et al., 2003).</p><!><p>Geometries of the main and high-energy conformers and transition states (TSs) of their mutual conformational transformations, as well as their harmonic vibrational frequencies have been calculated at the B3LYP/6-311++G(d,p) level of theory (Hariharan and Pople, 1973; Krishnan et al., 1980; Lee et al., 1988; Parr and Yang, 1989; Tirado-Rives and Jorgensen, 2008), using Gaussian'09 package (Frisch et al., 2010). Applied level of theory has proved itself successful for the calculations of the similar systems (Brovarets' and Hovorun, 2010a,b, 2015c; Matta, 2010; Brovarets' et al., 2015b). A scaling factor that is equal to 0.9668 has been applied in the present work for the correction of the harmonic frequencies of all conformers and TSs of their conformational transitions (Palafox, 2014; Brovarets' and Hovorun, 2015c; Brovarets' et al., 2015b; El-Sayed et al., 2015). We have confirmed the local minima and TSs, localized by Synchronous Transit-guided Quasi-Newton method (Peng et al., 1996), on the potential energy landscape by the absence or presence, respectively, of the imaginary frequency in the vibrational spectra of the complexes. We applied standard TS theory for the estimation of the activation barriers of the tautomerisation reaction (Atkins, 1998).</p><p>All calculations have been carried in the continuum with ε = 4, that adequately reflects the processes occurring in real biological systems without deprivation of the structurally functional properties of the bases in the composition of DNA and satisfactorily models the substantially hydrophobic recognition pocket of the DNA-polymerase machinery as a part of the replisome (Bayley, 1951; Dewar and Storch, 1985; Petrushka et al., 1986; García-Moreno et al., 1997; Mertz and Krishtalik, 2000; Brovarets' and Hovorun, 2014d,e).</p><!><p>We continued geometry optimizations with electronic energy calculations at the single point at the MP2/aug-cc-pVDZ level of theory (Frisch et al., 1990; Kendall et al., 1992).</p><p>The Gibbs free energy G for all structures was obtained in the following way:</p><p>where Eel-electronic energy, while Ecorr-thermal correction.</p><!><p>Electronic interaction energies ΔEint have been calculated at the MP2/6-311++G(2df,pd) level of theory as the difference between the total energy of the base pair and energies of the monomers and corrected for the basis set superposition error (BSSE) (Boys and Bernardi, 1970; Gutowski et al., 1986) through the counterpoise procedure (Sordo et al., 1988; Sordo, 2001).</p><!><p>The time τ99.9% necessary to reach 99.9% of the equilibrium concentration of the reactant and product in the system of the reversible first-order forward (kf) and reverse (kr) reactions was estimated by the formula (Atkins, 1998):</p><p>The lifetime τ of the conformers has been calculated using the formula 1/kr, where the values of the forward kf and reverse kr rate constants for the tautomerisation reactions were obtained as (Atkins, 1998):</p><p>where quantum tunneling effect has been accounted by Wigner's tunneling correction (Wigner, 1932), successfully used for the double proton reactions in DNA base pairs (Brovarets' and Hovorun, 2013, 2014c):</p><p>where kB–Boltzmann's constant, h–Planck's constant, ΔΔGf,r–Gibbs free energy of activation for the conformational transition in the forward (f) and reverse (r) directions, νi–magnitude of the imaginary frequency associated with the vibrational mode at the TSs.</p><!><p>Bader's quantum theory of Atoms in Molecules (QTAIM) (Bader, 1990; Matta and Hernández-Trujillo, 2003; Matta et al., 2006a; Cukrowski and Matta, 2010; Matta, 2014; Lecomte et al., 2015), using program package AIMAll (Keith, 2010), was applied to analyse the electron density distribution. The presence of the bond critical point (BCP), namely the so-called (3,−1) BCP, and a bond path between hydrogen donor and acceptor, as well as the positive value of the Laplacian at this BCP (Δρ > 0), were considered as criteria for the H-bond formation (Bader, 1990; Matta and Hernández-Trujillo, 2003; Matta et al., 2006a; Cukrowski and Matta, 2010; Matta, 2014; Lecomte et al., 2015). Wave functions were obtained at the level of theory used for geometry optimisation.</p><!><p>The energies of the intermolecular uncommon H-bonds (Brovarets' et al., 2013, 2015a) in the base pairs were calculated by the empirical Espinosa-Molins-Lecomte (EML) formula based on the electron density distribution at the (3,−1) BCPs of the specific contacts (Espinosa et al., 1998; Matta, 2006; Matta et al., 2006b; Mata et al., 2011; Brovarets' et al., 2014a):</p><p>where V(r) – value of a local potential energy at the (3,−1) BCP.</p><p>The energies of all other conventional AH···B H-bonds were evaluated by the empirical Iogansen's formula (Iogansen, 1999):</p><p>where Δν—magnitude of the frequency shift of the stretching mode of the AH H-bonded group involved in the AH···B H-bond relatively the unbound group. The partial deuteration was applied to minimize the effect of vibrational resonances (Brovarets' and Pérez-Sánchez, 2016a, 2017; Brovarets' et al., 2016, 2017a,b, 2018; Brovarets' and Hovorun, in press).</p><p>The atomic numbering scheme for the DNA bases is conventional (Saenger, 1984).</p><!><p>For the first time we have detected on the potential (electronic) energy surface of each of the four biologically important A·T(WC), A·T(rWC), A·T(H) and A·T(rH) DNA base pairs the shallow local minima (ΔΔE < kT under normal conditions) corresponding to the dynamically stable A·T(wWC), A·T(wrWC), A·T(wH) and A·T(wrH) conformers, correspondingly, with shifted, wobble (w) architecture (Figure 1). These conformers possess significantly non-planar structure (see Table 1 with the selected angles of the non-planarity) and C1 point group of symmetry. At this, the piramidalized amino group of the A DNA base is involved into the intermolecular H-bonding with T base through two anti-parallel N6H…O4/O2 and N3H…N6 H-bonds in the A·T(WC)/A·T(rWC) base pairs and N6H′…O4/O2 and N3H…N6 H-bonds in the A·T(H)/A·T(rH) DNA base pairs. In all conformers and TSs without exception the N3H…N6 H-bonds with significantly increased ellipticity are weaker than the N6H/N6H′…O4/O2 H-bonds (Table 2). These interactions should be attributed to the weak and medium H-bonds according to the existing classification (Saenger, 1984). Their most important characteristics are presented in Table 2. It should be noted that each of the four investigated A·T DNA base pairs in the basic plane-symmetric conformation is stabilized by the participation of the three intermolecular H-bonds, one of which, namely, the C2H/C8H…O4/O2 is non-canonical (Brovarets' et al., 2013, 2015a). For all A·T DNA base pairs without exception the middle N3H…N1/N7 H-bonds are the strongest (~7 kcal·mol−1). At this, the total energy of the intermolecular H-bonds in each complex consists only some part of the total electronic energy of the interaction between the bases (Figure 1, Table 2). The same regularity is observed for the other DNA base pairs (Brovarets' et al., 2014b; Brovarets' and Hovorun, 2015d,e,f,g, 2016b). For all conformers without exception the amino H or H' atom of the A DNA base, that directly takes part in the H-bonding with T DNA base, significantly deviates from the plane of the purine ring in comparison with the other H′ or H hydrogen atom (Table 1). In all cases the high-energy conformers of the biologically important A·T base pairs are more polar than main conformers (Table 2).</p><!><p>Reaction pathways of the discovered conformational transitions of the four biologically important A·T DNA base pairs. Electronic energies of the interaction ΔEint (MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory, in kcal·mol−1), relative Gibbs free energies ΔG and electronic energies ΔE (in kcal·mol−1), imaginary frequencies νi at the TSs of the conformational transitions (MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of theory in the continuum with ε = 4 at T = 298.15 K) are presented below pathways in brackets. Dotted lines indicate AH···B H-bonds – their lengths are presented in angstroms (for more detailed physico-chemical characteristics of the H-bonds see Table 2); carbon atoms are in light-blue, nitrogen – in dark-blue, hydrogen – in gray and oxygen – in red.</p><p>Selected geometrical parameters characterizing the non-planarity of the discovered conformers of the four biologically important A·T DNA base pairs and TSs of their conformational transitions to the main conformers with plane symmetry, obtained at the B3LYP/6-311++G(d,p) level of theory in the continuum with ε = 4.</p><p>Electron-topological, geometrical and energetic characteristics of the intermolecular H-bonds in the investigated conformers of the A·T DNA base pairs and TSs of their conformational transformations obtained at the B3LYP/6-311++G(d,p) level of theory (ε = 4) (see Figure 1).</p><p>The electron density at the (3,−1) BCP of the H-bond, a.u.</p><p>The Laplacian of the electron density at the (3,−1) BCP of the H-bond, a.u.</p><p>The ellipticity at the (3,−1) BCP of the H-bond.</p><p>The distance between the A (H-bond donor) and B (H-bond acceptor) atoms of the AH…B H-bond, Å.</p><p>The distance between the H and B atoms of the AH…B H-bond, Å.</p><p>The H-bond angle, degree.</p><p>The energy of the H-bonds, calculated by Iogansen's or Espinose-Molins-Lecomte (marked with an asterisk) formulas, kcal·mol−1.</p><p>The dipole moment of the complex, D.</p><!><p>We have also localized the non-planar transition states of the A·T(WC)↔A·T(wWC), A·T(rWC)↔A·T(wrWC), A·T(H)↔A·T(wH) and A·T(rH)↔A·T(wrH) conformational transitions - TSA·T(WC)↔A·T(wWC), TSA·T(rWC)↔A·T(wrWC), TSA·T(H)↔A·T(wH) and TSA·T(rH)↔A·T(wrH), respectively, with low values of imaginary frequency (7.1, 11.4, 9.4 and 14.6 i cm−1). These wobble structures (Table 1) are supported by the couple of the anti-parallel intermolecular H-bonds - N6H…O4/O2 and N3H…N6 H-bonds (A·T(WC)↔A·T(wWC) and A·T(rWC)↔A·T(wrWC), respectively), N6H′…O4/O2 and N3H…N6 H-bonds (A·T(H)↔A·T(wH) and A·T(rH)↔A·T(wrH), respectively) (Figure 1, Table 2). Characteristically, that all revealed conformational transitions without exception are dipole-active, since they are accompanied by the changing of the dipole moment of the initial and terminal base pairs. At this, TSs of each conformational transition have maximal value of the dipole moment (Table 2).</p><p>Main characteristics of the investigated conformational transitions are presented in Table 3. Analysis of these data points that short-lived conformers are dynamically-stable structures with the lifetimes (1.4–3.9) · 10−12 s. Really, for all of them the energy of zero vibrations, which frequency become imaginary in the TS, is less than the electronic energy of the electronic energy barrier ΔΔE for the reverse conformational transition and Gibbs free energy barrier for the reverse conformational transition ΔΔG > 0 under normal conditions. Notably, the range of the six low-frequency intermolecular vibrations of the discovered conformers is significantly shifted to the low-frequency region comparably with the main conformational states. These data points on the fact that revealed conformers are quite soft structures, that could be easily deformed under the influence of the external forces, in particular, caused by the stacking interactions with the neighboring DNA bases.</p><!><p>Energetic and kinetic characteristics of the discovered conformational transitions of the four biologically important A·T DNA base pairs obtained at the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) (marked by the asterisk) and MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) (marked by the double asterisk) levels of theory in the continuum with ε = 4.</p><p>The imaginary frequency at the TS of the conformational transition, cm−1.</p><p>The Gibbs free energy of the product relatively the reactant of the conformational transition (T = 298.15 K), kcal·mol−1.</p><p>The electronic energy of the product relatively the reactant of the conformational transition, kcal·mol−1.</p><p>The Gibbs free energy barrier for the forward conformational transition, kcal·mol−1.</p><p>The electronic energy barrier for the forward conformational transition, kcal·mol−1.</p><p>The Gibbs free energy barrier for the reverse conformational transition, kcal·mol−1.</p><p>The electronic energy barrier for the reverse conformational transition, kcal·mol−1.</p><p>The forward rate constant for the conformational transition, s−1.</p><p>The reverse rate constant for the conformational transition, s−1.</p><p>The time necessary to reach 99.9% of the equilibrium concentration between the reactant and the product of the conformational transition, s.</p><p>The lifetime of the product of the conformational transition, s.</p><!><p>The methyl group of the T DNA base does not change its orientation during the process of the conformational transformations. Moreover, the heterocycles of the bases remain planar, despite their ability for the out-of-plane bending (Govorun et al., 1992; Hovorun et al., 1999; Nikolaienko et al., 2011).</p><p>Special attention should be payed to the characteristic specificities of the A·T(WC)↔A·T(wWC), A·T(rWC)↔A·T(wrWC), A·T(H)↔A·T(wH) and A·T(rH)↔A·T(wrH) conformational transformations. These reactions are non-dissociative, since they are accompanied by the transformation of the H-bonds and rupture of only some of them. Intermolecular N6H/N6H′…O4/O2 H-bonds exist along all intrinsic reaction coordinate opposite the N3H…N1/N7 H-bonds, that initially weaken and then rupture with a time delay in order to transform into the N3H…N6 H-bond. In other words, in the process of the conformational transformations the N3H group of the T DNA base as proton donor remain for some time free from the intermolecular H-bonding. This comes up with an opinion that discovered conformational transitions could be used for the explanation of the occurrence of the hydrogen-deuterium exchange in the A·T DNA base pairs. It is not excluded that revealed by us novel corridor of the spontaneous thermal fluctuations of the A·T DNA base pairs accompanied by the transformation of the base pair from the plane-symmetric geometry into the significantly non-planar wobble conformation could be useful for the explanation of the specificities of the blurriness of the transition at the DNA pre-melting enriched by the A·T DNA base pairs, that could not be explained in details in the framework of the two-states model.</p><p>We would continue to work in the direction of the elucidation of the biological importance of the revealed unusual conformers of the biologically important A·T DNA base pairs.</p><!><p>In general, in this work at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of theory in the continuum with ε = 4 for the first time we have revealed the A·T(WC)↔A·T(wWC), A·T(rWC)↔A·T(wrWC), A·T(H)↔A·T(wH) and A·T(rH)↔A·T(wrH) conformational transformations in the biologically important A·T DNA base pairs and characterized their structural, energetic, polar and dynamical features. These data open new perspectives for the understanding of the physico-chemical mechanisms of the opening of the base pairs preceding DNA melting and also to describe in details the breathing of DNA, that has been experimentally registered. Moreover, it is also the subject for the investigation by using modern spectroscopic techniques such as two-dimensional fluorescent spectroscopy (2DFS) (Widom et al., 2013), time-resolved single molecule fluorescence resonant energy transfer (smFRET) (Lee et al., 2013), single molecule fluorescent linear dichroism (smFLD) (Phelps et al., 2013) and THz spectroscopy (Alexandrov et al., 2013).</p><!><p>OB, performance of calculations, discussion of the obtained data, preparation of the text of the manuscript. DH, proposition of the task of the investigation, discussion of the obtained data, preparation of the text of the manuscript. KT, preparation of the numerical data for Tables and graphical materials for Figures, preparation of the text of the manuscript. All authors were involved in the proofreading of the final version of the manuscript.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>

PubMed Open Access

The \xcf\x80 Configuration of the WWW Motif of a Short Trp-rich Peptide Is Critical for Targeting Bacterial Membranes, Disrupting Preformed Biofilms and Killing Methicillin-resistant Staphylococcus aureus

Tryptophan-rich peptides, being short and suitable for large-scale chemical synthesis, are attractive candidates for developing a new generation of antimicrobials to combat antibiotic-resistant bacteria (superbugs). Although there are numerous pictures for membrane-bound structure of a single tryptophan (W), how multiple Trp amino acids assemble themselves and interact with bacterial membranes are poorly understood. This communication presents the three-dimensional structure for an eight-residue Trp-rich peptide (WWWLRKIW-NH2 with 50% W) determined by the improved 2D NMR method, which includes the measurements of 13C and 15N chemical shifts at natural abundance. This peptide forms the shortest two-turn helix with a distinct amphipathic feature. A unique structural arrangement is identified for the Trp triplet, WWW, that forms a \xcf\x80 configuration with W2 as the horizontal bar and W1/W3 forming the two legs. Arginine scan reveals that the WWW motif is essential for killing methicillin-resistant Staphylococcus aureus USA300 and disrupting preformed bacterial biofilms. This unique \xcf\x80 configuration for the WWW motif is stabilized by aromatic-aromatic interactions as evidenced by ring current shifts as well as nuclear Overhauser effects. By maintaining the WWW motif, a change of I7 to R led to a potent antimicrobial and antibiofilm peptide with four-fold improvement in cell selectivity. Collectively, this study elucidated the structural basis of antibiofilm activity of the peptide, identified a better peptide candidate via structure-activity relationship studies, and laid the foundation for engineering future antibiotics based on the WWW motif.

the_\xcf\x80_configuration_of_the_www_motif_of_a_short_trp-rich_peptide_is_critical_for_targeting_ba

<p>Naturally occurring antimicrobial peptides (AMPs) are endogenous defense molecules of living organisms.1 They remain potent for millions of years, making them appealing templates for developing the next generation of antimicrobials to combat superbugs and difficult-to-kill viruses. The amino acid use in such peptides is biased so that they have the desired sequence feature to recognize invading pathogens. In α-helical amphibian AMPs, glycine, leucine, alanine, and lysine are rich, whereas glycine, arginine, and cysteine are abundant in β-sheet peptides. Such abundant amino acids can determine the peptide scaffold.2 In contrast, certain amino acids, such as histidine (His) and tryptophan (Trp), are less frequently deployed on average in natural AMPs (http://aps.unmc.edu/AP; Figure S1 in Supporting Information). However, Trp and His can be abundant in some peptides. Interest in Trp-rich peptides remains high because they are relatively short yet potent against superbugs. Usually, these Trp-rich peptides are also accompanied by arginine (Arg).3 The combination of hydrophobic Trp and cationic Arg are sufficient to generate amphipathic sequences. Such amphipathic sequences lay the foundation for AMPs to target bacterial membranes. While cationic residues can recognize anionic surfaces, hydrophobic Trp amino acids can anchor the entire peptide to the membrane. Structural determination of membrane-bound AMPs enables us to view the active conformation and provides a basis for structure-based peptide design. Most of the structures of small AMPs are determined by the classic 2D NMR method,4a which typically requires the recording of TOCSY, DQF-COSY, and NOESY spectra. Due to the complex nature of biological membranes, most of the structural characterization of Trp-containing AMPs was conducted in membrane-mimetic environments such as micelles. The structures of indolicidin and tritrpticin are extended with multiple turns.3 How the multiple aromatic rings bind to membranes is not always obvious because they are not all located on the same hydrophobic surface. The story is further complicated since those Trp-rich peptides can also interact with bacterial DNA.5</p><p>Here we present a unique structure for a newly designed Trp-rich peptide TetraF2W-RK (WWWLRKIW-amide),6 which contains 50% Trp, one of the highest percentages known to date. This peptide appears to act on bacterial membranes. First, Staphylococcus aureus USA300 can be rapidly killed. Second, the peptides synthesized using L- or D-amino acids give an identical minimal inhibitory concentration (MIC) against S. aureus, suggesting a chiral protein-like interface is not involved. Third, after peptide treatment, a non-permeable dye fluorescein isothiocyanate (FITC) can enter S. aureus in 30 sec, suggesting membrane damage by the peptide.6 These results laid a solid basis for us to determine the 3D structure of TetraF2W-RK bound to membrane-mimetic micelles. We chose to utilize the improved 2D NMR method7a for structural determination because recent studies show the importance of this method for peptides rich in certain amino acids (e.g., leucine and cysteine).7b,7c In this method, five 2D NMR spectra are recorded (1H-1H TOCSY, DQF-COSY, NOESY, 1H-13C HSQC, and 1H-15N HSQC). The 15N and 13C resonances have a broader range of chemical shifts, enabling the validation of proton assignments. In addition, these heteronuclear chemical shifts also contain structural information and can be used to refine the nuclear Overhauser effect (NOE)-derived structure to achieve high quality.7c Different from indolicidin and tritrpticin, this membrane-targeting Trp-rich peptide, TetraF2W-RK, forms a regular two-turn α-helix with a clear amphipathic nature, enabling us to decipher the role of each residue via single residue substitutions. Our results reveal that the π configuration of the N-terminal Trp triplet (WWW) of this short cationic peptide plays a critical role in disrupting the preformed biofilms of S. aureus USA300 based on quantification of both biomass and live bacteria.</p><p>The NMR spectra of TetraF2W-RK bound to perdeuterated dodecylphosphocholine (DPC-d38) are well dispersed (Figure 1, A&B), leading to complete assignments. Based on these 1H chemical shifts (Table S1), a total of 225 NOEs were assigned and converted to distance restraints for structural calculations. The number of NOEs per residue is 28, which is high for a micelle-bound peptide.3 Multiple (i, i+3) and (i, i+4) types of NOEs indicate a helical conformation, consistent with the secondary 1Hα chemical shift analysis (Table S2). Moreover, peptide backbone dihedral angles derived from a set of heteronuclear chemical shifts also indicate a helical structure.4e All the distance and angle restraints were utilized for structural calculations. Figure 1C presents an ensemble of 20 energy minimized 3D structures with backbone atoms superimposed. The backbone structure of TetraF2W-RK was well defined (rmsd = 0.06 Å). The 3D structure of the peptide consists of a two-turn helix. This can be seen in the Ramachandran plot, where nearly all the backbone angles (ϕ and φ) are clustered into the helical region (Figure S2). Remarkably, the side chains of the peptide also superimpose well and each Trp side chain is clearly visible (Figure 1, D & E). The aromatic rings of three Trp residues (W1, W3, and W8), as well as L4 and I7, occupy the same side, constituting the hydrophobic surface (Figure 1, D–F). W2, however, is located between the hydrophilic and hydrophobic interface where it is adjacent to the side chain of K6 (Figure 1, D–F). R5 is the only side chain that is clearly on the hydrophilic surface. Hence, TetraF2W-RK forms one of the shortest amphipathic helices after binding to membrane-mimetic micelles.</p><p>To provide evidence for the binding of the amphipathic helix to the micelles, we also compared the water exchange cross peaks of the side chain NH of the four Trp residues (Figure S3). The exchange peak for W2 is strongest, indicating that it was most exposed to water. The exchange cross peaks for W1 and W3 side chains reached 50% of the peak intensity of W2, implying that these two aromatic rings inserted deeper into the micelles. There is no clear cross peak with water for the side chain of W8, implying an even deeper penetration into the micelles. Such a Trp side chain proton exchange picture of the peptide with water is fully consistent with the amphipathic structure of TetraF2W-RK with the hydrophobic side chains buried into the micelles (Figure 1D).</p><p>There are multiple NOE cross peaks between the aliphatic portion of K6 and the aromatic ring of W2, suggesting a possible aromatic-charge interaction. However, it did not appear to matter much whether it is lysine or arginine (Arg) at position 6, because a swap of the positions between R5 and K6, leading to TetraF2W-KR, did not influence bacterial killing efficiency.6 When we compared all possible dibasic pairs in the same peptide context, the order of S. aureus USA300 killing efficiency is RR > RK ~ KR > KK. This means that arginines at both positions of this peptide template are more effective than lysines in bacterial killing (based on both colony count and membrane permeation experiments). Indeed, results in the literature reveal the preference of Trp-rich peptides to bind anionic membranes, indicating that these arginines are involved in interaction with anionic phosphatidylglycerols (PGs).3 We have observed direct Arg-PGs contacts between a human cathelicidin LL-37 peptide and dioctanoylphosphatidylglycerol by intermolecular NOE spectroscopy.8 Interestingly, the double arginine variant, TetraF2W-RR, is less cytotoxic to human HEK293 and HaCaT cells.6 It appears that the Arg-Trp combination, frequently observed in Trp-rich peptides, offers an evolutional advantage by optimizing the desired antimicrobial effect and minimizing toxicity to the host.</p><p>We then asked whether insertion of additional arginines to the peptide would be helpful to the peptide in terms of potency and selectivity. For this purpose, we have made six single arginine variants based on the above double arginine variant TetraF2W-RR template (wild type WT). The sequences of these peptides are given in Table 1. The quality of these synthetic peptides can be seen from the HPLC chromatograms (Figure S4) as well as correct masses. Antimicrobial activities of these peptides were evaluated by the microdilution method as described6 using both Gram-negative Escherichia coli and Gram-positive S. aureus (Table 1). In the case of E. coli ATCC 25922, there was a four-fold increase in the minimal inhibitory concentration (MIC) for all the variants, except for the I7R peptide (viz, I7 is substituted by R), which retained the same MIC. When methicillin-resistant S. aureus (MRSA) USA300 was tested, we also observed a four-fold loss in activity for the W1R, W3R, L4R, and W8R variants compared to the WT (Table 1). However, the anti-MRSA activity of W2R was less reduced, suggesting that W2 is less important here than other Trps in the peptide. Interestingly, like the case of E. coli, the activity of I7R against S. aureus USA300 also remained the same (MIC 3.1 μM).</p><p>To further appreciate these activity differences, we also compared the growth inhibition as well as membrane permeation power of these peptide variants in Table 1. I7R is almost equally effective to the WT in inhibiting the growth of S. aureus USA300 at 3.1 μM (Figure 2A). The L4R variant is also potent followed by the W2R peptide. The substitution of W1, W2, or W8, however, made the peptide less inhibitory in this experiment. A similar trend was also observed with membrane permeation by propidium iodide (PI) (Figure 2B). The WT is most potent followed by the I7R and W2R variants. These results reinforce the significance of aromatic amino acids W1, W3, and W8 in anchoring the peptide to the membranes of S. aureus USA300.</p><p>In nature, most bacteria live in the biofilm form, and the formation of biofilms makes it more resistant to traditional antibiotics. To further understand the activity of these peptides in Table 1, we also compared their ability in disrupting the 24-h biofilms of S. aureus USA300. After peptide treatment (3.1 to 25 μM), biofilms were stained with either crystal violet (Figure 3, left panels) or the XTT-based cell proliferation kit (right panels). Crystal violet staining measures the amount of biofilms. When the C-terminal hydrophobic residues were replaced, the peptide variants showed only slightly reduced antibiofilm capability (Figure 3, E–G). However, substitution of any of the N-terminal Trp residues (panels B–D) had a detrimental effect on antibiofilm ability, although W2R became more effective at 12.5–25 μM (panel C). These plots underscore the importance of the N-terminal Trp triplet in disrupting the S. aureus biofilms.</p><p>Whether S. aureus is killed in biofilms remains to be tested. We estimated dead bacteria by using the XTT assay that reports the amount of live cells. At a low concentration of 3.1 μM, none of the peptides were effective. At 6.2 μM, only the WT was active. When the peptide reached 12.5 μM, nearly all S. aureus USA300 was eliminated by the WT (Figure 3H). In addition, the W2R, L4R, I7R, and W8R variants also displayed some effects, with W2R and I7R (80% killing in Figure 3J&M) being much more effective than L4R and W8R (20% killing in Figure 3, L&N). At 25 μM, these four peptide variants killed nearly all S. aureus in the biofilms based on the XTT staining. The stronger effect of the WT may result from its higher hydrophobicity (longest HPLC retention time in Table 1). Notably, neither W1R nor W3R was able to kill the S. aureus USA300 strain in the biofilms even at 25 μM, indicating that W1 and W3 are critical in both biofilm disruption and bacterial killing based on a combined use of two biofilm staining dyes (Figure 3).</p><p>We also compared the cytotoxicity of these peptides using human red blood cells. A concentration dependent hemolysis enabled us to estimate HL50, the concentration that causes 50% hemolysis. The results are included in Table 1. Based on HL50 and MIC values, we also calculated cell selectivity index, which is defined as the ratio of HL50 and MIC. While the cell selectivity index for the WT peptide is 35/3.1=11, it is 150/3.1=48 for I7R. Thus, the hemolytic ability of the I7R variant was reduced (Table 1) by four fold compared to the WT. Therefore, through this structure-activity relationship studies, we have obtained a more selective peptide, the I7R variant, that retains antibacterial activity (the same MIC), making it a more suitable candidate for development of novel antimicrobials to combat MRSA.</p><p>To further verify that the I7R variant remains membrane targeting, we also conducted a live cell experiment by following the entry of a non-membrane permeable dye FITC into S. aureus USA300. Only after peptide treatment, we saw green fluorescent bacteria in 28 sec (Figure S5), indicating membrane permeation by the peptide. To view the dead bacteria directly, we also treated the 24 h preformed biofilms of S. aureus USA300 with the I7R peptide followed by confocal microscopy. Without treatment with the I7R variant, we observed green cells (live). After treatment, the majority of cells are red, indicating that this peptide killed MRSA in biofilms as well (red cells in Figure S6). Similar results were obtained using the WT peptide, indicating I7R, like the WT, killed S. aureus USA300 in the biofilms.</p><p>We then attempted to correlate the peptide activity (Table 1) with the 3D structure. W2, being located in the interface of the amphipathic helix, is less significant for membrane binding, consistent with only a two-fold loss in activity (Table 1). Likewise, I7 is close to the interface and its change to an arginine probably has compensated the loss, leading to the same MIC in Table 1 for both bacteria. These two peptide variants also retained antibiofilm capability similar to the WT (Figure 3 right). In contrast, residues W1, W3, L4, and W8 are all located on the hydrophobic surface, critical for anchoring the peptide into bacterial membranes (Figure 1D). It is not surprising that a change of any of these residues into an arginine caused a substantial drop in antibacterial activity (Table 1). The same is true of the biofilm cases, especially when XTT was utilized (Figure 3 right). It is worthwhile to point out that W1, W3, L4 and W8 are also important for hemolysis since their arginine variants became much less toxic (Table 1).</p><p>In disrupting the MRSA biofilms, however, W1 and W3 of the peptide play an even more critical role than L4 or W8. When stained with crystal violet, there are more biofilms left for the cases of W1R and W3R variants (Figure 3, B & D) than those of L4R and W8R (Figure 3, E & G). Remarkably, the peptide variants of W1 and W3 completely lost their killing ability when stained with XTT (Figure 3, I & K), indicating that biofilm disruption based on crystal violet did not entirely reflect bacterial killing based on XTT. Interestingly, the three Trp residues at the N-terminus of TetraF2W-RK of the structure assemble into a π configuration (Figure 1F, red), where W2 is the horizontal bar, while W1 and W3 constitute the two legs critical for interdigitation into the membranes. These aromatic rings of the WWW motif stack with each other. For instance, a cross peak between W2 and W3 can be seen in Figure 1B, while a cross peak between W1 and W3 can be seen in Figure S3. Also, W1 and W3 are perpendicular to each other, leading to the ring current shifts of multiple protons (Table S1). It seems that aromatic-aromatic interactions play a role in stabilizing the π-configuration of the WWW structural motif.</p><p>In conclusion, we determined the 3D structure for a newly designed Trp-rich peptide (50% Trp). This structure nicely correlates with antibacterial and antibiofilm activities. It also revealed a novel π-configuration for the Trp triplet stabilized by both aromatic-aromatic and aromatic-aliphatic interactions. This π-configuration of the WWW motif, especially the two legs (W1 and W3), is critical for biofilm disruption and bacterial killing in the biofilms. Such a critical structural motif may be utilized to guide our design of novel peptides. To obtain better peptides, the WWW motif at the N-terminus may be retained by varying the C-terminal residues. Our identification of the I7R variant, which retains antimicrobial and antibiofilm activities but with reduced toxicity to human cells, provides an excellent example for this. Consequently, I7R constitutes a better candidate for further development than the WT peptide. In addition, the WWW motif can be applied to peptide ends to enhance antimicrobial activity as demonstrated by Schmidtchen and colleagues.9 Collectively, our study has not only obtained a more promising peptide candidate, but also uncovered a novel π-configuration for a tryptophan triplet via studying the structure-activity relationship of a new Trp-rich peptide that yields novel insight into membrane targeting, MRSA killing, and biofilm disruption. This unique WWW motif is of general interest for engineering potent antibiofilm peptides to combat drug-resistant pathogens.</p>

PubMed Author Manuscript

Oligodendrocytes Do Not Export NAA-Derived Aspartate In Vitro

Oligodendroglial cells are known to de-acetylate the N-acetylaspartate (NAA) synthesized and released by neurons and use it for lipid synthesis. However, the role of NAA regarding their intermediary metabolism remains poorly understood. Two hypotheses were proposed regarding the fate of aspartate after being released by de-acetylation: (1) aspartate is metabolized in the mitochondria of oligodendrocyte lineage cells; (2) aspartate is released to the medium. We report here that aspartoacylase mRNA expression increases when primary rat oligodendrocyte progenitor cells (OPCs) differentiate into mature cells in culture. Moreover, characterising metabolic functions of acetyl coenzyme A and aspartate from NAA catabolism in mature oligodendrocyte cultures after 5 days using isotope-labelled glucose after 5-days of differentiation we found evidence of extensive NAA metabolism. Incubation with [1,6-13C]glucose followed by gas chromatography–mass spectrometry and high performance liquid chromatography analyses of cell extracts and media in the presence and absence of NAA established that the acetate moiety produced by hydrolysis of NAA does not enter mitochondrial metabolism in the form of acetyl coenzyme A. We also resolved the controversy concerning the possible release of aspartate to the medium: aspartate is not released to the medium by oligodendrocytes in amounts detectable by our methods. Therefore we propose that: aspartate released from NAA joins the cytosolic aspartate pool rapidly and takes part in the malate–aspartate shuttle, which transports reducing equivalents from glycolysis into the mitochondria for ATP production and enters the tricarboxylic acid cycle at a slow rate.

oligodendrocytes_do_not_export_naa-derived_aspartate_in_vitro

Introduction<!>Materials<!>Primary Cultures of Rat Oligodendrocytes<!>Quantitative Reverse Transcriptase PCR<!>Incubations with 13C Labelled Glucose and NAA<!>Glucose and Lactate Analyses<!>High Performance Liquid Chromatography (HPLC)<!>Gas Chromatography–Mass Spectrometry (GC–MS)<!>Statistical Analysis<!>Results<!><!>Results<!><!>Results<!><!>Results<!><!>Results<!><!>Discussion<!>Tri-Cellular Compartmentation of NAA Metabolism?<!><!>Conclusion

<p>The brain is an organ with exceptionally high energy demands and relies on an uninterrupted supply of substrates for oxidative phosphorylation in mitochondria. Around 25 % of the body's total glucose budget is spent on processes in the brain, including the generation of action potentials and synaptic transmission [1]. Glucose-derived energy is therefore of utmost importance for maintaining physiological function of the brain. In contrast, many neurodegenerative diseases including Alzheimer's disease [2] are associated with compromised glucose metabolism and markers of low energy status. Quantification of N-acetylaspartate (NAA) has often been used to assess the metabolic integrity of neurons [3, 4]. NAA can be detected by 1H-magnetic resonance spectroscopy, and has been applied as a non-invasive quantitative method for detecting progression, recovery, and remission in an ever-increasing catalogue of disorders of the brain [5]. However, the fundamental role of NAA in the brain remains elusive and the available evidence for its function is limited to a role in providing acetyl groups for lipid synthesis [6].</p><p>NAA is amongst the most abundant amino acid derivatives in the brain and is synthesized from aspartate and acetyl coenzyme A (CoA) by aspartate N-acetyltransferase [7]. After birth, NAA content in the brain is subject to a rapid increase to reach concentrations of 5 to 10 mM [8, 9], being especially concentrated in gray matter-rich regions [9–12]. NAA synthesis is dependent on mitochondrial integrity [7, 13] and fluctuations in concentration can occur in parallel with changes in adenosine triphosphate (ATP) [14], suggesting an intimate relationship with metabolic energy.</p><p>NAA is produced by and released from neurons. These depend on the supply of precursor molecules provided by astrocytes to synthesize the aspartate necessary for NAA production [15, 16]. NAA is metabolized in oligodendrocytes, which contain aspartoacylase (ASPA) [17–19], the only known NAA-catabolizing enzyme in the brain. The importance of ASPA to myelination is highlighted by the severely dys-myelinated phenotype of the inherited human paediatric leukodystrophy, Canavan disease (CD), which results from the loss of ASPA function. The abnormally high levels of NAA in CD are in contrast to abnormally low levels of NAA that typically are seen in practically all other neurodegenerative diseases. Considering the importance of NAA as a prognostic marker of metabolic function across a wide pathological range the view that NAA solely acts as a shuttle for acetyl groups during lipid synthesis may therefore be insufficient. Multiple roles have been suggested for NAA such as: being an osmolyte that acts as a "molecular water pump" to remove metabolically produced water from neurons [20, 21]; in addition to moving acetate groups across the mitochondrial membrane system, NAA also acts by moving nitrogen groups to the cytoplasm [22]; NAA is involved in facilitating glutamine/glutamate oxidation in neuronal mitochondria, while bypassing the glutamate dehydrogenase reaction and therefore avoiding ammonia production [23]; being a storage and transport form of acetate [24]. Other proposed roles include the involvement in histone and protein acetylation reactions [25]; altering metabolism (aerobic glycolysis and Warburg effect) in cancer cells [26, 27] and, more recently, it was linked to neuronal differentiation [28].</p><p>In order to investigate the role of NAA in intermediary metabolism, we incubated cultures enriched for mature oligodendrocytes in medium containing [1,6-13C]glucose for 8 and 24 h in the presence and absence of NAA and analysed cell extracts and media using gas chromatography–mass spectrometry (GC–MS) and high performance liquid chromatography (HPLC). We found that the acetyl CoA produced by hydrolysis of NAA does not enter mitochondrial metabolism. We propose that the aspartate joins the aspartate pool active in the malate–aspartate shuttle and we established that aspartate is not released to the medium in amounts detectable by our methods.</p><!><p>Cell culture reagents were purchased from Sigma (Dorset, UK)—Dulbecco's modified Eagle's medium (DMEM), minimum essential medium Eagle (MEM), l-glutamine, poly-l-lysine (PLL), papain, NAA—or Life Technologies (Paisley, UK)—fetal bovine serum (FBS), penicillin–streptomycin (pen–strep), trypsin–EDTA, phosphate buffered saline (PBS). 13C-labelled compounds were obtained from Cambridge Isotope Laboratories, MA, USA. The mass spectrometry derivatization reagents MTBSTFA (N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide), MSTFA (N-methyl-N-(trimethylsilyl) trifluoroacetamide) and the t-BDMS-Cl (tert-butyldimethylchlorosilane) were purchased from Regis Technologies, Inc. (Morton Grove, IL, USA). Recombinant human PDGF-AA and Recombinant human FGF-basic were purchased from PeproTech (Rocky Hill, NJ). All other chemicals were of the purest grade available from Sigma (Dorset, UK).</p><!><p>Primary mixed glia cultures were isolated from neonatal Sprague Dawley rat (postnatal day 0–2) forebrains following established protocols [29]. Briefly, pups were euthanized according to "Schedule 1" regulations from the Home Office Animal Procedures Committee UK. Cells were cultured for 10–15 days in DMEM supplemented with 10 % FBS, 1 % pen-strep and 4 mM glutamine, and kept under a humidified atmosphere at 37 °C and 7 % CO2. Oligodendrocyte precursor cells (OPCs) were subsequently isolated using a step-based shake-off protocol and cultured in Sato's medium on PLL-coated plates [29]. To obtain immature OPCs, cells were cultured for 1 day in Sato's medium supplemented with human PDGF-AA and human recombinant FGF at 10 ng/ml. To induce differentiation, OPCs were cultured in Sato's medium supplemented with 0.5 % fetal calf serum (FCS) for 1 or 5 days. The cell culture medium was replaced by fresh medium at day two of differentiation. For all experiments only cultures with >93 % purity (determined based on O4 immunostaining) were used [30].</p><!><p>To determine the mRNA levels of ASPA during OPC differentiation, cells were cultured either for 1 day in proliferation medium, or for 1 or 5 days in differentiation medium. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Manchester, UK). cDNA was synthesized from 20 ng RNA per sample using the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA USA). Quantitative PCR (qPCR) was conducted as previously outlined [29] on an Applied Biosystems 7500HT Fast Real-time PCR system. The primers used were: ASPA F-AGACGTGGCTGCTGTTATCC; ASPA R-GATCTCCAGGGTGCAATGGT; beta actin F-CATGGCATTGTGATGGACT; beta actin R-ACGGATGTCAACGTCACACT. Values are represented as ASPA/beta actin ratios. Measurements were made on 8–11 samples obtained from three independently generated cultures.</p><!><p>OPCs isolated from mixed glia cultures were cultured in 6 well plates at a cell density of 4 × 105 cells/well and allowed to differentiate for 5 days (see above for details). Prior to incubation, cells were washed once with PBS and incubated with 2 ml Sato's medium prepared from a glucose, glutamine and pyruvate-free DMEM (Sigma D5030, Dorset, UK) supplemented with 0.5 % FCS and 2 mM [1,6-13C]glucose, 2 mM glutamine and 2 mM NAA (controls did not have NAA in the incubation medium) for 8 and 24 h. Samples of medium were collected before and after the incubation period and subsequently analysed by mass spectrometry. To stop the incubation, cells were washed twice with cold PBS and the intracellular metabolites extracted with 70 % ethanol [31]. Experiments were performed in four independently generated cultures with, at least, two replicate wells per condition.</p><!><p>Glucose and lactate levels in the cell culture medium were analysed at the Core Biochemical Assay Laboratory, Clinical Biochemistry, Addenbrooke's Hospital using automated assays on a Siemens Dimension RxL analyser. The rate of glucose and lactate net change relative to cells over time (µmol/106 cells/24 h) was calculated by subtracting the value measured at the end of the experiment (T = 24 h) from the one measured in a sample of medium collected at the onset of the incubation, and dividing the resulting value by the amount of cells in each experiment, multiplied by the experimental volume (2 ml). The cell number considered in the calculations was the cell number at plating since oligodendrocytes do not proliferate. Analyses were performed on 9–12 samples, which derived from four independently generated cultures.</p><!><p>HPLC was used to quantify the total amounts of amino acids in samples of cell extracts. Samples were lyophilized and re-suspended in 0.01 M HCl and subsequently derivatised with o-Phthaldialdehyde [32] using an automated method prior to injection into the HPLC column. Amino acid concentrations were determined by comparison to a calibration curve of standard solutions of amino acids run after every 12 samples. Analyses were performed on 6–11 samples derived from three independently generated cultures. For details see Amaral et al. [32].</p><!><p>For analysis of percent enrichment with 13C in lactate, amino acids (aspartate, glutamate and glutamine) and TCA cycle intermediate (citrate) after incubation with [1,6-13C]glucose ± NAA, cell extracts and samples of medium were lyophilized and re-suspended in 0.01 M HCl. Derivatisation with MTBSTFA in the presence of 1 % t-BDMS-Cl [33] was performed as described in Amaral et al. [30]. The samples were analysed on an Agilent 6890 gas chromatograph connected to an Agilent 5975B mass spectrometer (Agilent Technologies, Palo Alto, CA). The parent ion (M) and atom percent excess for one 13C atom (M + 1) values for lactate, citrate, glutamate, glutamine and aspartate were calculated from the GC–MS data using the MassHunter software supplied by Agilent (Agilent Technologies, Palo Alto, CA) and correcting for the naturally abundant 13C by using non-enriched standards [34]. Analyses were performed on 6–12 samples of medium and 8–11 samples of extracts derived from four independently generated cultures.</p><!><p>Statistical analysis was conducted using unpaired two-tailed Student t tests (confidence interval = 95 %) to compare the effect of NAA on the intracellular amounts of amino acids, on enrichment in 13C in extracellular and intracellular metabolites and on the glucose consumption and lactate release rates. To evaluate differences in the expression of ASPA mRNA levels at different stages of OPC maturation in culture, a one-way ANOVA (alpha = 0.05) followed by Tukey's multiple comparison test was performed.</p><!><p>To investigate how NAA is metabolised by mature oligodendrocytes, we incubated primary OPC cultures maintained in differentiation medium for 5 days with [1,6-13C]glucose in the presence or absence of NAA. By assessing differences in metabolite enrichment in 13C, this strategy enabled us to study aspartate and acetate, both metabolic products of NAA, in metabolic pathways of glucose. For details on 13C labelling Fig. 1a. [1,6-13C]Glucose is taken up by oligodendrocytes and converted to 2 molecules of [3-13C]pyruvate via glycolysis. Lactate dehydrogenase converts [3-13C]pyruvate to [3-13C]lactate or [3-13C]pyruvate, which can enter the mitochondria to be further metabolized to [2-13C]acetyl CoA and enter the TCA cycle. The subsequent TCA cycle metabolites and amino acids derived from oxaloacetate and α-ketoglutarate, aspartate and glutamate, respectively, will be labelled with one 13C atom, resulting in mass M + 1. When a labelled acetyl CoA molecule condenses with a labelled oxaloacetate molecule the subsequent metabolites will contain two 13C atoms which will result in mass M + 2. Apart from its role in the TCA cycle, citrate is used to funnel acetyl CoA units into lipid synthesis. Figure 1b provides an overview of the metabolic pathways assessed in the present study. We postulated that oligodendrocytes possess distinct mitochondria or TCA cycles that are specialised for lipid synthesis and others that prioritise energy and amino acid production (Fig. 1b). When NAA enters the cell it is hydrolyzed into acetyl CoA and aspartate by ASPA. Whereas acetate is used for lipid synthesis (Fig. 1b), aspartate can then participate in the malate–aspartate shuttle (for details see [32]) or/and enter the TCA cycle after conversion to oxaloacetate.</p><!><p>a 13C glucose labelling patterns in oligodendrocytes. [1,6-13C]Glucose is taken up by oligodendrocytes and converted to 2 molecules of [3-13C]pyruvate via glycolysis. Lactate dehydrogenase converts [3-13C]pyruvate to [3-13C]lactate (LAC) and alanine aminotransferase to alanine (ALA). Alternatively, [3-13C]pyruvate can enter the mitochondria to be metabolized to [2-13C]acetyl CoA which can enter the TCA cycle. The subsequent TCA cycle metabolites such as: citrate, malate (MAL) and oxaloacetate (OAA) and amino acids such as aspartate (ASP), glutamate (GLU) and glutamine (GLN), will be labelled with one 13C atom, resulting in an increase in the parent ion M mass to M + 1, which can be detected by GC–MS. b Schematic presentation of glucose metabolism in oligodendrocytes in the presence of NAA. We propose that at least two distinct TCA cycles exist: (1) one TCA cycle (depicted on the left) in which most of the energy is produced and in which only a fraction of citrate leaves the cycle and contributes to lipid synthesis. This "energy producing" TCA cycle is halted by the presence of NAA, which results in increased labelling of e.g. glutamate and glutamine and, to a lesser degree, citrate. The aspartate derived from NAA does not enter into this (1) TCA cycle during the initial phase (up to 8 h), since this would reduce labelling of glutamate and glutamine. In contrast, we found that labelling of glutamate and glutamine increased but labelling of aspartate did not increase. These findings indicate that the aspartate derived from NAA enters the malate–aspartate shuttle and dilutes aspartate labelling. (2) A second TCA cycle is implied by the increased 13C labelling of citrate in the medium, which occurred much more extensively than labelling of other metabolites after 24 h incubation with [1,6-13C]glucose. The citrate producing TCA cycle for lipid synthesis was not affected by the presence of NAA as the unchanged labelling of the major pool of citrate suggests</p><!><p>In order to verify that the oligodendrocyte cultures used in the present study expressed the enzyme responsible for NAA hydrolysis, we quantified the ASPA mRNA levels. This demonstrated an increase in ASPA mRNA expression from day 1 to day 5 of differentiation (Fig. 2). All subsequent experiments were performed on 5-day-old cultures.</p><!><p>Aspartoacylase (ASPA) mRNA levels during OPC differentiation in vitro. Cells were cultured either for 1 day in proliferation medium, or for 1 or 5 days in differentiation medium. Values are represented as ASPA/beta actin ratios. Measurements were made on 8–12 samples obtained from three independently generated cultures (for details see "Methods" section)</p><!><p>Cells were incubated in medium containing [1,6-13C]glucose for 8 and 24 h in the presence and absence of NAA. The presence of NAA increased glucose consumption and lactate production (Fig. 3a, b). The theoretical maximum enrichment of lactate from [1,6-13C]glucose is 100 %. However, after 8 h only approximately 50 % of lactate was 13C labelled, reaching approximately 65 % after 24 h (Fig. 3c, d). The addition of NAA did not affect 13C enrichment in lactate (Fig. 3c, d). Similarly, 13C-enriched citrate was released to the medium, but its enrichment was not affected by the addition of NAA (Fig. 3e, f).</p><!><p>Effect of NAA on glucose consumption, lactate release, and enrichment with 13C of extracellular metabolites in mature oligodendrocyte cultures. Oligodendrocytes were incubated in medium containing [1,6-13C]glucose, 2 mM glutamine and 2 mM N-acetyl aspartate (NAA). Controls did not have NAA in the incubation medium. Samples of medium were collected and subsequently analysed by gas chromatography–mass spectrometry. % 13C enrichment above natural abundance is given for lactate (LAC) and citrate (CIT) after 8 or 24 h of incubation. Experiments were performed on 6–12 samples, which derived from four independently generated cultures. Glucose consumption and lactate release were measured in 9–12 samples obtained from four independently generated cultures after 24 h of incubation (for details see "Methods" section)</p><!><p>The intracellular amounts of aspartate, glutamine and glutamate after 8 and 24 h incubation are provided in Fig. 4. Aspartate was increased in the presence of NAA whereas glutamine and glutamate were not (Fig. 4).</p><!><p>Effect of NAA on the intracellular levels of amino acids in mature oligodendrocyte cultures. HPLC was used to quantify the amounts of aspartate (ASP), glutamate (GLU) and glutamine (GLN) in samples of cell extracts of oligodendrocytes cultured for 5 days in differentiation medium and incubated for 24 h in the presence or absence of 2 mM N-acetyl aspartate (NAA). Analyses were performed on 6–11 samples, which derived from three independently generated cultures. Amounts are given in nmol/106 cells (for details see "Methods" section)</p><!><p>Analysis of 13C incorporation from [1,6-13C]glucose into intracellular metabolites was conducted after 8 and 24 h in the absence or presence of NAA (Fig. 5). No differences were detected in 13C enrichment in any metabolite analysed after 24 h (Fig. 5) and aspartate and citrate were also not affected at 8 h (Fig. 5). All other metabolites displayed an increase in 13C enrichment after 8 h incubation with [1,6-13C]glucose and NAA as compared to cultures that did not receive NAA (Fig. 5).</p><!><p>Effect of NAA on [1,6-13C]glucose-derived enrichment with 13C in intracellular metabolites of mature oligodendrocytes. Oligodendrocytes differentiated for 5 days were incubated in medium containing [1,6-13C]glucose, 2 mM glutamine and 2 mM N-acetyl aspartate (NAA). Controls did not have NAA in the incubation medium. Cell extracts were collected and subsequently analysed by gas chromatography–mass spectrometry. % 13C enrichment above natural abundance is given for aspartate (ASP), glutamate (GLU), glutamine (GLN), citrate (CIT) and malate (MAL) after 8 or 24 h of incubation. Experiments were performed on 8–11 samples, which derived from four independently generated cultures</p><!><p>Glucose metabolism in neurons and astrocytes has been studied extensively and much is known about the interaction between these two cell types in the brain [35]. However, until very recently oligodendrocytes have not been studied in this context [30, 35–38].</p><p>It was evident that the metabolism of N-acetylaspartylglutamate (NAAG) has a tri-cellular compartmentation, however, this was not clear in regard to NAA [38–40]. Both NAA and NAAG are synthesized by neurons in the brain. Neurons release both NAA and NAAG to the extracellular space upon stimulation, where astrocytes, the target cells for NAAG, hydrolyse it, releasing NAA, which is subsequently catabolised in oligodendrocytes. It has been proposed that oligodendrocytes release the aspartate obtained from NAA for recycling to neurons [39]. This hypothesis assumes that aspartate produced from NAA is passed from oligodendrocytes back to neurons to be reutilized for re-synthesis of NAA and NAAG. This exchange of metabolites would be in analogy with the well-established glutamine–glutamate cycle between glia and neurons [35]. Existing evidence suggests that the aspartate derived by catabolism of NAA in the brain may not be used for the re-synthesis of NAA [22]. Studies demonstrated rapid transamination of doubly labelled (3H and 15N) aspartate from [3H2 15N]NAA, in which the amino group was transferred to glutamate, in the absence of 15N-NAA production. However, Miller et al. [22] could not follow the carbon skeleton of NAA since the nitrogen was labelled, not the carbon atoms. Our results, using a 13carbon labelled precursor ([1,6-13C]glucose), demonstrate that aspartate is not released to the medium by oligodendrocytes in culture, disproving the hypothesis of aspartate recycling between neurons and oligodendrocytes [39]. What we observed was an increase in aspartate content in the oligodendrocytes but the % 13C labelling remained unchanged. It is likely that aspartate is converted to oxaloacetate and further to malate in the cytosol, steps in the energy producing malate-aspartate shuttle for transporting reducing equivalents from NADH from glycolysis into the mitochondria [35]. The malate–aspartate shuttle is tightly coupled to glycolysis in that reducing equivalents are transported into the mitochondria for oxidative phosphorylation. Therefore, ATP is produced and NAD, which is necessary for glycolysis, is re-generated in the cytosol. The increased glucose consumption and lactate production observed in the presence of NAA in the present study is indicative of an increased aerobic glycolysis (Warburg effect) also seen in cancer cells. In this context it is noteworthy that exogenously applied NAA has been found to promote growth in several cancer cell lines [41]. A potential explanation consists in a more efficient NAD regeneration due to an increase in the capacity of the malate–aspartate shuttle due to aspartate generation from NAA. Entry of aspartate from NAA into the TCA cycle appears to be slow due to the expected dilution of 13C labelling of compounds derived from this cycle, and consequently % enrichment, was not decreased after 8 h of incubation. Aspartate labelling would be expected to increase together with glutamate and glutamine at 8 h, but this was not observed. Indeed, % 13C labelling of aspartate was unchanged, indicating that aspartate synthesis from NAA took place in a restricted compartment, which we hypothesise to be the malate-aspartate shuttle. However, after 24 h incubation with NAA the labelling of glutamate and glutamine was not found to be different from the one observed in cells cultured in the absence of NAA, indicating that aspartate was participating in the TCA cycle. This observation may also be related to the slow turnover of NAA detected in vivo. The reported turnover rates of NAA C6 and C3 in rat brain range between 0.7 ± 0.1 and 0.6 ± 0.1 µmol/(g h) with the time constants 14 ± 2 and 13 ± 2 h, respectively, with an estimated pool size of 8 µmol/g [42]. These results suggest that complete label turnover of NAA from glucose occurs in approximately 70 h. This is in agreement with a slow incorporation of aspartate into the oligodendrocyte TCA cycle in order to maintain homeostasis between synthesis and degradation. A potential explanation for the increase in % 13C labelling in glutamate and glutamine is a decrease in the amount of 13C labelled citrate leaving the TCA cycle as the acetyl CoA content in the cytosol is increased due to the catabolism of NAA. The reduced leakage of labelled substance is expected to cause an overall increase in labelling.</p><p>Percent 13C labelling of glutamate and glutamine were increased in the presence of NAA. This may be caused by a decrease in 13C labelled citrate efflux from the TCA cycle to the cytosol. Citrate is the most common precursor for acetyl CoA production in the cytosol, and also acts as a substrate for lipid synthesis. High levels of NAA in the cytosol of oligodendrocytes incubated with NAA could support acetyl CoA production from acetate generated by hydrolysis of NAA. This is a possibility since ACSS2, the gene for the nuclear-cytosolic form of acyl-CoA synthetase short-chain family member, is expressed in oligodendrocytes to a high extent as reported in transcriptomics studies of the mouse brain [43, 44]. This would decrease the need for citrate catabolism for fatty acid synthesis. In contrast to the increased enrichment of glutamate and glutamine, no differences in the labelling of citrate inside the cells and in the medium were detected. This indicates that the majority of citrate was produced in a separate compartment. The notion of compartmentation is also supported by the much larger % 13C enrichment in citrate in the medium compared to that of intracellular metabolites.</p><p>It is unlikely that the acetate moiety of NAA released by ASPA entered the mitochondria and was converted to acetyl CoA since this would have resulted in decreased 13C labelling of the metabolites, and this was not observed. This is in agreement with a report that oligodendrocytes do not have the mitochondrial enzyme AceCS2 [4]. However, in the mitochondria of oligodendrocytes in culture, we have shown that acetate from the medium is indeed converted to acetyl CoA [30]. As previously mentioned, the most likely fate of the resulting acetyl CoA is its entry into lipid synthesis [6].</p><!><p>Tri-cellular compartmentation is necessary for catabolism of NAAG but not for NAA. For the latter, interactions between neurons and oligodendrocytes appear to be sufficient. However, it is important to note that aspartate production in neurons is only possible with the help of glutamine from other cells. Neurons do not express pyruvate carboxylase, the anaplerotic enzyme in the brain [15] and, thus, cannot produce "de novo" aspartate. In order to export NAA, neurons have to import glutamine from external sources [45]. Aspartate is synthesized in the TCA cycle as a result of multiple conversion steps involving glutamate and oxaloacetate. So far only astrocytes are known to synthesise glutamine "de novo" and to release it to the medium [35]. We have recently demonstrated that oligodendrocytes are capable of anaplerosis [32] and therefore meet one of the requirements for supporting neurons. However, it remains unknown whether they are capable of exporting glutamine and, so, support transfer of NAA. Our present findings indicate a tri-cellular compartmentation of NAA metabolism, which starts with astrocytes releasing glutamine, which is taken by neurons, and converted into NAA. Subsequently, NAA released into the extracellular space is taken up and metabolized by oligodendrocytes. Figure 6 summarizes the findings described in the present work. A similar multicellular metabolism of NAA has been proposed depicting some of the metabolite trafficking between various cell types in the brain associated with NAA synthesis and breakdown [4, 46]. The authors stressed that the slow rate of NAA synthesis in neurons and breakdown and utilization in oligodendrocytes is suggestive of non-energy derivation roles in lipid synthesis and protein acetylation reactions under normal conditions, with a shift to much more rapid metabolism in response to injury or disease. The rate of synthesis and breakdown would also be much greater during postnatal myelination than in the normal adult brain and the oligodendrocyte cultures used in the present study might reflect this.</p><!><p>Schematic overview of the metabolic interactions involving N-acetyl aspartate (NAA) and N-acetyl aspartate glutamate (NAAG) between neurons, oligodendrocytes and astrocytes. Glucose from the blood is taken up by neurons, astrocytes and oligodendrocytes and can be metabolized via glycolysis giving rise to pyruvate formation. Pyruvate can be carboxylated in astrocytes via pyruvate carboxylase and glutamine (GLN) can be formed eventually (for details see [26]). In brain, the predominant cell type for NAA synthesis is neurons that synthesize it from aspartate (ASP) and Acetyl-CoA. NAAG is also synthesized in neurons and converted to NAA in astrocytes. NAA is taken up and metabolized by oligodendrocytes. The acetate moiety produced by hydrolysis of NAA does not enter mitochondrial metabolism in the form of Acetyl-CoA it is used for lipid synthesis. Aspartate is not released to the medium by oligodendrocytes in amounts detectable by our methods. We propose that aspartate released from NAA joins the cytosolic aspartate pool rapidly and takes part in the malate–aspartate shuttle, which transports reducing equivalents from glycolysis into the mitochondria for ATP production and enters the tricarboxylic acid cycle at a slow rate</p><!><p>In addition to the results from a previous study [47], this study provides new evidence that cultured oligodendrocytes hydrolyse NAA. The resulting aspartate and the acetate moieties remain within the cells. As the acetate is not incorporated into TCA cycle intermediates, it is likely to be used in lipid synthesis. The aspartate entity is likely to affect the malate–aspartate shuttle, glycolysis and energy production.</p>

PubMed Open Access

7,8‐Dihydro‐8‐oxoguanosine Lesions Inhibit the Theophylline Aptamer or Change Its Selectivity

AbstractAptamers are attractive constructs due to their high affinity/selectivity towards a target. Here 7,8‐dihydro‐8‐oxoguanosine (8‐oxoG) has been used, due in part to its unique H‐bonding capabilities (Watson–Crick or Hoogsteen), to expand the “RNA alphabet”. Its impact on the theophylline RNA aptamer was explored by modifying its binding pocket at positions G11, G25, or G26. Structural probing, with RNases A and T1, showed that modification at G11 leads to a drastic structural change, whereas the G25‐/G26‐modified analogues exhibited cleavage patterns similar to that of the canonical construct. The recognition properties towards three xanthine derivatives were then explored through thermophoresis. Modifying the aptamer at position G11 led to binding inhibition. Modification at G25, however, changed the selectivity towards theobromine (K d≈160 μm), with a poor affinity for theophylline (K d>1.5 mm) being observed. Overall, 8‐oxoG can have an impact on the structures of aptamers in a position‐dependent manner, leading to altered target selectivity.

7,8‐dihydro‐8‐oxoguanosine_lesions_inhibit_the_theophylline_aptamer_or_change_its_selectivity

<!>Introduction<!><!>Introduction<!><!>Introduction<!>Results<!><!>Results<!><!>Results<!>Small‐molecule binding<!><!>Small‐molecule binding<!>Discussion<!><!>Discussion<!>Conclusion<!>Experimental Section<!><!>Experimental Section<!>Conflict of interest<!>

<p>C. Kiggins, A. Skinner, M. J. E. Resendiz, ChemBioChem 2020, 21, 1347.</p><!><p>An aptamer is the smallest fragment of a biopolymer that is able to recognize a particular target with high affinity and specificity, often with a sub‐micromolar/sub‐nanomolar dissociation constant.1 These constructs display recognition of a wide array of different molecules and their targets range from large biopolymers such as proteins2 to small molecules3 or ions.4 In addition, their small size, high flexibility, and ease of manufacture make them attractive candidates for various applications, including as sensors, materials, or replacements for antibodies.5 The potential for functionalization of nucleic acid aptamers with a plethora of groups at various positions has enabled the development of these systems into functional structures with promise in therapeutic applications.6 However, a potential disadvantage in comparison with their analogous protein structures (antibodies) is the lack of diversity in the structural set, which is restricted to the four nucleobases (G, A, C, U).7 This has prompted efforts to diversify the nature of the nucleobases with other groups.8 Of note are examples in which a selection process led to chemically modified aptamers with dissociation constants in the nanomolar to sub‐picomolar range, and with potential for therapeutic applications, with selectivity towards biologically relevant targets such as cancer cell lines, aspartyl protease β‐secretase 1 (BACE1), proprotein convertase subtilisin/kexin type 9 (PCSK9), vascular endothelial cell growth factor‐165 (VEGF‐165), or interferon‐γ (IFN‐γ).9 Specifically, the modified nucleobases that were used in these works include 7‐(thiophen‐2‐yl)imidazo[4,5‐b]pyridine, 5‐chlorouracil/7‐deazadATP mixtures, or C5‐modified pyrimidine systems containing hydrophobic moieties such as naphthyl, phenyl, or morpholino groups. We are interested in probing the H‐bonding capabilities of modified nucleobases to control the function of aptamers and to diversify the "toolkit" to generate constructs with distinct selectivity and, ideally, increased affinity.</p><p>We opted to explore a modification that has been widely studied in other contexts: 7,8‐dihydro‐8‐oxoguanosine (8‐oxoG). Of the many oxidative lesions that have been characterized in DNA or RNA, purine‐derived modifications are expected to be the most abundant [redox potential trend=G<A<C<U].10 Two important features that make 8‐oxoG a unique structural building block are: 1) its preference to undergo an anti→syn conformational change around the glycosidic bond, and 2) the distinct H‐bonding patterns arising from each of these isomers (or, put another way, its ability to form stable base pairs with cytosine or adenine, Scheme 1). The nature of the conformational change to the syn isomer is known to be independent of solvent (DMSO or water) or pH (5–8) and is due to steric hindrance between the C5′‐position and the atom at the C8‐position.11 Thus, it is expected that, in a single‐stranded RNA, this will be the preferential conformation. However, because both faces can be involved in H‐bonding, this context may vary for cases in which 8‐oxoG is involved in intra‐ or intermolecular interactions, such as in its folding to other secondary structures,12 or in H‐bonding with other biopolymers.13 These factors are known to present a challenge from a biological point of view, if the modification is generated in vivo, and have been characterized in monomers14 and in oligonucleotides (ONs).15 On the other hand, this behavior can be attractive and promising from a design perspective: that is, in the use of 8‐oxoG as an ON building block to target small molecules or proteins of interest. The 8‐oxoG unit offers the possibility of altered function, due to its inherent capability to generate H‐bonding networks that are distinct from those generated by its canonical analogues, yet structurally similar. This property has been explored in the design of compounds that fit the H‐bonding pattern of 8‐oxoG for its detection,16 to control DNA structure,17 in the formation of supramolecular helices with potential uses for electronic biodevices,18 or in the use of C8‐substituted guanosine analogues to understand biological mechanisms.19 Of note is an example in which a DNA aptamer was designed to detect 8‐oxoG through interactions similar to those shown in Scheme 1.20 In addition, the presence of 8‐oxoG has also been shown to affect the overall structure of DNA and to lead to its deformation.21 These examples provide evidence that, although the effects of this oxidative modification on RNA have not been explored in detail, there is great potential in using it as a handle to control the structure and function of RNA.</p><!><p>Oxidation of G at the C8‐position leads to 8‐oxoG. The group at this position experiences steric hindrance with the C5′‐hydrogen atoms, and this induces a conformational change to the syn isomer.</p><!><p>To probe for the impact that 8‐oxoG has on the structure and function of RNA, the aptamer for theophylline was used as model. This construct was originally selected with the development of SELEX over two decades ago.22 Since then, it has been used as a model system for various applications,23 such as to explore new sensing methodologies,24 to establish theoretical models,25 or to detect theophylline in serum.26</p><p>The construct is an RNA strand, 33 nt in length, with high selectivity for theophylline27 (Scheme 2). This represents a good model because of existing detailed knowledge on the interactions between the RNA and the small molecule. Specifically, it is known that four guanosine units (G4, G25, G26, G29) are involved in the recognition of theophylline whereas three more are present in the vicinity of the active site (G11, G19, and G31), thus making these positions attractive candidates for probing the impact that the 8‐oxoG lesion has on structure and small‐molecule recognition. This aptamer is able to recognize the small molecule over other targets, containing minor structural differences, with up to 10 000‐fold increased affinity.28 It has been established, for example, that the canonical aptamer can recognize theophylline (1,3‐dimethylxanthine) over theobromine (3,7‐dimethylxanthine), which differs in the position of a single methyl group, or caffeine (1,3,7‐trimethylxanthine), which contains an additional methyl group (Scheme 2 A).</p><!><p>A) Sequence of theophylline aptamer and structures of xanthine derivatives. B) Intrastrand/intermolecular interactions (color‐coded) that are directly involved in theophylline recognition.</p><!><p>In brief, three levels of scaffolding provide support for the aptamer's binding pocket (Scheme 2 B), which is composed of 14 nucleotides that form a platform/lower loop, as well as a ceiling, that fits the theophylline and recognizes it through hydrogen‐bonding interactions with C22 and U24. The aptamer contains an S‐turn and maintains a shape that brings the lower and upper loops of the conserved region into proximity to intercalate, thus generating the binding pocket.29 Specifically, U23 H‐bonds to A28, due to the S‐turn in its tertiary structure, and this allows a base triple interaction with U6 to form the floor of the binding pocket, whereas a base triple interaction between A7, C8, and G26 provides the "ceiling" of the binding pocket. One side of the recognition site is generated through π–π stacking interactions between C21, A7, C22, and U6, whereas the other side is formed by G26 intercalation between G25 and U24, thought to stabilize the sharp bend in the tertiary structure to allow C22 to intercalate between U6 and A7. In addition, providing further stabilization of the binding core, A10 π‐stacks with G25 and G11. An important factor to note is that A7 is displaced from its typical A‐form helical position, thus generating space that facilitates binding to the target. With regard to the high affinity towards theophylline, C22 and U24 are involved in its discrimination from other xanthine derivatives through H‐bonding interactions with N7−H and the O6 carbonyl group of the small molecule.</p><!><p>We set out to explore the impact of the 8‐oxoG unit on the theophylline RNA aptamer and incorporated the lesion into ONs of RNA via solid‐phase synthesis. The RNA construct (strand 1), 33 nucleotides in length, was modified with 8‐oxoG at positions G25, G26, and G11, chosen on the basis of their roles in target recognition within the binding pocket, to yield RNA strands 2, 3, and 4 respectively (Figure 1). Importantly, the construct for which more structural information is available was chosen.28</p><!><p>Sequences of RNA strands 1–4 (top) and folding of canonical strand 1, together with 20 % denaturing PAGE of RNAs 1–4 after treatment with (left) RNase A or (right) RNase T1. A hydrolysis ladder (NaHCO3) was used for fragment assignment. Pyrimidine and guanine rings in which cleavage was observed are highlighted in blue and green, respectively.</p><!><p>To understand the impact of the modification on the structure, all strands were radiolabeled and treated with RNase A, which cleaves single‐stranded RNAs at both pyrimidine and 8‐oxoG sites into fragments containing 5′‐OH and 3′‐phosphate ends.30 The results were then compared with those obtained with canonical strand 1. As shown in Figure 1 (left), RNA strands 1–3 displayed hyperreactivity at positions U6 and C9, and to a lower extent some cleavage in the C20–C22 region (in the cases of modified strands 2 and 3), thus suggesting that these positions are in environments available for ribonuclease access and not restricted to H‐bonding interactions with other nucleobases. Cleavage at position C9 was unexpected; however, this marks the beginning of the internal loop (4×1), thus presumably making it more accessible. Interestingly, incorporation of 8‐oxoG at positions G25 or G26 gave rise to similar cleavage patterns, thus suggesting that modification at these sites does not affect the overall structure of the aptamer, or that these positions are cleaved because they are also exposed in another structural arrangement. The only observed difference was that cleavage around the C20–C22 region was increased slightly in the case of RNA 3 (G26‐modified), thus indicating that interactions amongst nucleobases in this section of the construct might be altered. The most striking difference was observed in the case of strand 4, modified at position G11, which displayed prominent cleavage at additional positions (C12 and C13), consistent with disruption of the stem in the upper hairpin and exposure of these nucleobases to the ribonuclease. Assignment of the bands was carried out by comparing the results against a hydrolysis ladder (NaHCO3, pH 9.1). The ladder works through cleavage of every nucleobase from the 3′‐end, thus allowing a band to appear at every position of the aptamer. A pattern in which doublet bands are observed in cases of shorter fragments is consistent with the formation of the 3′‐phosphate, along with the corresponding cyclic phosphate derivative that has been characterized/observed previously.31</p><p>To complement these observations, experiments were also carried out with RNase T1, a ribonuclease that specifically cleaves at all G‐sites in a single‐stranded context. Interestingly, the only aptamer that showed some hypersensitivity to this ribonuclease was aptamer 4, which displayed cleavage at positions G14, G18, and G19. Consistent with the results obtained in the case of the RNase A cleavage, this observation points to disruption of the upper hairpin within the construct. Although the intensity of the cleavage bands was weak, the pattern was reproducible. Furthermore, in agreement with alteration at the binding site, cleavage at position G29 took place in the case of RNA strand 2, whereas position G25 (G26 was ruled out because of the reported inability of RNase T1 to cleave at 8‐oxoG sites) was accessible for ribonuclease activity in the case of modified aptamer 3.</p><p>In an attempt to interpret these changes and to explain the reactivity patterns, the UNAFold server was used to explore possible changes in structure (Figures S5–S8 in the Supporting Information).32 As expected, the model for the canonical RNA 1 matched relatively well with the known structure, with the only exception being the prediction of an A11:U24 base pair (Figure S5). Because 8‐oxoG is not available in this hybridization package, we reasoned that substituting the modification with uridine could provide a valid model, on the basis of the H‐bonding similarities between syn‐8‐oxoG and U. However, this approach predicted structural changes in the case of RNA strands 2/3 (modified at G25 and G26) that do not explain the observed RNase A cleavage patterns (Figures S6 and S7). The same analysis was carried out by substituting G11 with U, which resulted in disruption of the hairpin stem in this region but did not fully explain the RNase A cleavage pattern at positions C12 and C13. Overall, substitution of U in place of 8‐oxoG did not provide a good model in this context.</p><p>To assess the impact that 8‐oxoG might have in the upper stem, RNA strands 5 and 6, containing G or 8‐oxoG (Scheme 3), were prepared in order to mimic this structural motif. The substitution was incorporated at position 2 of this construct and all strands displayed bands consistent with formation of a hairpin (bands with positive and negative ellipticity at 260 and 210 nm, respectively). Thermal denaturation transitions were obtained by measuring the hypochromic shift in the dichroic signal at 260 nm as a function of temperature and were independent of concentration, thus suggesting unimolecular transitions. Incorporation of 8‐oxoG, as in strand 6, resulted in a large thermal destabilization relative to canonical RNA hairpin 5. This is consistent with other cases in which an oxidative lesion has a large impact on hairpin stability.33</p><!><p>Sequences of hairpins 5–8 mimicking the upper scaffold of the aptamer and their corresponding T m values (experimentally measured and calculated with UNAFOLD). Conditions: RNA 3.5 μm, NaCl 1 mm, MgCl2 5 mm, Na2P2O7 10 mm, pH 7.2.</p><!><p>To confirm that the 8‐oxoG base pairs with C in this context, hairpins 7 (containing U) and 8 (lacking a nucleotide) were also prepared, and displayed larger thermal destabilization in both cases. This result indicates that 8‐oxoG does interact with C at this position, albeit more weakly than in the case of the corresponding WC pair. The observed trends were in agreement with those predicted by use of UNAFOLD, albeit with values that were consistently lower than those observed experimentally (Figures S9–S11 and Scheme 3, inset). Overall, the difference in the thermal stability between hairpins 5 and 6 (ΔT m ≈−9 °C) suggests that positioning 8‐oxoG in the aptamer at this position (G11, Figure 1) leads to a drastic structural change, yet to be fully characterized.</p><!><p>We then explored the impact that the 8‐oxoG lesion has on small‐molecule recognition and used microscale thermophoresis (MST) to establish the selectivity and affinity of each construct towards the three xanthine derivatives. This technique was chosen because it allows for the determination of the dissociation constant (K d) between an aptamer and its cognate ligand in free solution and with minimal sample consumption.34 This technique relies on recording the thermophoretic effect of the aptamer in the absence and in the presence of the target molecule,35 and is carried out by measuring changes in fluorescence as the bound/unbound ON migrates across a heat gradient inside a capillary.36 To this end, ONs (modified at the same positions as in the previous strands: G11, G25, G26) containing a cyanine‐5 dye (λ abs=646 nm, λ em=662 nm) at the 3′‐end were prepared via solid‐phase synthesis to yield RNA strands 9–12 (Figure 2 A). To interpret the data from the binding thermophoretic assays, the recommendations from a recent report were followed.37 In addition, because interactions between nucleic acids and their targets can be affected by salt and buffer concentrations38 we used buffer systems that have been reported previously.28b</p><!><p>A) Sequences of Cy‐5‐labeled RNAs 9–12 and their corresponding thermal denaturation transitions (T m), obtained in a 10 mm sodium phosphate buffer (pH 7.5) and a 50 mm Tris⋅HCl saline (TBS) buffer (pH 7.6). MST binding check, binding traces, and K d‐fit curve of B) construct 9 with theophylline, and C) construct 10 with theobromine (additional curves are included in Figure S14, displaying the same binding check and K d calculation with a different buffer). The blue trace corresponds to unbound RNA, the green trace to RNA bound to the small molecule, and the red traces correspond to the RNA titrated with the small molecule (some curves at different concentrations were omitted for clarity). D) K d‐fit curve of constructs 10/11 with theophylline and 9 (no binding) with theobromine. All curves were obtained in triplicate.</p><!><p>Binding checks were performed in the presence of each target to validate interactions between the small molecule and the aptamer, or to rule out aptamers that do not display specific binding; these experiments were carried out by measuring the ON mobility either in the absence or in the presence of high concentrations of the small molecules. As depicted in Figure 2 B (left) the canonical aptamer 9 displayed binding to the expected target, theophylline, whereas no binding occurred in the presence of theobromine or caffeine. Solutions containing the small molecule were then prepared by making sixteen twofold dilutions, to obtain a dissociation constant (K d) of (29±35) μm, a value consistent with the literature.28b, 29 In addition, isothermal titration calorimetry (ITC) was used to validate binding of the canonical RNA aptamer 1 to theophylline, to obtain a K d value of 1.35 μm (Figure S24). Interestingly, modified aptamers 10 and 11 also displayed binding affinity towards theophylline, albeit with affinities approximately two orders of magnitude weaker (K d>1.5 mm, Figure 2 D). An exact quantity could not be established, due to the low solubility of the small molecules in the buffer system at higher concentrations. The fact that these two aptamers recognize theophylline, to some extent, is not surprising in view of their similar structural characteristics (as observed through enzymatic cleavage experiments). On the other hand, as shown in Figure 2 C, aptamer 10 (modified at G25) displayed a higher binding affinity towards theobromine, with K d>160 μm (a complete sigmoidal curve for this pair could not be obtained, due to the poor solubility of theobromine), and with no binding of caffeine. This result highlights the ability of 8‐oxoG to have an impact on structure as well as on small‐molecule affinity and selectivity. Furthermore, consistent with large structural changes, aptamer 12 (modified at G11) did not display any binding affinity towards the three xanthine derivatives tested in this work. To provide better understanding of the impact of 8‐oxoG as a function of position, the thermal denaturation transitions were obtained (by CD, Figure S15) to show values for aptamers 9 and 10 that are equivalent, whereas aptamers 11 and 12 have increased thermal stability. As established previously, stabilization of structure can, in some cases, be directly related to decreased affinity in target binding.12 The large stabilization observed in the case of aptamer 11 might be due to increased interactions between 8‐oxoG and other nucleobases, arising from extended H‐bonding networks formed from both Watson–Crick and Hoogsteen faces.</p><p>Attempts to record changes in structure by CD in the absence and in the presence of the xanthine derivatives did not show any appreciable differences (Figure S23). All aptamers displayed dichroic bands consistent with folding into A‐form duplexes. Although the structural probing experiments indicate that a different structure might be formed in the cases of RNAs 4/12 (modified at position G11), the CD spectra showed that the extent of the duplex region is comparable to those of the other modified RNA strands, as well as that of the canonical aptamer (Figure S15).</p><!><p>The impact of 7,8‐dihydro‐8‐oxoguanosine (8‐oxoG) on RNA structure and function was explored by using the aptamer for theophylline as a model. The 8‐oxoG motif was independently incorporated onto ONs of RNA and its structural impact was assessed by electrophoretic analyses and circular dichroism, whereas its function was established by assessing the ability of the modified aptamers to bind three different xanthine derivatives: theophylline, theobromine, and caffeine. Thermophoretic analyses showed that the selectivity was altered in the case of modified aptamer 8 (8‐oxoG at position 25), which displayed K d values in the micromolar range for theobromine and in the millimolar range for theophylline.</p><p>The results from the RNase A/T1 cleavage studies, along with results obtained from MST experiments, indicate that a modification at G11 has the most drastic impact on the aptamer's structure, whereas modifications at G25 and G26 alter the structure slightly and still allow binding of theophylline. Although RNA 2/10 has features that suggest a structure similar to that of the canonical analogue 1/9, based on enzymatic degradation and T m analyses, a change in the H‐bonding pattern of 8‐oxoG25 might induce a change in the binding pocket. It is plausible that the anti‐to‐syn flip for 8‐oxoG25 allows it to hydrogen bond with A10, in turn allowing the formation of WC base pair G26:C9, breaking interactions with C8, which ultimately disrupts the ceiling for the theophylline binding pocket (Scheme 4 A). C8 is necessary for the canonical aptamer's high‐affinity interaction with theophylline. The 8‐oxoG25 and A10 base pairing also disrupts the π‐stacking interaction between G25 and A10; this could then destabilize the binding core and might displace conserved residues such as C21 and C22. The large increase in K d is also consistent with a disruption in the binding pocket. The slight degradation seen at G25 could indicate that the H‐bond to A10 is relatively weak. Furthermore, the higher affinity towards theobromine can be explained by the displacement of C22, which is integral in discriminating for theophylline. The conservation of U24 does not provide the ability to discriminate theophylline from xanthine derivatives because N9 is the same in both theophylline and theobromine, and this could thus explain why this aptamer can bind to theobromine.</p><!><p>Representations of RNA aptamer modified at position A) G25 (2/8), or B) G26 (3/9) and proposed models of how 8‐oxoG might induce a unique tertiary structure that disrupts the binding pocket. C) Plausible structures after modification at G11, showing how 8‐oxoG is likely to have a different, more pronounced, impact on the overall structure.</p><!><p>The results obtained with RNAs 3/11, modified at position 26, showed an increase of over 100‐fold in K d towards theophylline, and a lack of recognition for theobromine or caffeine. It is possible that, as depicted in Scheme 4 B, interactions with U6, which is pivotal in forming the floor of the binding pocket, are disrupted by the expected 8‐oxoG26:A7 base pair. This could also displace or prevent U6's interaction with its corresponding upper‐loop residues and impart stability, evident from a larger T m (74.5 °C). As mentioned above, A7 needs the ability to move out of the A‐form helix to create space for the small molecule, and this is unachievable if it is H‐bonding with 8‐oxoG26. This distinct H‐bond might also aid in the formation of a new ceiling for the small molecule, because G25 can now H‐bond with C8. The H‐bond between the 8‐oxoG and A7 might also disrupt the π‐stacking interactions of C21, A7, C22, and U6 by abolishing its ability to intercalate between C21 and C22; this could decrease the interaction between the upper and lower loop residues. Both RNAs 2/10 and 3/11 retain the ability to bind to theophylline, although weakly, because U24 is still conserved in the core and is potentially not affected by adverse H‐bonding or π‐stacking interactions, while still having an impact on the binding pocket.</p><p>We initially hypothesized that modification at G11 (strands 4/12) would not greatly impact small‐molecule recognition. However, this RNA did not bind to any of the xanthine derivatives. This can be explained in terms of the large structural changes observed in the upper loop, evident from the distinct RNase A/T1 cleavage pattern and suggesting that 8‐oxoG11 disrupts the upper scaffolding of the aptamer overall. It is possible that 8‐oxoG11 could be involved in H‐bond interactions with A16 or A17, which would explain why both G18 and G19 are exposed to degradation. In addition, this could result in C21 and C22 being prevented from intercalating with A7 and U6, in turn affecting the role of U24 within the binding core and intercalation between G26 and A28, which would ultimately destabilize the S‐turn that is needed to maintain the integrity of the binding pocket. Attempts to explain the overall structure with the assistance of the UNAFold engine were carried out by substituting 8‐oxoG with U; however, the predicted structures, as well as other possibilities (Scheme 4 C), do not match well with the cleavage patterns. Overall, although the structures of the G11‐modified aptamers were not established, this suggests that the presence of 8‐oxoG at this position has a large impact on the structure and function of this aptamer. Other positions that are potential candidates for G‐to‐8‐oxoG substitution involve G14 and G29. However, two aspects deterred us from probing these positions: 1) they have not been reported to be involved in the recognition of the xanthine derivatives,29 and 2) given their proximity to adenosine units, they might potentially generate base‐pair interactions that stabilize the overall structure at the expense of selectivity and/or affinity.12</p><p>Lastly, another point of relevance relates to the biological impact that oxidative stress has on RNA. Oxidized RNA has been shown to be present in various types of RNA, including rRNA,39 mRNA,40 or miRNA,41 and this also makes riboswitches42 prone to oxidative damage. Although this relationship has not been established, it is plausible that such processes might play a role in dysfunction or altered regulatory mechanisms. Thus, understanding the potential structural/functional changes arising from the formation of oxidative lesions is of importance to assess their biological implications.</p><!><p>The impact that 8‐oxoG has on structure and function, through small‐molecule recognition, was probed with use of the theophylline aptamer as a model. It was shown that the effect of a single 8‐oxoG modification varies as a function of position and that it can result in decreased, or abolished, affinities towards the cognate target molecule, or that it can also change the selectivity of the aptamer towards a different target. Specifically, modification at G25 led to preferential binding of theobromine over theophylline. This result suggests that 8‐oxoG might be usable as a modification in the discovery of aptamers with distinct selectivity/affinity. Although this is a promising strategy, obstacles remain to overcome, including 1) the development of sequencing technologies that would enable a selection process that includes 8‐oxoG in RNA (although recent advances show promise in this respect),43 and 2) deeper understanding of the impact of 8‐oxoG on RNA structures within various structural motifs—aspects that are not trivial and that we are working to address.</p><!><p>General: 8‐OxoG phosphoramidite was synthesized out according to a previous report.12 All experiments were carried out in triplicate.</p><p>RNA synthesis: ONs were synthesized with a 394 ABI DNA/RNA synthesizer and use of CPG supports and 2′‐O‐TBDMS phosphoramidites (purchased from Glen Research). 5‐Ethylsulfanyl‐1H‐tetrazole (0.25 m) in acetonitrile was used as the coupling reagent, dichloroacetic acid in dichloromethane (3 %) was used for deblocking, a 2,6‐dimethylpyridine/acetic anhydride solution was used for capping, and an iodine/THF/pyridine solution was used in the oxidation step. Coupling times of 10 min were used. ONs were deacetylated/debenzoylated/deformylated and cleaved from the CPG support in the presence of 1:1 aq. methylamine (40 %) and aq. ammonia (40 %) with heating (60 °C, 1.5 h). A mixture of 1‐methylpyrrolidin‐2‐one/triethylamine/HF (3:2:1) was used for removal of the TBDMS groups (60 °C, 1 h), followed by purification by electrophoresis (20 % denaturing PAGE). C18‐Sep‐Pak cartridges were obtained from Waters and used to desalt the purified oligomers with NH4OAc (5 mm) as the elution buffer. ONs were dissolved in H2O and used as obtained for subsequent experiments. Unmodified ONs were purchased from IDT‐DNA or ChemGenes and, after quantification by UV/Vis, used without further purification. ONs containing a fluorescent Cy‐5 probe at the 3′‐end were synthesized on resin (purchased from Glen Research) containing this moiety and purified as described above.</p><p>RNA characterization: MS (MALDI‐TOF) was used in the characterization of all modified ONs, with use of C18 Zip Tip pipette tips to desalt and spot each ON as follows: 1) wash tip with acetonitrile (50 %, 10 μL×2), 2) equilibrate tip with trifluoroacetic acid (TFA, 0.1 %, 10 μL×2), 3) load tip with sample (typically 100–150 pmol), 4) wash tip with TFA (0.1 %, 10 μL×2), 5) wash tip with water (10 μL×2), 6) elute sample into matrix [10 μL of 2,4,6‐trihydroxyacetophenone monohydrate (25 mm), ammonium citrate (10 mm), ammonium fluoride (300 mm) in aq. acetonitrile (50 %)], and 7) spot directly onto MALDI plate. All analyses were carried out with an ABI 4800 Plus MALDI‐TOF/TOF mass spectrometer in positive mode (see the Acknowledgements).</p><p>UV/Vis spectroscopy: Concentrations of all ONs were determined by UV/Vis with a PerkinElmer λ‐650 UV/Vis spectrometer and quartz cuvettes (1 cm pathlength). General UV/Vis spectra were also taken with a 1 mm pathlength and 1 μL volumes (Thermo Scientific Nano Drop Nd‐1000 UV/Vis spectrometer). Origin 9.1 was used to plot and normalize spectra of monomers and ONs for comparison.</p><p>Circular dichroism (CD) spectroscopy and thermal denaturation transitions (T m ): CD spectra were recorded at various temperatures (PTC‐348W1 Peltier thermostat) with use of quartz cuvettes with a 1 cm pathlength. Spectra were averaged over three scans (325–200 nm, 0.5 nm intervals, 1 nm bandwidth, 1 s response time) and background‐corrected with the appropriate buffer or solvent. Solutions containing the RNA strands had the following compositions: RNA (1.5 μm), MgCl2 (5 mm), NaCl (10 mm), sodium phosphate [pH 7.3 (or other pH values whenever appropriate), 1 mm]. All solutions that had been prepared in order to record thermal denaturation transitions (T m) were hybridized prior to the recording of spectra by heating to 90 °C followed by slow cooling to room temperature. T m values were recorded at 270 nm with a ramp of 1 °C min−1 and step size of 0.2 with temperature ranges from 4 to 95 °C. A thin layer of mineral oil was added on top of each solution to keep concentrations constant at higher temperatures. Origin 9.1 was used to determine all T m values and to plot CD spectra of RNAs with/without small molecules.</p><p>ON labeling: T4 polynucleotide kinase (PNK) and γ‐32P‐ATP‐5′‐triphosphate were obtained from PerkinElmer. ONs were labeled by mixing PNK, PNK buffer, ATP, DNA, and water (final volume 50 μL) according to the manufacturer's procedure followed by incubation at 37 °C for 45 min. Radiolabeled materials were passed through a G‐25 Sephadex column followed by purification by electrophoresis (20 % denaturing PAGE). The bands of interest (slowest) were extruded and eluted over a saline buffer solution (0.1 m NaCl) for 36 h at 37 °C. The remaining solution was filtered and concentrated to dryness under reduced pressure followed by precipitation over NaOAc and ethanol. Supernatant was removed and the remaining ON was concentrated under reduced pressure and dissolved in water. Activity was assessed with a Beckmann LS 6500 scintillation counter.</p><p>Microscale thermophoresis (MST): This technique was used to establish binding affinities and potential for binding with the three xanthine derivatives. RNA strands modified with the Cy‐5 fluorophore at the 3′‐end were used.</p><p>MST—small‐molecule binding studies: The small‐molecule binding studies were performed with 16 twofold dilutions of each small molecule as can be seen in Table 1. The goal was to measure binding at varying concentrations of small molecule with a constant concentration of RNA to determine the corresponding dissociation constants (K d). The Monolith NT.115 MST system was used for these studies with concentrations of small molecules as shown in Table 1. Theobromine did not readily dissolve in the buffers used here and was dissolved in DMSO (100 %). This solution was then diluted in the corresponding buffer to achieve solutions with a DMSO content lower than 5 %. Precipitated theobromine was observed in the cases of solutions in which the small molecule was present in the 5 mm range, so experiments were set such that the concentration of theobromine was 1 mm maximum.</p><!><p>Small molecules used in the binding affinity studies and the corresponding concentration ranges.</p><p>Small molecule</p><p>Conc. range</p><p>theophylline</p><p>11.25 mm–343 nm</p><p>theobromine</p><p>1 mm–30 nm</p><p>caffeine</p><p>11.25 mm–343 nm</p><!><p>Binding checks were accomplished for all RNAs with each small molecule prior to K d determination. The binding checks were carried out to validate binding of the small molecule and aptamer. Eight samples were prepared—four that contained the RNA aptamer and four that contained the RNA and the small molecule. Both the RNA and the small molecule were kept constant at 10 nm and at the highest small‐molecule concentration (e.g., the theophylline concentration in the presence of RNA 9 was 11.25 mm). The prepared mixtures were incubated at room temperature for ≈10 min, captured in a capillary tube (as described below), and placed on the plate, with the RNA sample in rows 1–4 and the RNA/small molecule complex in rows 5–8.</p><p>Dissociation constants were obtained as follows. First, 16 microtubes (0.6 mL) were filled with 10 μL of the buffer containing the appropriate small molecule. For theophylline and caffeine, the buffer was 1× TBS [pH 7.5, Tris⋅HCl (50 mm), NaCl (150 mm)], whereas for theobromine the buffer was 1× TBS with DMSO (10 %). This was carried out in such a manner that sequential twofold dilution, with respect to the small molecule, was present in each tube. This was followed by addition of RNA (20 nm, previously denatured and hybridized by heating to 90 °C and slow cooling to RT, 10 μL) to each tube to accomplish 10 nm [RNA] (while keeping the content of DMSO at 5 % DMSO max.). The mixtures were then incubated in ice for 20 min in the cases of the mixtures containing theophylline or caffeine and at room temperature in those of the mixtures containing theobromine. Next, ≈10 μL of the mixture was captured in a capillary tube, placed on the plate, and covered with the magnetic strip to prevent movement. Each mixture was withdrawn and handled one at a time, with the solutions containing the highest concentration of small molecule placed at position "1". The microtube and the capillary were held horizontally to prevent bubble formation, and the capillary was handled by the end of the tube. Each programmed experiment ran for approximately 20 min.</p><p>RNA structural probing</p><p>RNase A: A cocktail solution of RNA (3000–5000 counts) in phosphate buffer (pH 5.5, 10 mm) was made. A mixture of the RNA and the enzyme (1:1) was prepared and incubated at RT for 1 h. After incubation, loading buffer [LB: formamide (90 %), EDTA (1 mm), 7 μL] was added, and the mixture of interest (9–10 μL) was added to a denaturing PAGE. For shorter oligomers (<20 nt) a short gel was used. For longer oligomers a long gel was used. The gels were run until the methylene blue dye ran halfway to three quarters of the way down the gel.</p><p>RNase T1: A cocktail solution of RNA (3000–5000 counts) in phosphate buffer (pH 5.5, 10 mm) was made. A mixture of the RNA and the enzyme (1:1) was prepared and incubated at 50 °C for 45 min. After incubation, LB (7 μL) was added, and the mixture of interest (9–10 μL) was loaded onto a denaturing PAGE. For shorter oligomers (<20) a short gel was used. For longer oligomers (>20) a long gel was used. The gels were run until the methylene blue dye ran halfway to three quarters of the way down the gel.</p><p>The degradation patterns for all aptamers were compared against a hydrolysis ladder (0.5 m NaHCO3, pH 9.1), which was used for band assignment. The ladder works through hydrolysis of every nucleobase from the 3′‐end and was produced by incubating the RNA of interest and the hydrolysis buffer at 90 °C for 12 min (68:32 RNA/NaHCO3 ratio, by volume) followed by addition of loading buffer (formamide, 90 %) prior to loading onto gel.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Catalytic oxidative desulfurization of a 4,6-DMDBT containing model fuel by metal-free activated carbons: the key role of surface chemistry

Commercial micro/mesoporous activated carbons were utilized as metal-free catalysts for the desulfurization of a model fuel, i.e. 4,6-dimethyldibenzothiophene (4,6-DMDBT) in hexadecane under ambient conditions. Both adsorption and catalytic oxidation were investigated as means of 4,6-DMDBT removal.The effect of chemical modification/oxidation of the carbon surface via treatment with two different acids (HNO 3 or H 2 SO 4 ) aiming to introduce additional functional groups was also investigated. The catalysts were characterized by FT-IR spectroscopy, N 2 porosimetry, potentiometric titration, Boehm titration, and SEM-EDX, while adsorption and catalytic oxidation activity towards sufloxides and sulfones were assessed by GC-MS and UV-Vis analysis. The surface chemistry of the carbons, expressed by the density of the acidic functional groups, was found to be the most critical parameter with regard to adsorption or to catalytic oxidative performance. The surface modification of carbons by oxidation had a positive impact on the catalytic oxidation activity, leading to a 100% conversion of 4,6-DMDBT towards the corresponding sulfoxide and sulfone, compared to 67% with the parent non-oxidized carbon. Reusability tests showed that the oxidation activity of the carbons can be maintained for at least 5 cycles. † Electronic supplementary information (ESI) available. See

catalytic_oxidative_desulfurization_of_a_4,6-dmdbt_containing_model_fuel_by_metal-free_activated_car

Introduction<!>Materials and reagents<!>Modification of the carbon catalyst<!>Materials characterization methods<!>Characterization of activated carbon<!>Desulfurizationadsorption and catalytic oxidation results for the commercial carbons<!>Effect of oxidation reaction parameters<!>Reusability cycles<!>Adsorptive/catalytic performance of oxidized carbons<!>Characterization of oxidized carbons<!>Effect of contact timekinetics of the 4,6-DMDBT removal<!>Activated carbons<!>Characterization of the carbons after reaction<!>UV-Vis and GC-MS analysis of the liquid products and extracts from the used catalysts<!>Mechanistic insights<!>Conclusions

<p>Nowadays, great interest has been focused on the mitigation of sulfur compounds in fuels in order to comply with stringent regulations, as they cause environmental problems and human health issues. [1][2][3] According to the Environmental Protection Agency of USA (EPA), the admissible sulfur concentration in diesel fuel has been 15 ppm since 2006. 1,4 The European Parliament and the Council of the European Union have established in 2009 an even lower concentration of sulfur in fuels, 10 ppm. 5 The industrial process used for decades to remove sulfur from fuels is hydrodesulfurization (HDS), a catalytic process in which organic sulfur compounds are converted to hydrogen sulfide and sulfur-free hydrocarbons by reaction with hydrogen over CoMo/Al 2 O 3 or NiMo/Al 2 O 3 catalysts. 6,7 An important drawback of HDS is the required high hydrogen pressure, 4 leading to a very costly process, especially when deep desulfurization is aimed. 8,9 Furthermore, HDS is not effective in the removal of sulfur heterocyclic hydrocarbons such as dibenzothiophene (DBT) and its derivatives, especially 4,6-dimethyldibenzothiophene (4,6-DMDBT), which is not so reactive due to steric hindrance effects. 4,[9][10][11][12][13] Within this context, the development of more efficient and greener/sustainable desulfurization methods which have the potential to produce extremely ultra-low-sulfur fuels, so as to replace or complement the HDS process, is of great importance. Adsorption, 14,15 biodesulfurization, 16,17 extraction by ionic-liquids, 18 photocatalytic oxidation 19 and oxidative desulfurization (ODS) 20,21 are some new processes that have been introduced for efficient fuel desulfurization. Among these approaches, oxidative desulfurization constitutes a promising method, as it is simple and of higher efficiency compared to HDS. 22,23 In addition, 4,6-DMDBT is expected to exhibit higher reactivity in ODS. 24,25 The ODS method involves the oxidation of the sulfur-containing compounds, followed by the extraction/removal of the oxidized products from the fuel due to their polarity. In the ODS process, sulfur-containing compounds are oxidized using selective oxidants such as nitric acid, 26 nitrogen oxides, 27 organic hydroperoxides, 28 hydrogen peroxide 24,29,30 or/and ozone 31 in the presence of a catalyst to produce sulfone compounds which can be extracted preferably due to their increased polarity. 32 The most commonly used oxidant is hydrogen peroxide (H 2 O 2 ), due to the fact that it is environ-mentally friendly, it has low cost and it is commercially available. 29 Various homogeneous and heterogeneous catalysts like polyoxometalates (POMs), 33 mono-or bimetallic alloys, 34 organic acids, 35 ionic liquids, 18,36 multi-walled carbon nanotubes 37 and activated carbons 38 have been shown to be active in the desulfurization reactions. In particular, the latter ones have been previously utilized as supports for active phases like metal oxide, metal or bimetallic nanoparticles, [39][40][41][42][43] as for example in the deposition of molybdenum cobalt nanocatalysts on a carbon support which led to 34% increment of dibenzothiophene hydrodesulfurization from a model oil, compared to pure MoCo. 44 Even though carbons are often used as catalyst supports, they can also be utilized as catalysts on their own, because of their physicochemical properties, such as the rich surface chemistry due to the presence of oxygen containing functional groups, the large micro/mesopore surface area, and their relatively stable structure and morphology at high temperatures and/or various liquid reaction media. [45][46][47] One of the most known reactions catalyzed by carbonaceous materials, the decomposition of hydrogen peroxide, is highly dependent on the nature of the surface functional groups. Chemical reactions on carbon's surfaces usually follow a free radical mechanism. 48,49 The produced radicals are stabilized on the surface of the carbon so that they can act as adsorption/oxidation sites of sulfur compounds. 48,50 Despite the wide application of carbons in environment related reactions and processes, limited investigation has been devoted to the use of activated carbons as metal-free catalysts in desulfurization processes and especially for ODS of 4,6-DMDBT. In most cases, the carbons were studied as adsorbents for the removal of smaller DBT or other derivatives. 22,50 Considering that the production of carbons with different morphology, texture and surface properties can be achieved by utilizing the most abundant and renewable source, biomass, [51][52][53][54] the use of biomass derived carbocatalysts will be of great importance towards a sustainable future. In the present study, we examined five activated carbons, with or without prior treatment with acids, for the oxidation of 4,6-DMDBT within the process of fuel desulfurization. Emphasis was given to the investigation of the role of carbon's textural characteristics and surface chemistry features towards maximization of desulfurization activity.</p><!><p>Five commercial activated carbons, obtained from Cabot Norit activated carbon and CPL activated carbons, were chosen to be tested as carbocatalysts: (i) Norit SX-PLUS, (ii) Norit SAE-SUPER, (iii) Norit D-10, (iv) Norit SAE-2, and (v) CPL. 4,6-DMDBT (4,6-dimethyldibenzothiophene), hexadecane, commercially available 30% wt% H 2 O 2 , methanol, HNO 3 , and H 2 SO 4 99.999% purity were purchased from Sigma Aldrich.</p><!><p>The SX PLUS carbon was chemically treated either with HNO 3 or H 2 SO 4 , targeting to modify its surface chemistry.</p><p>2.2.1. Oxidation with HNO 3 . For the preparation of the HNO 3 oxidized activated carbon sample, 10 g of the activated carbon SX PLUS was oxidized in a 70% HNO 3 solution (100 mL) for 4 hours under vigorous stirring at room temperature. The excess of acid and the possibly formed soluble products upon the oxidation process were removed by filtration and extensive washing of the carbon sample in a Soxhlet apparatus, until constant pH. 4 The obtained material was oven dried at 60 °C for 24 h. This carbon sample was named SX PLUS N-ox.</p><p>2.2.2. Oxidation with H 2 SO 4 . The H 2 SO 4 modified activated carbons were prepared by oxidizing 10 g of the activated carbon with concentrated H 2 SO 4 (100 mL) at 60 °C for 4 hours under stirring. The oxidized carbons were recovered by filtration, washed thoroughly in a Soxhlet apparatus until constant pH and dried at 60 °C for 24 h. 55 This sample is referred to as SX PLUS S-ox.</p><!><p>2.3.1. Textural/porosity characterization of activated carbons. The textural characterization of the activated carbons was carried out by measuring the N 2 adsorption/desorption isotherms with an AS1Win (Quantachrome Instruments, FL, USA) porosimeter. In a typical measurement, 0.05 g of the activated carbon were initially outgassed under vacuum at 150 °C overnight, followed by determination of the N 2 adsorption and desorption isotherms at −196 °C. The BET surface area was calculated from the isotherm data using the Brunauer, Emmett and Teller (BET) equation, while the pore size distribution curves were estimated using the DFT method. 56,57 2.3.2. pH measurement. The pH of the activated carbons provides information about the acidity and basicity of their surface. For the pH measurement, 0.4 g of the carbon was added to 20 ml of deionized water and the suspension was kept under magnetic stirring at room temperature for about 24 hours to achieve equilibrium. The pH of the solution was then measured using a CRISON basic-20 pH meter.</p><p>2.3.3. Boehm titration. The oxygen containing groups located on the surface of the activated carbons were determined by Boehm titration. This method relies on the different acidity of the surface groups, where each group is neutralized with a different reagent of similar activity. 58 In particular, sodium bicarbonate (NaHCO 3 ) neutralizes only carboxyls belonging to the strong acidic groups on the carbon surface. Sodium carbonate (Na 2 CO 3 ) neutralizes carboxyls and lactones and sodium hydroxide (NaOH) neutralizes carboxyls, lactones and phenols belonging to the weaker acidic groups. The determination of the basic surface groups was performed with hydrochloric acid solution, HCl. 59 In a typical measurement, 1 g of sample was placed in 50 ml of 0.05 N solution of each base (NaOH, NaHCO 3 , Na 2 CO 3 ) or the acid (HCl) in sealed conical flasks under stirring for 24 h at ambient temperature.</p><p>Then, the solution was filtered, and the specific volume of the filtrate was titrated. Thus, the excess base or acid in the solution was neutralized with HCl or NaOH, respectively. The numbers of total surface acidic and basic sites, as well as the content of carboxylic, phenol and lactone groups, were thus determined.</p><p>2.3.4. Potentiometric titration. Potentiometric titration measurements were carried out with a Mettler Toledo T50 automatic titrator. In a typical measurement, 0.1 g of activated carbon was placed in a conical flask with 50 mL KNO 3 solution (0.1 mol L −1 ) under stirring for 24 h, at 25 °C. The solution was then titrated with NaOH solution (0.1 mol L −1 ) under a N 2 atmosphere over a wide pH range. The total surface charge, Q (mmol g −1 ), was calculated as a function of pH from the following equation: 4</p><p>where C A and C B (mol L −1 ) are the acid (HCl) and base (NaOH) concentrations (mol L −1 ), respectively, [H + ] and [OH − ] are the equilibrium concentrations of these ions (mol L −1 ) and W is the concentration of the solid (g L −1 ). 2.3.5. Point of zero charge. A certain volume of 0.01 M NaCl solution was placed in titration vessels at constant temperature (25 °C) and 0.05 g of activated carbon was added to each vessel. The pH initial of the dispersions was adjusted to values between 3 and 9 (3, 5, 7, 9) and the suspensions were allowed to equilibrate, under stirring, for 48 h. The final pH was measured and was plotted for each dispersion against the initial pH. The pH at which the curve crossed the line pH initial = pH final was taken as the point of zero charge (PZC). 60 2.3.6. Fourier transform-infrared spectroscopy (FTIR). Fourier transform-infrared (FTIR) spectra (10 scans per measurement) were recorded on a PerkinElmer 2000-FTIR spectrophotometer (Dresden, Germany) in the wavenumber range of 4000-450 cm −1 , by applying the KBr-pellet technique.</p><p>2.3.7. Energy-dispersive X-ray spectroscopy (EDX). Elemental analysis was performed by EDX using a scanning electron microscope, model Zeiss Supra 55VP, Jena, Germany. The hermetically sealed conical flasks were placed in a shaking bath for 48 h at constant temperature. After the equilibrium time (evaluated from kinetic studies), the remaining concentration of 4,6-DMDBT was determined by using a UV-vis spectrophotometer (Hitachi U2000) at a wavelength of 313 nm using a quartz cuvette. The removal percentage (R%) of 4,6-DMDBT was calculated by the equation:</p><p>where C 0 and C e ( ppmw of sulfur) are the initial and the equilibrium concentrations, respectively. 61 Kinetic experiments were performed by dispersing 0.01 g L −1 of carbon in 20 mL of 4,6-DMDBT solution in hexadecane (C 0 = 20 ppmw of S), under stirring at 60 °C for different intervals of time. 62 For each separate experiment, filtration through a 50 μm pore size membrane was performed and the 4,6-DMDBT concentration was measured by using a UV spectrophotometer as mentioned above.</p><p>The experimental results were fitted by Lagergren's pseudofirst order kinetic model, 63 given by the equation lnðq e À q t Þ ¼ ln q e À k 1 t where q t and q e are the amounts of 4,6-DMDBT (mg g −1 ) adsorbed at time t and at equilibrium, respectively and k 1 is the rate constant of the pseudo-first order adsorption process (min −1 ), as well as by the pseudo-second order kinetic model, 64 given by the following equation in linear form, where k 2 , the rate constant (g mg −1 min −1 ):</p><p>1 q e t 2.4.2. Catalytic oxidation of 4,6-DMDBT/oxidative desulfurization. The model 4,6-DMDBT solution in hexadecane was of 20 ppmw concentration in sulfur. In a 50 ml round-bottom flask with a reflux condenser, 0.025 g of the carbon catalyst were weighed, and then 10 ml of the model solution and 1 ml H 2 O 2 (30 wt%) were added. The catalytic reactions were conducted at 60 °C for 24 h, under mechanical stirring. After the reaction, the mixture was filtered and the residual amount of 4,6-DMDBT in the solution was determined using UV-Vis spectroscopy at λ = 313 nm.</p><p>The liquid products were also analyzed by GC-MS on a 7890A/5975C system by Agilent (electron energy 70 eV, helium flow rate: 0.7 cc min −1 , Column: HP-5MS 30 m × 0.25 mm ID × 0.25 μm). Identification of mass spectra peaks was performed by the use of the scientific library NIST11s. Extraction solutions of the spent samples were also analyzed with the same instrument.</p><!><p>The porosity characteristics and surface chemical features of the different activated carbons were initially determined and correlated with the adsorption and catalytic oxidation of 4,6-DMDBT. The N 2 adsorption-desorption isotherms of all the carbon samples (Fig. s1a †) showed a bimodal type of shape based on IUPAC classification. 65 At low relative pressures, there is a clear typical type I adsorption isotherm for all parent commercial carbons, revealing the abundance of micropores. At intermediate and higher relative pressures, the adsorption isotherms tend to adopt a type IV shape with the corresponding hysteresis loop, which was more pronounced for the CPL and SAE SUPER carbons, due to capillary condensation in narrow slit-shaped mesopores. 66,67 The pore size distribution curves (Fig. s1b †) revealed that there is a relatively narrow distribution in the low micropore range, i.e. <1 nm, and a broader distribution for pores >1 nm, except for the case of CPL which showed a relatively narrow distribution between 1 and 2 nm. The BET surface area values and porosity parameters for the activated carbons are presented in Table s1. † It is seen that CPL (1702 m 2 g −1 ) presented the highest surface area, followed by SX PLUS (1280 m 2 g −1 ), SAE SUPER (1182 m 2 g −1 ), and SAE2 (892 m 2 g −1 ), while D10 showed a relatively low surface area (515 m 2 g −1 ). The total pore volumes (Table s1 †) were found to follow the same trend as the SSA BET . The values of the microand meso-pore volume are shown in Fig. 1a (also listed in Table s1 †). CPL revealed also the highest ratio of mesopore to micropore or total pore volume, while SX PLUS the lowest.</p><p>Considering that the molecular size of 4,6-DMDBT is 0.59 × 0.89 nm, 4 it could be expected that micropores of ≤1 nm, as well as bigger micropores and small mesopores (up to ca. 2-3 nm), would play an important role regarding the adsorption and oxidation activity of the carbons.</p><p>The surface chemistry characteristics of the activated carbons are reported in Table s2. † From the surface pH measurements, it can be seen that D10, SAE SUPER, and SAE 2 activated carbons possess a relatively basic surface, SX PLUS almost neutral, while CPL exhibits a more acidic nature. The results from the point of zero charge ( pzc) determination (Fig. s2 †) are in-line with the proton binding curve potentiometric titration results (Fig. s3 †). The most interesting observation can be derived from the ratio of acidic to basic groups, with CPL having only acidic groups, followed by SX PLUS (0.70), while SAE SUPER has the lowest ratio (0.08). The calculated density of acidic surface functional groups (d ASFG ) and of the basic (d BSFG ) per surface area is presented in Fig. 1b. The Boehm titration results revealed mainly two kinds of oxygen functional groups on carbon's surface, i.e. lactonic and phenolic, while no carboxyl groups were detected. The amount of each type of group (mmol g −1 ) as well as their density per specific surface area unit (μmol m −2 ) is collected in Table s2. † SX PLUS shows the highest number of lactones, while SAE2 the highest amount of phenol groups.</p><!><p>The 4,6 DMDBT (20 ppmw of sulfur) adsorption results in hexadecane as a solvent in the dark, at 60 °C, without H 2 O 2 are presented in Fig. 2a. SX PLUS showed by far the best performance, achieving 60% removal while the lowest adsorptive capability was observed for the carbon with the lowest surface area and pore volume, D10. On the other hand, the activated carbon CPL with the highest BET surface area exhibited a moderate adsorption efficiency. Furthermore, SX PLUS and SAE SUPER have a similar surface area, but the former showed substantially higher adsorption of 4,6-DMDBT. It can thus be concluded that the BET surface area of the carbons is not the sole critical parameter that would define the adsorption performance. If one considers the correlation between the size of 4,6-DMDBT and the size of the pores, it appears that the carbons with a higher abundance of smaller micropores, i.e. with a size of ≤1 nm, such as SX-PLUS, SAE-2 and SAE SUPER, are more efficient possibly due to increased confinement effects due to size similarity.</p><p>With regard to the effect of the functional groups and correlation with the surface pH, SX PLUS and CPL provided a neutral/slightly acidic pH, compared to SAE-2 and SAE SUPER which were basic, and furthermore, they exhibited the highest ratio of acidic to basic surface groups, i.e. 1.0 for CPL and 0.7 for SX PLUS. In order to further investigate the possible combined effects of the surface area and acidic sites, the density of the acidic surface functional groups, per surface area unit, was estimated and it was plotted against the removal efficiently (Fig. 2b). As can be seen in Fig. 2b, the maximum adsorption capacity increases with the increment of the density of acidic surface functional groups (d ASFG ) up to an optimum value followed by a decrement upon further increase of d ASFG . This fact can be assigned to an enhanced steric effect when the active surface functional groups are close to each other, as well as to a blockage of the pores' entrance by adsorbed molecules, hindering the penetration of other molecules towards the active adsorption sites at the interior of the pores. This behavior leads to the suggestion that the density and dispersion of the active sites may be an important parameter for the adsorption efficiency of the carbons. However, when comparing the properties and performance of SX PLUS and CPL, it can be seen that the former contains a high portion of lactones while phenolic groups are predominant in the latter (Fig. 1c). This may also lead to the assumption that the more acidic lactones compared to phenols are more prone to interact with 4,5-DMDBT, considering also the relatively (i.e. compared to pyrrole for example) moderate Lewis basicity strength and enhanced aromatic stabilization of DMDBT. Overall, it can be suggested that both the type of acidic functional group and their density are key factors towards increasing the adsorption efficiency of the carbons.</p><p>Moreover, the Langmuir 68 and Freundlich 69 isotherms were fitted to the experimental results, as presented in Fig. s4, † and models' parameters are presented in Table s3. † Both models exhibit relatively good fitting indicating the initial monolayer formation of sorbed 4,6-DMDBT as well as the formation of secondary layers, due to the presence of meso/macroporosity as well as due to the inhomogeneity of the adsorption sites. The models' parameters are presented in Table s3, † from where it is seen that SX PLUS presented the highest adsorption capacity between all carbons.</p><p>The potential of fuel desulfurization via catalytic oxidation was investigated by the use of H 2 O 2 as the oxidation agent with the parent commercial activated carbons as catalysts (Fig. 2a</p><!><p>The effect of different parameters i.e. catalyst's dose, reaction temperature, and H 2 O 2 relative amount, on the catalytic oxidation was also studied, using SX PLUS being identified above as the most efficient. As mentioned above, no conversion of 4,6-DMDBT was observed in the presence of H 2 O 2 without using carbons at 60 °C, showing that the activated carbon actually acts as a catalyst in the reaction. 70 The 4,6-DMDBT removal efficiency with different amounts of catalysts is shown in Fig. 3a. It is observed that by increasing the amount of carbocatalyst, the removal extent is enhanced. More than 90% removal is achieved by a relatively low amount of carbon (100 mg). 25 mg can be assumed as the optimum one, since the removal extent by a further increase of the carbon amount is minimal.</p><p>The effect of temperature was also investigated by carrying out catalytic experiments at three different temperatures, 25, 60 and 90 °C and the results are shown in Fig. 3b. From 25 to 60 °C, a clear improvement in the removal efficiency can be observed from ∼39% to ∼76%. This can be linked to a variety of factors, like activation of the acidic surface functional groups and increase of the thermodynamics of the system, resulting in enhanced mass transfer (diffusion inside the pores towards the active sites) and decrement of adsorption/ decomposition activation energy. In contrast, a further temperature rise to 90 °C had a negative effect, decreasing the removal performance to ∼47%. This can be assigned to an increase of the hydrogen peroxide decomposition rate, which may also lead to undesirable secondary species other than the targeted hydroxyl radicals. Besides, oxidation of useful components in the fuel may also occur at elevated temperatures. This result is in agreement with other research results reported in the literature for the same desulfurization via adsorption/ oxidation methods. 30,[70][71][72] In view of these results, the optimum reaction temperature was set at 60 °C.</p><p>The results of the effect of the amount of H 2 O 2 on the removal of 4,6-DMDBT are shown in Fig. 3c. It is observed that by increasing the volume ratio, and therefore the molar ratio of H 2 O 2 , the removal percentage of 4,6-DMDBT is enhanced during the catalytic oxidation. The maximum removal was for 1 mL of H 2 O 2 solution (25 mg SX PLUS, 20 ppmw of sulfur PLUS) and the results are consistent with the literature. 70 An important conclusion was gained for the test in which only one 1 mL of water was used (no H 2 O 2 addition); the removal efficiency was limited to ∼44%, a value even lower than the maximum adsorptive removal without hydrogen peroxide (adsorption tests, Fig. 2a). This is strong evidence that the presence of water has a negative impact on the removal of 4,6-DMDBT from hexadecane (or a real fuel) by blocking/hindering the active adsorptive/reactive centers.</p><p>The above experiments (referring to the results of Fig. 3) were also carried out in the dark in order to explore if the presence of ambient light has an effect of the adsorption/oxidation capability. The catalytic oxidation removal under ambient light exposure was 10% higher than that in the dark. This can be linked either to the fact that the light plays a positive role in the reactive and/or adsorptive sites or that light power can enhance the formation of reactive oxygen species during the decomposition of H 2 O 2 on the carbon's surface. These two aspects will be discussed herein after in more detail.</p><!><p>To investigate the possibility of regeneration and reusability of the catalyst, the carbocatalyst SX PLUS was washed with water and methanol (dried for 2 h at 60 °C) after the oxidation experiments, in order to remove any product or reactant from its surface. From the results presented in Fig. 4, a small decrease of 4.5% in the removal efficiency occurred after the first cycle of reuse, while after 5 cycles the removal efficiency loss was not higher than 10.8%. It can be concluded that the catalytically active sites are maintained during reaction/regeneration. It is worth mentioning that the removal extent (∼63%) even after 5 cycles is higher than the maximum adsorptive capability of the rest of the commercial carbons tested (Fig. 2a).</p><!><p>Taking into account the high adsorption and catalytic oxidation efficiency of the parent commercial SX PLUS carbon, this carbon was further used to investigate the effect of chemical modification of its surface by increasing the density of the acidic surface functional groups. To this end, two different counterparts of modified SX PLUS were prepared. The first one was obtained after treatment with HNO 3 , herein-after referring to as SX PLUS-Nox, while for the second one H 2 SO 4 was used, with the prepared sample named SX PLUS-Sox. The catalytic oxidation results of the HNO 3 and H 2 SO 4 oxidized counterparts, as well as of the adsorption data (included for the sake of comparison), are presented in Fig. 5. A significant increase of the removal efficiency via catalytic oxidation can be observed for both treated SX PLUS samples, reaching 100% removal, being an additional indication of the positive effect of the surface acidity on the catalytic oxidation by activated carbons as metal-free catalysts. A small positive effect can also be identified in the adsorption removal, verifying further the enhanced interaction between 4,6-DMDBT and the surface acidic sites of carbon.</p><!><p>The porosity characteristics of the parent and oxidized SX PLUS carbons are shown in Table 1. Both the BET surface area and total pore volume were decreased upon oxidation. The decrement of the S BET was slightly more pronounced after treatment with HNO 3 (−27%) rather than after H 2 SO 4 treatment (−22%). Similar trends of decrease upon oxidation were found for the pore volumes. It is worth pointing out that although the oxidized counterparts showed inferior porosity characteristics in comparison with the parent SX PLUS, their adsorptive performance was superior, a fact that confirms a more crucial role of carbon's surface chemistry against porosity.</p><p>The surface pH as well as the amount of acidic and basic surface groups of the SX PLUS carbons is shown in Fig. 6, while the densities per surface area unit can be seen in Table s4. † The general observation is that the amount of the acidic surface groups was increased upon oxidation and the amount of the basic surface groups was decreased, thus resulting in a decrease of surface pH. The increment of the acidic groups was substantially more pronounced in the case of SX PLUS-Nox, while the decrement of the basic groups was more pronounced for SX PLUS-Sox. Boehm titration experiments (results not shown) revealed that the phenol groups were totally eliminated, lactones were increased, and carboxylic acids were formed by the oxidative treatment.</p><p>With regard to the correlation between adsorptive/catalytic performance and the surface acidity, the above derived volcano-type trend between adsorption of 4,6-DMDBT and the density of surface acid groups (Fig. 2b) was found valid also for the parent SX PLUS and its oxidized counterparts, since SX PLUS-Nox showed a significantly higher d ASFG but a slightly lower adsorption performance compared to SX PLUS-Sox. On the other hand, in the case of catalytic oxidation performance, both treated carbons reached the maximum removal efficiency, as shown in Fig. 5, at least for the experimental conditions used in this study.</p><!><p>In order to examine the effect of the adsorption/oxidation reaction time on the removal efficiency of the carbon catalysts, the remaining sulfur in the solution, expressed as C/C 0 , was plotted against the contact time and the derived curves are presented in Fig. 7. A different 4,6-DMDBT removal rate can be observed between the adsorption and the catalytic oxidation process. In the adsorption experiments, 4,6-DMDBT removal Table 1 Porosity characteristics of SX PLUS and its counterparts oxidized by HNO 3 and H 2 SO 4 (in parenthesis the % differences for the oxidized samples compared to SX PLUS)</p><!><p>S BET , m 2 g −1 V tot , cm 3 g −1 V mic , cm 3 g −1 V mes , cm 3 increased rapidly in the first 5-10 min followed by a gradual equilibration and a plateau after ca. 50 min, for both the parent SX PLUS and the two oxidized counterparts. The pseudo-first and the pseudo-second order kinetic models in their linear form were applied for the fitting of the experimental results. The linear fitting curves and the corresponding kinetic parameters are shown in Fig. s5 and Table s5, † respectively. The pseudo-second kinetic model found to fit better the adsorptive removal results, as concluded by the R 2 values presented in Table s5, † indicative of a physical adsorption onto the carbon surface, results consistent with findings by other researchers. 73,74 The kinetic experiments of the 4,6-DMDBT catalytic oxidation showed that 100% removal was achieved for the SX PLUS-Nox and SX PLUS-Sox carbons within 10 and 16 min, respectively. Interestingly, more than half of the 4,6-DMDBT was converted within less than 2 min. Since two separate slopes were observed (a steep one for 0 to 2 min and one for 2 to 10 or 16 min), it can be suggested that initially the catalytic oxidation/removal occurs on the large internal surface of the micropores where the majority of the active sites exist, and is being enhanced by the favorable confinement effects due to size similarity, as discussed above. As the micropores are filled by the first reactant molecules, in combination with the decrease of the concentration and abundance of H 2 O 2 , the reaction takes place at the meso/macropore and external surface of the carbons at lower rates. This theory can be supported from the fact that the catalytic oxidative removal follows both the pseudo-first and the pseudo-second order kinetics (Table s5 †), although with a slightly better fitting indicative of an oxidation process. 3,30,70,75 The importance of the oxidation treatment and enrichment of the carbon's surface with acidic groups can be also concluded from the fact that in the case of pristine SX PLUS, 4,6- DMDBT was not completely eliminated (Fig. 7). Taking also into account that the adsorptive performance between the parent and the oxidized SX PLUS carbons is more or less similar, then it can be further suggested that the formed acidic groups, i.e. carboxyls and lactones, have a higher impact on the oxidation mechanism of 4,6-DMDBT than on just increasing the chemical interaction/sorption.</p><!><p>In order to examine the surface chemistry alterations and the possibility of strongly sorbed reaction products on the carbon catalysts' surface, FTIR spectra analysis was conducted (Fig. 8a-c). The comparative study of the parent and oxidized SX PLUS samples, as well as of the corresponding carbons after exposure to 4,6-DMDBT, shed light on the chemical interactions between 4,6 DMDBT and the catalysts' surface functional groups and on the related oxidation mechanism. In all spectra, the bands presented at about 1610-1630 cm −1 can be attributed to C-C stretching vibrations of the aromatic rings. The bands at 1100-1150 cm −1 can be attributed to CvO and O-H bonds of alcoholic, phenolic and carboxyl oxygen groups while the peak at about 1700-1725 cm −1 can be attributed to CvO stretching vibrations of the carboxyl groups. For the modified activated carbons, (SX PLUS-Nox and SX PLUS-Sox) prior to the exposure to 4,6-DMDBT in hexadecane, an increase in the intensity of the bands at 1720 and 1100 cm −1 was presented, representing carboxylic acids and lactones, due to their oxidation with HNO 3 or H 2 SO 4 acids. The bands at 1330-1370 cm −1 , which appeared on the spectra for the carbons after catalytic oxidation tests and after heating to 280 °C to remove the solvent, can be attributed to the sulfur compounds formed on the carbon surface, i.e. to sulfoxides, sulfones or sulfonic acid. The sulfones are specified by absorption at 1300-1350 cm −1 and 1120-1160 cm −1 . The bands at 1166, 1076 and 1020 cm −1 are characteristic of the molecular vibrations of C-S bonds. The increase in intensity of the bands at 1730 cm −1 may be due to hydrogen bond interactions of the 4,6-DMDBT oxidation products with carboxyl groups.</p><p>The FTIR results indicated that 4,6-DMDBT was oxidized onto the carbon surface to the corresponding sulfones, sulfoxides or/and sulfonic acids as well as that these products were strongly adsorbed on the carbon's surface. Besides, EDX analysis, presented in Fig. 8d and e, supported further the FTIR results and proved the presence of sulfur on the surface of the carbocatalysts' after the catalytic experiment. The slight decrease of oxidation/removal performance observed in the reusability tests can be linked to the formation and strong retention of these sulfur compounds which were not totally removed/desorbed by washing from the active sites.</p><!><p>At the end of the catalytic oxidation tests, the hexadecane solution was spectroscopically (UV-vis) analyzed, while the oxidation products accumulated on the carbons' surface were extracted with methanol for analysis. Methanol was selected because it is a polar solvent and could dissolve the formed sulfones and sulfoxides, compounds with enhanced polarity. The UV-Vis spectra of the liquid phase as well as the spectrum of the methanol extract after a catalytic oxidation experiment with SX PLUS-Nox as a catalyst are shown in Fig. s6. † In the spectra of the reaction product, the intensity decrement of the bands attributed to 4,6 DMDBT was obvious (Fig. s6a †), while in the spectrum of the methanol extract (Fig. s6b †), new peaks appeared corresponding to 4,6-DMDBT derived sulfoxides and/ or sulfones. 76 From the spectra it can be concluded that the oxidation products were accumulated/strongly sorbed on the carbon surface after the oxidation of 4,6-DMDBT.</p><p>The liquid phases were also analyzed by GC-MS. From the results presented in Fig. 9a, the decrease of the peak's intensity corresponding to 4,6-DMDBT (at a retention time of 31.996 min) by the use of the parent activated carbon SX PLUS and the disappearance of this peak for SX PLUS-Nox and SX PLUS-Sox verify the UV-Vis analysis results and the obtained high or 100% removal of 4,6-DMDBT (Fig. 5 and 7). GC-MS analysis of the extracts of the used carbon with another polar solvent (acetonitrile) also showed the presence of 4,6-DMDBT products, such as sulfone and sulfoxide, as can be seen in Fig. 9b.</p><!><p>From the above results it can be suggested that the aromatic ring of 4,6-DMDBT interacts with the carbon surface through the π-π stacking and/or through the donor acceptor mechanism, and these interactions are responsible for the high adsorptive capability of the studied activated carbons. The activity of the carbons as oxidation catalysts is associated with the concentration of oxygen surface functional groups, i.e. lactones or/and carbonyl groups. 50 These surface groups can also be responsible for the strong retention of the formed oxidation products, like sulfoxide and sulfones. The most crucial and still not clearly elucidated aspect is the catalytic role of the carbon surface. The major and most well-established pathway is that the above functional groups on the carbon's surface catalyze the decomposition of H 2 O 2 to free radicals such as • OH or • OOH, which are powerful oxidants for the oxidation of sulfur compounds to their corresponding sulfoxide and/or sulfones. These radicals can also be responsible for the formation of active molecular oxygen anions that also can act as an oxidant of 4,6-DMDBT. And all these reactions occur inside the micropores of carbon that act as "microreactors".</p><p>In order to conclude if the catalytic capability can be linked to the formation of free radicals, methanol was used as a scavenger (Fig. 10). In the presence of methanol, the conversion/ removal percentage of 4,6-DMDBT was decreased for all carbons. In the case of SX PLUS-Sox, the decrement of the removal was 40% by the addition of 10% methanol per volume of the solvent. This removal efficiency is even lower from the measured adsorptive performance of this sample, a fact that suggests the blockage of both adsorption and catalytically active sites. This can be assigned to the water moieties, which remain adsorbed on the surface of the carbon by polar forces or/and hydrogen bonds, forming a film that hinders the interaction with 4,6-DMDBT (Fig. 11a). This is in perfect agreement with the above presented results when pure water was added, leading to a decrement of the oxidation/removal efficiency. The negative effect upon methanol addition may be attributed to the reaction of methanol with the free radicals, which are generated by the decomposition of hydrogen peroxide on the surface of the activated carbon, 50,77,78 Since hydrogen peroxide can undergo a fast decomposition in the presence of activated carbon, the most crucial aspect is the simultaneous presence of 4,6-DMDBT and H 2 O 2 inside the pores in order for all the consecutive reactions to occur (Fig. 11a). The presence of water in a high amount can decrease the reaction kinetics by blocking/occupying the adsorption/reaction sites. The oxidation of 4,6-DMDBT undergoes through a redox cycle of carbon with the participation of superoxide anionic radicals and H 2 O 2 (Fig. 11b). Analogous redox cycle based catalytic oxidation pathways were reported for metal oxides. 79 In general, peroxide has a triple role. The first one is to activate carbon's surface (Fig. 11c), the second to act as a pool of molecular oxygen (Fig. 11d), and the third to participate in the formation of superoxide anionic radicals (Fig. 11e).</p><p>In more recent publications, free radical species ( • OH/ • OOH) have been postulated as the main intermediates in the reaction mechanism, whose formation would take place through an electron-transfer reaction similar to the Fenton mechanism, with AC and AC + as the reduced and oxidized catalyst states. The recombination of free radical species ( • OH/ • OOH) in the liquid phase and/or onto the activated carbon surface will produce water and oxygen according to reactions in Fig. 11. When 4,6-DMDBT is adsorbed on the surface of activated carbon, in which also H 2 O 2 is decomposed into radicals H + and HOO t , the latter reacts with O 2 to superoxide, which further oxidizes 4,6-DMDBT to sulfoxides and/or sulfones. It is also feasible that the free • OH or • OOH radicals attack directly and oxidize 4,6-DMDBT.</p><!><p>In the current work, we presented the potential use of activated carbons not only as adsorbents of sulfur compounds but also as metal-free oxidation catalysts for the desulfurization of fuels via oxidation of 4,6-DMDBT to the corresponding sulfones and sulfoxides in a reusable manner and under ambient conditions. The comparison of five commercial porous carbons revealed that the removal capability depends predomi-nately on the density of acidic surface functional groups, while the adsorption is governed by the formation of donor-acceptor complexes between the adsorbent and the adsorbate. Further chemical modification of the carbon's surface via treatment/ oxidation with HNO 3 or H 2 SO 4 was also investigated. In both cases, the basic surface groups were decreased, the phenolics were eliminated, while the lactones were increased, and carboxyl groups were formed. The modification with the former acid had a more pronounced effect on the formation of acidic groups and induced faster catalytic oxidation/removal kinetics, while treatment with the latter acid led to a significant decrease of the basic sites and to the highest adsorptive capability. Although the oxidation of carbons had a relatively moderate negative effect on porosity characteristics, the oxidized counterparts showed a 100% desulfurization capability, a value being 33% higher compared to the performance of the parent non-modified commercial carbon, confirming the predominant role of the surface chemistry. In addition, it was shown that the abundance of micropores with a size of ≤1 nm is beneficial for the adsorptive/catalytic reactivity due to confinement effects considering the similar size of 4,6-DMDBT. The reaction of 4,6-DMDBT in the presence of H 2 O 2 led to the formation of the corresponding sulfoxide and sulfone, as a result of the formation of superoxide and free • OH and • OOH radicals upon the decomposition of H 2 O 2 on the carbocatalyst surface. These results can boost the research activities and exploitation of activated porous carbons, without the use of any external metals with redox activity, as highly efficient green catalysts in oxidative deep desulfurization of fuels or other related processes.</p>

Royal Society of Chemistry (RSC)

Development of dual casein kinase 1\xce\xb4/1\xce\xb5 (CK1\xce\xb4/\xce\xb5) inhibitors for treatment of breast cancer

Casein kinase 1\xce\xb4/\xce\xb5 have been identified as promising therapeutic target for oncology application, including breast and brain cancer. Here, we described our continued efforts in optimization of a lead series of purine scaffold inhibitors that led to identification of two new CK1\xce\xb4/\xce\xb5 inhibitors 17 and 28 displaying low nanomolar values in antiproliferative assays against the human MDA-MB-231 triple negative breast cancer cell line and have physical, in vitro and in vivo pharmacokinetic properties suitable for use in proof of principle animal xenograft studies against human cancers. 2009 Elsevier Ltd. All rights reserved.

development_of_dual_casein_kinase_1\xce\xb4/1\xce\xb5_(ck1\xce\xb4/\xce\xb5)_inhibitors_for_treatmen

1. Introduction<!>2.1. Synthetic Chemistry<!>2.2. Structure-activity relationship (SAR) studies<!>2.3. Physicochemical and ADME Properties<!>2.4. Molecular Modelling<!>2.5. Kinase selectivity<!>2.6. In Vivo Pharmacokinetics<!>3. Conclusions<!>4.1. Material and Methods<!>4.2. General procedure for preparation of compounds 11\xe2\x80\x9376<!>4.3. Biochemical Assays<!>4.4. Cell Culture and Proliferation Assays of CK1\xce\xb4/\xce\xb5 Inhibitors<!>4.5.1. Solubility<!>4.5.2. Permeability<!>4.5.3. Hepatic microsomal stability<!>4.5.4. P450 inhibition<!>4.5.5. Pharmacokinetics<!>4.6. LogD calculations

<p>The casein kinase 1 (CK1) family consists of six monomeric serine/threonine-specific protein kinases (α, δ, ε, γ1, γ2 and γ3).1 All six human CK1 isoforms are highly homologous in their active site, with the delta (CK1δ) and epsilon (CK1ε) isoforms sharing 98% sequence identity in their protein kinase domains. Elsewhere in the enzyme, such as in the C-terminal, and non-catalytic domains, significant variances exist between isoforms.2 CK1 kinases play crucial roles in regulating a variety of cellular growth and survival processes including circadian rhythm,3 membrane trafficking,4 DNA damage repair,5 cytoskeleton maintenance,6 and notably Wnt signaling.7 A recent study showed that CK1δ/CK1ε might be involved in the etiology of addictive behavior and that their inhibition prevents relapse-like alcohol drinking.8 Importantly, abnormal regulation of these two CK1 family members is implicated in human cancers and several known CK1δ and/or CK1ε substrates control tumor cell growth, apoptosis, metabolism and differentiation.9 For instance, both isoforms are overexpressed in pancreatic ductal adenocarcinoma,10 ovarian cancer11 and chronic lymphocytic leukemia.12 Interestingly, expression of constitutively active, myristoylated CK1ε in mammary epithelial cells is sufficient to drive cell transformation in vitro via stabilization of β-catenin and the activation of Wnt transcription targets.13 At the same time, forced expression of kinase defective CK1δ mutants blocks SV40-driven cellular transformation in vitro and mammary carcinogenesis in vivo.14</p><p>The Wnt/β-catenin pathway has known roles in breast cancer, where: (i) Wnt signaling can contribute to triple negative breast cancer; (ii) nuclear β-catenin connotes metastasis and poor outcome; and (iii) β-catenin contributes to mutant ErbB2-driven breast cancer.15 Importantly, gain-of-function (e.g., in β-catenin) and loss-of-function (e.g., in CK1α APC and AXIN) mutations prevalent in other cancers are not found in breast cancer. Recent discoveries from our laboratories establish casein kinase 1 delta (CK1δ) as an essential regulator of β-catenin activity that is overexpressed and amplified in human breast cancer.16</p><p>CK1δ and CK1ε are eminently tractable for small molecule targeted drug discovery.17 Among the small molecules reported to inhibit CK1δ/CK1ε are CKI-7,18 (R)-CR8 and (R)-DRF053,19 IC261,20 D4476,21 LH846,22 benzimidazole 5,23 PF480056724 and PF670462.3a, 24 Unfortunately, most of these compounds are not specific inhibitors of CK1δ/CK1ε and in some cases their pharmacological effects are now known to be (or are suspected to be) due to non-selective target engagement.20b, 25 At the same time, the contribution of CK1δ and CK1ε to human cancer is still not fully understood and the non-selective nature of previously reported CK1δ/ε inhibitors has impeded pharmacological validation of these kinases as anti-cancer targets.3a, 20b Nonetheless, available data clearly shows that targeted inhibition of CK1δ/CK1ε is a viable, highly attractive anticancer strategy yet to be fully developed.26</p><p>In research targeting the development of novel therapeutics for treatment of metastatic and resistant forms of cancer, the Roush laboratory at Scripps Florida identified highly potent and selective purine-derived dual inhibitors of CK1δ and CK1ε under the aegis of the NIH's MLPCN program (Figure 1).25 These agents induce proliferative arrest and rapid apoptosis of CK1δ/CK1ε-expressing human luminal B, HER2+ and triple negative cancer cells ex vivo and SR-3029 induces tumor regression in orthotopic xenograft models in vivo.26 Specifically, genetic knockdown or pharmacological inhibition of CK1δ using SR-3029 blocks β-catenin nuclear localization and TCF transcriptional activity, induces rapid apoptosis and provokes tumor regression in patient derived orthotopic models of triple negative breast cancer.25–26 Mechanistic studies are consistent with the premise that CK1δ controls breast cancer tumorigenesis via its effect on β-catenin, which plays known roles as an effector of aberrant Wnt signaling in human breast cancer.26</p><p>Noteworthy, profiling of 442 kinases with SR-3029 (and three close analogs) confirmed their high selectivity vs. the plethora of kinases whose activity is impaired by the purportedly specific Pfizer CK1δ inhibitor (PF670462).25 The four off-target kinases that are inhibited by SR-3029 largely have no known function. High selectivity of N9-arylsubstituted purine scaffold could be explained by the unique structural features of the CK1δ/ε active site. Specifically, relatively small size of the gatekeeper residue Met82 in case of both CK1δ/ε creates large hydrophobic pocket which is occupied by N9 aryl residue of the inhibitors. This structural feature of CK1δ/ε was also encountered by Shokat and colleagues when developing analog-sensitive kinase technology.27</p><p>Herein we report structure-activity and structure-property relationship (SAR and SPR) studies that led to identification of dual selective CK1δ/ε inhibitors (including SR-3029) with physicochemical properties and in vitro and in vivo pharmacokinetic parameters suitable for use in murine xenograft studies against breast cancer.26</p><!><p>The general procedure for the synthesis of purine-based CK1δ/ε inhibitors is based on a previously reported sequence 25, 28 which we improved by incorporating a more efficient method for arylation of the N9 nitrogen of dichloropurine (Scheme 1). Thus, N9 aryl purine intermediates (permutations of structure 6) were generated by copper (I) mediated coupling of commercially available dichloropurine 4 with a variety of symmetrical or unsymmetrical diaryliodonium salts 5.29 This reaction provides 6 in 65%–85% yield – a substantial improvement relative to the previously employed Chan-Lan coupling (yield 0–30%).25 The diaryliodonium salts were accessed via one-step procedures from aryl iodides 1 and arenes 2 or boronic acids 3.30 Subsequently, substituted benzimidazoles 9 and various amines were introduced at C6 and C2 of 6, respectively. This procedure provided the targeted CK1δ/ε inhibitors with excellent regioselectivity and good yields (70–90%).25 The substituted benzimidazoles (9) were accessed as previously described from o-dianilines, 7.25, 31</p><!><p>We synthesized analogs in an iterative fashion using the chemistry outlined in Scheme 1, refining our compound design on the basis of biochemical and biological data obtained from prior rounds of tested inhibitors. Compounds with low nanomolar biochemical affinity (IC50 < 100 nM, 20 μM ATP concentration) were assessed in cell proliferation assays vs. MDA-MB-231 breast cancer cell lines using 3-day MTT and longer term clonogenicity assays (see Experimental section for details).25</p><p>Our SAR optimization efforts commenced with the synthesis of agents with various N9 alkyl and aryl substitution (R1) (Table 1). Alkyl substitution of the purine N9 position (11, 12) resulted in the complete loss of CK1δ/ε activity. Consequently, subsequent SAR efforts focused on aryl R1 substituents. Since we looked at only one example of a N9-difluorophenyl substitution in our original publication,25 other mono- and disubstituted fluorophenyl analogs were explored. Notably, all the analogs with a N9 4-fluorophenyl substitution resulted in an approximately 10 fold loss of activity against CK1δ compared to SR-3029 (14, 15, 19, Table 1). Other difluorophenyl analogs 2,3-difluorophenyl 16, 3,5-difluorophenyl 17 and 2,5-difluorophenyl 18 showed similar activity to SR-3029 with 18 having a slightly better biochemical profile.</p><p>Introduction of other substituents at the ortho position of the N9 aromatic moiety (R1), such as in inhibitors 20 and 21, resulted in a modest reduction of biochemical activity (2–4 fold) but substantial loss of cell-based activity (EC50 = 6–10 μM, Table 1).</p><p>While performing the initial rounds of SAR optimization, we noticed a moderate correlation between the calculated logD (pH = 7.4) values of tested analogs and their antiproliferative properties (Figure 2, library of 224 inhibitors from Bibian et al25 and related patent32). In particular, the majority of poorly active analogs (EC50 > 750 nM) were more lipophilic (logD > 4) than those agents with significant activity. The logD distribution of compounds with EC50 < 750 nM and compounds with EC50 > 750 nM also suggests that partition coefficients peak around 2.5 for active and 3.8 for inactive analogs (Figures 2A, B).</p><p>In an effort to lower the lipophilicity of the CK1δ/ε inhibitors, we synthesized and tested a series of N9 heteroaryl-substituted analogs (Table 2). Although N9 thiophene substitution resulted in inhibitors with low nanomolar values in both biochemical and cell based assays (22 and 23), an unsubstituted thiophene is generally considered to be a metabolic liability.33 To avoid this potential problem, we selected alternate groups that could be used at the purine N9 position (R1) to lower lipophilicity while, hopefully, maintaining CK1δ/ε inhibitory activity. Replacement of the thiophene ring by a variety of pyridyl groups retained the logD values in the targeted range (≤ 3.5), however all these compounds were less potent against CK1δ/ε (IC50 > 143) compared to either 23 or SR-3029.</p><p>Contemporaneously, we sought to vary the substitution on the benzimidazole unit (R5). The data presented in Table 3 are for one series in which the purine N9 substituent was held constant as m-pyridyl. Additionally three different amines (N-methylpiperazine, morpholine and piperazine; Table 3) were utilized at purine C2 throughout this compound set. Compounds 36, 37 and 38, which have an unsubstituted benzimidazole ring, showed decreased CK1δ/ε inhibition (IC50 vs CK1δ = 824 – 1566 nM) compared to other analogs in this set (Table 3). Substitution of the benzimidazole with either 5-chloro (39–41) or 5-methoxy (45–47) groups led to a 2–3 fold increase in CK1δ/ε inhibition (IC50 vs CK1δ = 336 – 624 nM) relative to the unsubstituted benzimidazole, derivatives 36–38. Further improvement of CK1δ/ε inhibitor activity was achieved through the incorporation of two fluorine atoms into the 5 and 6 positions of the benzimidazole ring (48, 49). Finally, the most active compounds (IC50 vs CK1δ < 100 nM) in this series were obtained through the introduction of a 5,6-dichloro substitution pattern (42–44) into the benzimidazole unit. These three analogs were additionally tested in a cell based assay vs. MDA-MB-231 breast cancer cell lines, where they showed excellent antiproliferative properties (EC50 < 6 nM). Interestingly, compound 42 is almost 100-fold more potent in the cell-based assay than in in vitro tests against purified CK1δ. This observation suggests that 42 may possibly be engaging additional kinases that contribute to cell-based activity of this compound. This assumption, however, remains to be validated experimentally.</p><p>Lastly, inclusion of morpholine or piperazine units at C2 of the purine scaffold generally conferred greater CK1δ/ε inhibition activity than compounds with N-methylpiperazine units at this position.</p><p>In addition to SAR studies performed on the benzimidazole ring itself, we also examined the possibility of using benzimidazole isosteres or replacing the benzimidazoles with different heterocycles (Table 4). The benzimidazole unit (R5) was replaced with structurally similar benzoxazole (50) and benzothiazole (51) units.</p><p>Despite evincing excellent biochemical activity (51, IC50 vs CK1δ = 9 nM), these two compounds were inactive in the cell based assay (EC50 > 10 μM). Other inhibitors with benzimidazole replacements, such as phenyl-substituted imidazole (52), oxazole (53) and thiazole (54), were synthesized and tested in biological assays. Of these three compounds, 52 showed good activity against CK1δ/ε (IC50 vs CK1δ = 214 nM). Unfortunately 52 displayed markedly diminished potency in cell-based assays against MDA-MB-231 cancer cells (EC50 = 1400 nM). Replacing the benzimidazole with 1-methyl-4-(pyridin-3-yl)piperazine (55) or 1-methyl-4-phenylpiperazine (56) led to improvement in biochemical activity (IC50 of 56 vs CK1δ/ε = 50 nM).</p><p>Sadly, these compounds also displayed a significant disparity between biochemical and cell-based potency (EC50 of 56 vs. MDA-MB-231 = 7160 nM). Replacement of the benzimidazole unit with an imidazole decreased CK1δ/ε inhibition activity significantly (57). Finally, eschewing the benzimidazole in favor of a 2-pyridyl (58), 3-pyridyl (59), 4-methyl-2-pyridyl (60), 3-methyl-2-pyridyl (61) or 2-flurophenyl (62) groups resulted in a series of highly potent CK1δ/ε inhibitors, but which were inactive against MDA-MB-231 breast cancer cell lines (EC50 > 10 μM).</p><p>We tested a subset of eleven compounds for their ability to permeate cell membranes (PAMPA assay, see Experimental section for details). The majority of the analogs tested possessed low permeability of Papp ≤ 2.5 · 10−6 cm/s (Table 4), suggesting that this property could be a significant contributor to the disconnect between biochemical and cell-based activity for this analogs series.</p><p>We completed this series of SAR studies of the purine core by varying the substitution at the C2 position (Table 5). Analogs 63 and 64 retained good potency but lost antiproliferative activity. Next, the morpholine unit of SR-3029 was exchanged for a thiomorpholine (65) and its oxidized variant (66), which led to a 2–3 fold decrease in CK1δ/ε biochemical and cell based activities. We also introduced a number of different piperidines at the R4 position to obtain compounds 67, 68, 73, 75 and 76. Each of these agents displayed moderate biochemical activity against CK1δ/ε. The best compound in this series was 68, which evinced an IC50 of 69 nM against CK1δ and 133 nM in the MDA-MB-321 cellular assay. Extending the morpholine unit by two or three carbons from the purine core resulted in compounds 70 and 71, both of which were are approximately 20 fold less active than SR-3029. Finally, tetrahydropyran derivative 69 and butyl analog 72 did not show any improvement in activity (69, IC50 vs CK1δ = 321 nM; 72, IC50 vs CK1δ = 1031 nM).</p><!><p>In addition to testing inhibitory activity against CK1δ/ε and antiproliferative activity against MDA-MB-231 breast cancer cell lines, standard physicochemical properties including logP, logD7.4, and TPSA (total polar surface area) were assessed for all newly synthesized analogs. Compounds were prioritized on the basis of both their biological data and physicochemical properties, and high priority agents were assessed for in vitro drug metabolism and pharmacokinetics (DMPK, including aqueous solubility, microsomal stability and CYP450 inhibition; Table 6, PAMPA data for selected compounds are in Table 4).</p><p>Kinetic aqueous solubility in PBS buffer at pH 7.4 was determined using an HPLC-based protocol and reported as the average of two measurements (see Experimental Section for details). Apparent cell permeability was determined using the standard parallel artificial membrane permeability assay (PAMPA)34 using propanolol and ranitidine as controls and analyzed by HPLC-MS/MS. A large set of the most promising CK1δ/ε inhibitors was additionally tested for hepatic microsomal stability (human, mouse and rat) and cytochrome P450 inhibition as described previously.25 Briefly, the compounds were incubated together with hepatic microsomes and NADPH was used to initiate enzymatic oxidation. Acetonitrile was then added to quench the reaction at different time points and processed samples were analyzed using LC-MS/MS. Cytochrome P450 inhibition (CYP1A2, CYP2C9, CYP2D6, and CYP3A4) was evaluated in human liver microsomes using four selective marker substrates in the presence or absence of 10 μM test compound and reported as percentage inhibition (see Experimental section for details).</p><p>The calculated distribution coefficients (logD7.4) was in the acceptable range (1 < log D < 4) for the majority of new analogs, with the exception of 18, 51 and 62 (Table 6). This represents an improvement over the initial set of CK1δ/ε inhibitors,25 as the average logD value of our compound collection was reduced by approximately 1 order of magnitude. Despite improvements in lipophilicity, the majority of the compounds that were advanced to in vitro DMPK assessment possessed poor aqueous solubility (less than 0.5 μM). Fortunately, during our iterative analog synthesis efforts, we observed that aqueous solubility could be improved by appending a piperazine unit to the scaffold in lieu of a morpholine (i.e., 28, Table 6). Importantly, agent potency was not affected greatly by this substitution (though morpholine analogs are slightly more potent than their N-methylpiperazine counterparts).</p><p>Hepatic microsomal stability was considered to be an important factor when selecting candidates for in vivo testing. We sought compounds with half-lives resembling the FDA-approved small molecule tyrosine kinase inhibitor sunitinib, which was used as positive control (t1/2 = 46, 13 and 30 min in human, mouse and rat microsomes, respectively). Among the compounds advanced to metabolic stability studies, 43, 28, 31 and 51 emerged as the most stable candidates (Table 6). Agent stability was marginally redued or stayed comparable relative to reference compound SR-3029 for the thiophene (22, 23), benzylpyridine (61) and 2,3-difluorophenyl analogs 16 and 18.</p><p>Finally, CYP inhibition for a set of the most active compounds was assessed. It is well-known that 3-substituted pyridine-containing compounds can act as CYP3A4 inhibitors.35 Indeed, N9 m-pyridine-containing analogs 25, 40, 43 and 49 were the most potent inhibitors of all four cytochrome P450s tested (1A2, 2C9, 2D6, and 3A4, Table 6). However, it has also been reported that CYP3A4 inhibition can be decreased through the introduction of ortho-halogen or alkyl substitution into the pyridine.35 On this basis, we synthesized a series of 2-fluoro (30, 31), 2-chloro (32, 33) and 2-methyl (34, 35) substituted pyridines, which displayed significantly improved CYP inhibition profile (31, < 30% inhibition against all four tested CYP substrates at 10 μM, Table 6). However, these modifications led to reduced CK1δ/ε potency in some cases (32, 33, 35).</p><p>Based on the combination of biological activity and ADME properties, we selected 17 and 28 to advance into in vivo PK testing. While analog 28 is the most soluble compound in this series (28 μM, selected for PO administration), both compounds 17 and 28 possess acceptable stability in human, mouse and rat liver microsomes and a favorable CYP inhibition profile (Table 6) when compared to SR-3029 (13).</p><!><p>Docking studies were performed using the co-crystal structure of CK1δ and PF670462 (PDB ID:3UYT)36 to predict the binding modes of designed and synthesized inhibitors. The original co-crystal structure was refined using the Protein Preparation Wizard37 implemented in the Maestro 11.1 (Schrödinger Release 2017-2) interface, and invalid atom types were corrected using this same wizard. A receptor grid was generated from the refined structure using default values. The docked models for PF670462 were in good agreement with the reported crystal structures coordinates (see Supporting information for details). Designed inhibitors were docked into the grid using Glide 7.438 in standard precision (SP) mode, without any constraints. The proposed binding pose of 17 within the CK1δ active site is shown in Figure 3A, B.</p><p>As revealed by docking studies, the key contacts between 17 and the binding pocket of CK1δ are two hydrogen bonding interactions between the purine scaffold and Leu85 in the hinge region (Figure 3B). The benzimidazole moiety projects into the solvent-exposed area and also makes additional contacts with the same residue Leu85, (Figure 3A and B), while the N9 m-fluorophenyl ring occupies a relatively large hydrophobic pocket created by the gatekeeper residue Met82. An image of 28 docked in CK1δ active site is presented in Figure 3C. The binding mode of this agent closely mirrors the pose adopted by 17 (Figure 3B F 3C). Specifically, 28 is positioned in a manner analogous to 17 so as to maintain two key interactions with the hinge region (Leu85) of CK1δ. Additionally, as predicted by Glide, the N9 para-pyridyl ring of 28 projects into the kinase hydrophobic pocket and picks up additional stabilizing interactions with Tyr56 (Figure 3C). Despite this prediction, we observed a 4 fold decrease in CK1δ inhibition activity for 28 (Table 2). We hypothesize that reduced inhibitory activity compared to 17 could be the result of an undesirable repulsive interaction between gatekeeper Met80 and the pyridyl unit of 28.</p><!><p>In order to address the question of selectivity, kinase binding was performed using compound 17 (at 10 μM concentration) against a panel of 97 targets distributed across the kinome (DiscoverX scanEDGE Kinase Assay Panel). Analog 17 was exceptionally selective under the assay conditions, with only CK1δ being inhibited >90% of the 97 kinases tested (Figure 4, i.e., 5.2% of active kinase remaining at 10 μM). The only other off-target kinase, FLT3 (17% of active kinase remaining at 10 μM), was previously shown by us not to be responsible for the potent antiproliferative effects demonstrated by the purine-based CK1δ/ε inhibitors.25 The 95 remaining kinases in this assay showed greater than 35% control activity at 10 μM (see Supporting information for details).</p><!><p>The pharmacokinetic properties of 17 and 28 were assessed in male C57Bl6 mice following IP administration at 20 mg/kg (17) and IV and PO administration at 1 mg/kg and 10 mg/kg, respectively (28, Table 7, route of administration was selected based on solubility data, Table 6). Compound 17 showed good exposure after IP administration (Figure 5). The plasma concentration was maintained above 1 μM for more than 3 hours and above 100 nM for about 7 hours (EC50 MDA-MB-231 = 68 nM), albeit a short half-life (Figure 5, Table 7). At the same time, 28 exhibited a modest in vivo half-life (1.3 h) and a high volume of distribution (9.1 L/kg) following intravenous administration. The apparent oral bioavailability of 28 after PO dosing was approximately 16% (Table 7), marginally better than SR-3029 (13), despite improvement in aqueous solubility.</p><!><p>We have developed a series of potent and selective purine-based CK1δ/ε inhibitors with excellent antiproliferative activities. A set of the most active compounds was also subjected to extensive physicochemical testing for solubility, permeability, and microsomal stability in an effort to predict their in vivo profile. These efforts led to the identification of 17 and 28 that has physical, in vitro and in vivo PK properties suitable for use in xenograph studies of human cancer. Such studies in mice are planned and will be reported in due course.</p><!><p>All reagents were purchased from commercial suppliers and were used without further purification. Dichloromethane, diethyl ether, N,N-dimethylformamide and tetrahydrofuran were dried by being passed through a column of desiccant (activated A-1 alumina). Triethylamine and diisopropyl amine was purified by distillation from calcium hydride. Reactions were either monitored by thin layer chromatography or analytical LC-MS. Thin layer chromatography was performed on Kieselgel 60 F254 glass plates pre-coated with a 0.25 mm thickness of silica gel. TLC plates were visualized with UV light and/or by staining with ninhydrin solution. Normal phase column chromatography was performed on a Biotage Isolera automated flash system. Compounds were loaded onto pre-filled cartridges filled with KP-Sil 50 μm irregular silica. For microwave reactions, a Biotage Initiator Microwave system was used. Some of the final products were isolated by reverse-phase HPLC using Shimadzu Prep LC system with photodiode array detector, Waters SunFire C18 OBD Prep Column, 100Å, 10 μm, 30 mm × 250 mm. Compounds were eluted using a gradient elution of 90/10 to 0/100 A/B over 10 min at a flow rate of 50.0 mL/min, where solvent A was water (+0.1 % TFA) and solvent B was acetonitrile/methanol (1:1).</p><p>The structures of all compounds were verified via 1H NMR, 13C NMR, 19F NMR and HPLC/HRMS. The purity of isolated products was determined using an LC-MS instrument (Agilent 1260 Infinity series LC with 500 Ion Trap MS) equipped with Kinetex® 5 μm EVO C18 100 Å LC Column 100 × 4.6 mm (Phenomenex) column. Elution was performed using the following conditions: 2% (v/v) acetonitrile (+0.1% FA) in 98% (v/v) H2O (+0.1% FA), ramped to 98% acetonitrile over 8 min, and holding at 98% acetonitrile for 1 min with a flow rate of 1.75 mL/min; UV absorption was detected from 200 to 950 nm using a diode array detector. The purity of each compound was ≥95% based on this analysis.</p><p>NMR spectra were recorded at ambient temperature on a 400 or 700 MHz Bruker NMR spectrometer in DMSO-d6. All 1H NMR data are reported in parts per million (ppm) downfield of TMS and were measured relative to the signals for dimethyl sulfoxide (2.50 ppm). All 13C NMR spectra are reported in ppm relative to the signals for dimethyl sulfoxide (39.5 ppm) with 1H decoupled observation. 19F NMR experiments were performed with 1H decoupling. Data for 1H NMR are reported as follows: chemical shift (δ, ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), integration, and coupling constant (Hz), whereas 13C NMR analyses were obtained at 101 or 176 MHz and reported in terms of chemical shift. NMR data was analyzed and processed by using MestReNova software. High-resolution mass spectra were recorded on a spectrometer (ESI-TOF) at the University of Illinois Urbana-Champaign Mass Spectrometry Laboratory.</p><p>The synthesis and analysis of 2,6-dichloro-9-aryl-9H-purines 6 and benzimidazoles 9 were previously described.25, 28–29 Diaryliodonium salts 5 were prepared according to the reported procedures.30 Compounds 11–76 were newly synthesized and described in the Supporting Information.</p><!><p>A 5 mL Biotage microwave vial was charged with 2,6-dichloropurine-9-(aryl/alkyl)-purine (1 eq, 0.35 mmol), 6, 2-(aminomethyl)-benzimidazole, 9, (or the benzylamine corresponding to R5CH2NH2 in Scheme 1) (1.1 eq 0.39 mmol), freshly distilled N,N-diisopropylethylamine (5 eq, 1.77 mmol, 0.31 mL), and isopropanol (1.4 mL. 0.25M). The vial was sealed with a microwave cap, and then the reaction mixture was heated to 90 °C for 30 minutes in microwave reactor. The completion of the reaction was confirmed by LC-MS and resulting product 10 was collected by filtration. In cases where the product was soluble in isopropanol, the reaction mixture was concentrated and used in the next step without further purification. A large excess (>30 eq) of amine corresponding to R4 (Scheme 1, N-methyl piperazine, morpholine, etc) was then added to the vial containing the concentrated crude mixture or collected solid. The vial was resealed and the reaction was heated to 130 °C for 30 minutes in the microwave unit (monitored by LC-MS). The cooled reaction mixture was concentrated on a rotary evaporator, then the crude product was purified by flash chromatography using 10 g Biotage column (gradient, 0%–10% MeOH in DCM over 25 column volumes). Collected fractions were washed with solution of saturated NH4Cl to remove remaining amine (monitored by TLC using ninhydrin stain). In some cases (as specified), reverse-phase HPLC was used after the normal phase column chromatography to obtain product with purity of >95%.</p><!><p>CK1δ inhibitor IC50 values were measured by using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Briefly, final assay concentrations for CK1δ (Signal Chem), Ulight peptide substrate (ULight-Topo-Ila(Thr1342) peptide, Perkin Elmer) and ATP were 2 nM, 200 nM and 20 μM respectively. The reaction was performed at room temperature in a 10 μL final volume (384-well low volume plate, Greiner) containing: 50 mM Hepes, pH 7.5, 5 mM MgCl2, 0.1 mg/ml bovine serum albumin, 1 mM dl-dithiothreitol, 0.01% Triton X-100 and 1% DMSO (Sigma-Aldrich). After 10 min, the reaction was terminated by addition of 10 μL of 4 nM Eu-anti-p-Topo-Ila (Cat:TRF-0218, PerkinElmer) in Lance Detection Buffer (Cat: CR97-100, PerkinElmer). The fluorescent signal was detected using an EnVision plate reader (PerkinElmer). 10 point dose-response curves with 3–10 fold dilutions starting from 10 μM for each compound was generated in duplicate and data fit to a four parameter logistic.</p><!><p>Human MDA-MB-231 breast cancer cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, LifeTechnologies) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C, 5% CO2. To evaluate the anti-proliferative activity of newly synthesized CK1δ/ε inhibitors against MDA-MB-231 breast cancer cells, cells were plated into a 384-well plate. 10 point dose-response curves with 3–10 fold dilutions starting from 10 μM for each compound was generated in duplicate including DMSO control. Cell proliferation was measured 72 h after compound treatment using CellTiter-Glo (Promega) according to the manufacturer's instructions. EC50 values were determined by nonlinear regression and a four-parameter algorithm (GraphPad Prism 5).</p><!><p>Compounds from 10 mM DMSO stock solutions were introduced to pre-warmed pH 7.4 phosphate buffered saline in a 96-well plate. The final DMSO concentration was 1% and the plate was maintained at 37°C for 24 hours on an orbital shaker. The samples were centrifuged through a Millipore Multiscreen Solvinter 0.45 micron low binding PTFE hydrophilic filter plate and analyzed by HPLC with peak area compared to standards of known concentration.</p><!><p>An assessment of permeability was done using a commercial PAMPA (Parallel Artificial Membrane Permeability Assay) kit from BD Biosciences (Cat# 353015).34 Compound (5μM) was added to 300 μl PBS in the bottom donor plate and 200 μl of blank PBS was added to the top receiver plate. The plates were incubated in an orbital shaker temperature at 37°C for 5 h, aliquots were taken from the donor and receiver plates and the concentration of drug was determined. Compound permeability was calculated using the equation</p><p> Papp=-ln[1-CA(t)Ceq](A∗(1VD+1VA)∗t) where Papp is expressed in units of cm/s, CA(t) is drug concentration in the acceptor at time t, VD is donor well volume, VA is acceptor well volume, A is the area of the filter (0.3 cm2), t is time in seconds.</p><!><p>Microsome stability was evaluated by incubating 1 μM compound with 1 mg/ml hepatic microsomes (human, rat, or mouse) in 100 mM potassium phosphate buffer, pH 7.4. The reactions were held at 37° C with continuous shaking. The reaction was initiated by adding NADPH, 1 mM final concentration. The final incubation volume was 300 μl and 40 μl aliquots were removed at 0, 5, 10, 20, 40, and 60 min. The removed aliquot was added to 160 μl acetonitrile to stop the reaction and precipitate the protein. NADPH dependence of the reaction was evaluated in parallel incubations without NADPH. At the end of the assay, the samples are centrifuged through a 0.45 micron filter plate (Millipore Solventer low binding hydrophilic plates, cat# MSRLN0450) and analyzed by LC-MS/MS. The data was log transformed and results are reported as half-life.</p><!><p>Cytochrome P450 inhibition was evaluated in human liver microsomes using four selective marker substrates (CYP1A2, phenaceten demethylation to acetaminophen; CYP2C9, tolbutamide hydroxylation to hydroxytolbutamide; CYP2D6, bufuralol hydroxylation to 4′-Hydroxybufuralol; and CYP3A4, midazolam hydroxylation to 1′-hydroxymidazolam) in the presence or absence of 10 μM test compound. The reaction is initiated by the addition of 1 mM NADPH and stopped after ten min by the addition of 2-times volume of acetonitrile containing dextrorphan as an internal standard. The concentration of each marker substrate is approximately its Km.39 Furafylline, sulfaphenazole, quinidine, and ketoconazole were included to each run to validate that the assay could identify selective inhibitors of each isoform.</p><!><p>All procedures described are covered under existing protocols and have been approved by the Scripps Florida IACUC to be conducted in the Scripps vivarium, which is fully AAALAC accredited. Pharmacokinetics were determined in n=3 male C57Bl/6 mice. Compounds were dosed as indicated in the text via intravenous injection via tail vein or oral gavage. 25 μL of blood was collected via a small nick in the tail using heparin coated hematocrit capillary tubes which were sealed with wax and kept on ice until plasma was generated by centrifugation using a refrigerated centrifuge equipped with a hematocrit rotor. Dose levels are provided in the text. Time points for determination of pharmacokinetic parameters were 5m, 15m, 30m, 1h, 2h, 4h, 6h, and 8h. Plasma concentrations were determined via LC-MS/MS using a nine point standard curve between 0.4 and 2000 ng/ml prepared in mouse plasma. Pharmacokinetic analysis was done with WinNonLin, Pharsight inc. using a noncompartimental model.</p><!><p>cLogD values were calculated with Pipeline Pilot workflow application (Accelrys) at pH 7.4 as previously described.40</p>

PubMed Author Manuscript

Tunable-Deformed Graphene Layers for Actuation

Benefiting from unique planar structure, high flexibility, splendid thermal, and electric properties; graphene as a crucial component has been widely applied into smart materials and multi-stimulus responsive actuators. Moreover, graphene with easy processing and modification features can be decorated with various functional groups through covalent or non-covalent bonds, which is promising in the conversion of environmental energy from single and/or multi-stimuli, to mechanical energy. In this review, we present the actuating behaviors of graphene, regulated by chemical bonds or intermolecular forces under multi-stimuli and summarize the recent advances on account of the unique nanostructures in various actuation circumstances such as thermal, humidity, electrochemical, electro-/photo-thermal, and other stimuli.

tunable-deformed_graphene_layers_for_actuation

Introduction<!><!>The Deformation of Graphene by Chemical Modification<!>Oxygen-Carbon Bond in Graphene<!><!>Heteroatom-Carbon Bond in Graphene<!><!>Heteroatom-Carbon Bond in Graphene<!>The Deformation of Graphene by Intermolecular Interactions<!><!>The Deformation of Graphene by Intermolecular Interactions<!>The State-of-Art Applications<!>Temperature Stimuli<!>Humidity Stimuli<!><!>Humidity Stimuli<!>Electrical/Electrochemical Stimuli<!><!>Electrical/Electrochemical Stimuli<!><!>Light Stimuli<!><!>Conclusion and Outlook<!>Author Contributions<!>Conflict of Interest<!>

<p>Since the successful stripping of single-layer graphene by Geim and coworkers in 2004 (Novoselov et al., 2004), this unique two-dimensional carbon material has attracted considerable attention in the energy-related fields of energy storage and conversion, advanced electronic devices, biochemistry materials, and sensors. Graphene nanolayers in particular, are ideal active components in smart actuation applications because of their intrinsic properties such as excellent transparency, superior electron conductivity and mobility (>2 × 105 cm2 V−1 s−1 at an electron density of 2 × 1011 cm−2), large specific surface area (>2,500 m2 g−1), huge Young′s modulus (>0.5−1 TPa), and high thermal conductivity (over 3,000 W mK−1) (Liu et al., 2007; Moser et al., 2007; Lee et al., 2008; Morozov et al., 2008; Nair et al., 2008; Balandin, 2011; Mayorov et al., 2011; Novoselov et al., 2012). Up to now, great efforts have been devoted to the furtherance of smart graphene-based actuators by regulating the surface chemical and physical properties of graphene, leading to promising applications in robotics, sensors, mechanical instruments, microscopy tips, switches and memory chips (Osada et al., 1992; Baughman et al., 1999; Kim and Lieber, 1999; Fennimore et al., 2003; Ahir and Terentjev, 2005; Sidorenko et al., 2007; Jang et al., 2008; Park et al., 2010).</p><p>Various graphene-based active responsive materials modified with functional groups via covalent bonds or non-covalent bonds have been developed recently to achieve the conversion of one or more environmental stimuli, such as electrical, light, thermal, or chemical energy to mechanical energy (Cheng et al., 2017). It is necessary and crucial to excavate and summarize the essence of actuation behaviors of graphene-based functional materials, which has not been targeted in a report yet. In this review, we focus on the deformation behaviors of graphene's internal structure modified with chemical bonds and intermolecular forces (Figure 1), and summarize the recent developments of typical important fabrication methods for the deformation of graphene sheets within the fast-growing smart fields. The state-of-art applications of graphene-based actuators will also be presented, as well as conclusions and corresponding perspectives.</p><!><p>Schematic illustration of strategies toward tunable-deformed graphene materials.</p><!><p>It is the particular structure of graphene, an individual two-dimensional graphite composed of hexagonally network of sp2 carbon atoms that determine their unique properties, including high thermal/electrical conductivities, good light transparency, mechanical strength and flexibility. Most graphene actuation behaviors come from its inherent characteristics. For example, the electrochemical graphene actuator is mainly driven by electrical charging and discharging (Liu et al., 2012); the high photo-thermal conversion ability endows graphene with a promising light responsive component. However, the defect-free monolayer graphene shows very poor and negligible responsive behavior because of its perfect two-dimensional carbon-carbon structure with no energy gap in the electronic spectra, strong hydrophobic nature, and inert chemical reactivity, which hinders graphene from practical and widespread actuation applications. Nowadays, regulation of the carbon skeleton structure with other elements such as oxygen, nitrogen, sulfur, and phosphorus via a chemical method is an efficient way to change its sp2 bonded state and the location density of the electron cloud, in order to open the band gap and to increase the chemical active sites.</p><!><p>Introducing the oxygen element into the carbon-carbon structure is an efficient way to adjust the physical and chemical environment of graphene. In this regard, graphene oxide (GO) is recognized as the graphene covalently bonded with carboxyl, hydroxyl, and epoxy groups. Typically, GO sheets are synthesized by Hummer's method, in which graphite is oxidized using strong oxidants such as KMnO4, KClO3, and NaNO3 in the presence of nitric acid or its mixture with sulfuric acid (Kim and Lieber, 1999). The incorporation of these oxygen-related groups could change the intrinsic configurations of graphite sheets and induce the high polarization of electron density. The oxygen-containing groups bond with the carbon skeleton and distorts the structure of graphene from sp2 to sp3 hybridized states. The interlayer separation in graphene is about 0.34 nm while the inter sheet distance for GO varies from 0.63 to 1.2 nm, depending on the environmental relative humidity.</p><p>Compared with initial graphene, the existing oxygen groups in the GO system endow it with new features in advanced actuation fields. The oxygen-carbon bonds of GO nanosheets can form hydrogen bonds with water molecules in the surrounding environment, which induces the volume changes of GO materials under the heat or humidity stimuli. This hydrophilic property also causes the fast growing coefficient of thermal expansion in GO (130.14 × 10−6 K−1) compared to graphene (7 × 10−6 K−1), further enhancing the contraction amplitude as the temperature increases (Kelly, 1972; Kim and Lieber, 1999; Schniepp et al., 2006; McAllister et al., 2007).</p><p>Zhu's group confirmed this and found that the GO paper was able to present a reversible contraction/expansion with the stimulation of heating-cooling between 30 and 80°C (Zhu et al., 2012; Figure 2A). Unlike graphene, large negative coefficients of thermal expansion in GO is derived from the change of the water molecule between the GO sheet interlayers. With abundant oxygen-containing groups, temperature and humidity changes can induce the volume expansion or contraction of GO sheets because of the absorption or desorption of water molecules between its interlayers (Zhu et al., 2012; Figure 2B). During the adsorption (cooling) stage, water molecules percolated to the protruding islands in the hydrophilic regions and increased the overall interlayer spacing. The fast saturation of these hydrophilic regions and other areas with water caused GO sheets to slide apart from each other, resulting in the elongation of the GO sheets. On the contrary, the GO sheets will contract with the collapse of interlayer spacing in the desorption (heating) process. In addition, with the oxygen atoms of these groups via hydrogen bonding, GO systems can also show an actuation response under humidity stimuli due to their ability of fast absorption/desorption of water molecules. These water molecules could act as spacers for GO interlayers, and the distance between GO sheets can be regulated from 6 to 12 Å by controlling relative humidity (RH) from high water content to low content, therefore causing the deformation of GO materials (Zhu et al., 2012; Figure 2C). Cheng et al. discovered a similar elongation-contraction phenomenon in a rotational twisted GO fiber. The twisted GO fiber could rotate fast and reversibly once exposed to a humid environment. This is attributed to the strong expansion/contraction of GO layers, adsorbing and desorbing the water molecules. The twisted GO fiber can reach a large deformation of 5% (Cheng et al., 2013). Furthermore, a kind of simple focused-sun-light-induced photo reduction method has been developed to prepare reduced rGO and GO mixed bilayer paper by adjusting the sunlight radiation intensity (Han et al., 2015). The anisotropic GO/rGO showed bending curvatures from 0° to 168° under the moisture stimulus because of different water molecules absorption ability for GO and rGO layers. Overall, the unique performance of the oxygen-carbon bonds in graphene holds great promise for advanced graphene-based smart artificial electronic devices.</p><!><p>(A) Thermal behavior of GO paper for repeated cooling and heating cycles between 30°C and 80°C. (B) The process of the water adsorption/desorption process in GO paper (reprinted with permission from Zhu et al. (2012). Copyright 2012 American Chemical Society). (C) The process of absorption/desorption of water molecules in GO sheet (reproduced from Qiu et al. (2018) with permission from The Royal Society of Chemistry).</p><!><p>Apart from the oxygen-carbon bonding control in graphene, doping graphene with substituent heteroatoms is another efficient route to tune the electrical and chemical properties of graphene. Previous achievements have demonstrated that the graphene doped with nitrogen (N), boron (B), sulfur (S), phosphorus (P), and iodine (I) could efficiently create a disordered surface topography and modulates local surface and electric features of the conjugated carbon-carbon structures (Gong et al., 2009; Qu et al., 2010; Gao et al., 2013; Li et al., 2014; Cui et al., 2017; Figures 3A,B). Compared with the C atom, the electron-deficient B atom induces the charge polarization of the graphene basal plane. The doping of the B atom in graphene usually takes the place of the C atom on the plane to form the stable BC3 structure and/or out of the plane to create boric esters. While the N and P atoms are electron-rich donors, in which the P atom has an extra 3p orbital and larger atom radium, making it possible to increase the transformation of sp2 C to sp3 C (Cui et al., 2017).</p><!><p>(A) Heteroatom doped graphene. The balls colored gray, blue, and yellow are C, O, N atoms, respectively. (B) SEM image of BCN-graphene. Scale bar is 1 mm (reprinted with permission from Jung et al. (2014). Copyright 2014 Wiley-VCH). (C) Schematic electrochemical pathway of nitrogen-doped carbon materials (reproduced with permission. Copyright 2013, American Chemical Society).</p><!><p>It has been validated that graphene nanolayers possess electrostriction effects when injecting electrons into the carbon planar structure under the bias voltage, showing the electrochemical actuating behavior. Efficient heteroatoms-doping to graphene layers would increase the electron–ion activity and facilitate charge transfer at the electrode-electrolyte interface of graphene in the electrochemical process, facilitating the charge injection and C-C bond expansion of the graphene sheet at the interface. More importantly, the electrochemical responsive amplitude can be easily modulated by controlling the different heteroatoms doping or designing asymmetric doping in the graphene structure. In this regard, we experimentally verified the effect of different surface elemental doping on the graphene actuation ability, by constructing an asymmetrical surface modified graphene film (Xie et al., 2010). Two opposite sides of the graphene film were chemically treated with the hexane and O2 plasma, respectively. The side doped with oxygen groups exhibited a higher charge accumulation ability in the electrode-electrolyte interface than the other side, leading to the deformation of the graphene film.</p><p>At present, various methods have been developed to fabricate the heteroatoms doped graphene materials in the energy conversion field. In particular, the thermal annealing method is usually applied to fabricate heteroatom-doped graphene structures. Most of the precursors, such as B2O3, boric acid, urea, melamine, dicyanamide, aminoterephthalic acid, and hexachlorocyclotriphosphazene, are easily decomposed in a high temperature thermal annealing process, which are ideal candidates for constructing a single-/dual-heteroatom doped graphene (Sheng et al., 2011; Wang et al., 2012; Li et al., 2013; Fang et al., 2014; Vikkisk et al., 2014; Dong et al., 2015; Haque et al., 2015). In addition to the above method, other important routes have also been developed for the preparation of heteroatom-doped graphene, including discharge, plasma treatment, ball milling, chemical vapor deposition (CVD), and solvothermal reaction methods (Li et al., 2010; Qu et al., 2010; Deng et al., 2011; Jeong et al., 2011; Jeon et al., 2012; Dey et al., 2014; Jung et al., 2014; Hassani et al., 2016). These fabricating methods provide promising possibilities for the surface modification of graphene actuators, which are beneficial for improving the electrochemical performance in advanced energy conversion devices.</p><p>However, the exact actuation mechanism of the graphene actuator is still unclear, and requires further systematic studies focused on the electromechanical behaviors of graphene with surface modifications, leading to accurate control of the motion of the graphene actuator (Figure 3C). Nevertheless, there is no doubt that the graphene-based actuator holds great potential for applications in various electric/electrochemical responsive systems.</p><!><p>The unique properties of high electric-/photo-thermal and electrostriction effects make graphene an attractive active component for mechanical responsive devices (Novoselov et al., 2004, 2012; Stankovich et al., 2006). Apart from the chemical modification, graphene, especially for graphene oxide with a large 2D conjugated structure and rich oxygen-related groups, is easily combined with conjugated polymers, organic molecules, and inorganic constituents through intermolecular interactions, such as electrostatic interaction, hydrophobic interaction, chemisorption, π-π stacking, hydrogen bonding and so on. Effective contact of the active components directly determines the astute response, actuating ability, and the cycling life. Many efforts have been devoted to the combination of graphene and polymer, organic and inorganic constituents, showing great potential in energy-mechanical conversion applications (Stankovich et al., 2006; Compton and Nguyen, 2010; Dai, 2012; Cui et al., 2016; Azadmanjiri et al., 2018; Benzigar et al., 2018). In general, there are three typical responsive mechanisms for graphene-based functional composite actuators, including an electrostrictive response, electrothermal response, and a photothermal response (Figure 4).</p><!><p>(A) Schematic illustration of electrons charge or discharge into the graphene films. (B) The stretching or bending behaviors of graphene under the electrical stimulus. (C) The conversion of kinetic vibration energy to heat motion energy through infrared-phonon interactions.</p><!><p>As mentioned above, the expansion/contraction of the graphene sheet, caused by injecting electrons/holes into the carbon-carbon skeleton, can be regulated by chemical modifications. Similarly, this actuation behaviors (direction, curvature/displacement) of graphene, driven by electrical charging and discharging, can be further enhanced or precisely controlled through rational design of the graphene and active responsive components (Figure 4A). In order to achieve a large range of actuation displacement of graphene with good controllability, conducting polymers [such as polyaniline (PANI) and polypyrrole (PPy)] show the ability to convert electrical energy into mechanical energy in the electrochemical process, and are promising candidates for advanced actuators. In this case, we successfully constructed an asymmetric bilayer actuator by combining graphene and anions doped PPy in an ectropolymerization process (Liu et al., 2012; Cheng et al., 2017; Rasouli et al., 2018). Unlike the graphene deformation that operates by electron/hole injection, PPy deformation is mainly driven by the Faradaic doping and undoping process, in which the volume of the PPy structure increases or decreases with the embedding or expelling of the anions under negative or positive voltage. The discrepancy in the actuation mechanism of graphene and PPy would optimize the actuator configuration, leading to a larger bending ability than that of the graphene and PPy film itself. Such an asymmetric bilayer design can be structurally regulated into various actuation systems, including a 1D fiber actuator and a 3D framework actuator (Liu et al., 2012; Qiu et al., 2018), providing promising possibilities for the advanced actuation system.</p><p>Alternatively, as graphene has various stretching and bending vibrations in the perfect sp2-bonded carbon network, an appropriate electric current or infrared light applied to a graphene structure would induce Joule heating that generates the electric current passing through the graphene plane or induced the enhanced disordering degree by infrared–phonon interactions during the forced resonance vibration process, leading to the conversion of kinetic vibration energy to heat motion energy (Figures 4B,C). The higher electric power or light intensity inputs, the more thermal energy is converted. This outstanding electric-/photo-thermal conversion ability makes graphene the active component in various electric-/photo-stimuli actuators. Most thermoplastic polymers, such as polystyrene (PS), polyethyleneterephthalate (PET), poly (methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), and polyurethane (TPU), possess very weak photo- and electric-thermal conversion capacity, such that the generated thermal energy is not enough to convert into visible mechanical kinetic energy. Therefore, it is of practical significance for high-performance conventional polymer actuators to integrate with graphene sheets (Yan et al., 2010; Shi et al., 2015; Azadmanjiri et al., 2018). The functional groups of polymers can cross-link the carbon skeleton or oxygen-containing functional groups (epoxy, hydroxyl, carbonyl, and carboxyl) of the graphene or its derivatives through π-π stacking, electrostatic interaction, and hydrogen bonding. Consequently, multi-functional actuation devices are possibly designed and developed by a combination of specific polymers and graphene sheets or the structural regulation of graphene. Take the 3D graphene aerogel structure as an example, the spongy graphene aerogel with a special structure of a large area of folded material shows a larger negative coefficient of thermal expansion around −10−4 per °C at low voltage compared to other structured graphene materials, which makes achieving bimorph and stimulus-responsive actuators possible, by coupling with polymers.</p><!><p>The covalent and non-covalent functionalized graphene sheet endows it with biomimetic organism behaviors, which can achieve specific complex actions by combining functional responsive molecules or through a systematic design. These deformable actions are usually operated and controlled by external stimuli including thermal, light, moisture, and electric/electrochemistry; promising in applications ranging from sensors, switches, and artificial muscles to nano/micro electromechanical systems.</p><!><p>Considering the fact that graphene contracts upon heating because of the distinctive large negative coefficient of thermal expansion (Bao et al., 2009; Grigoriadis et al., 2010), thermal-induced actuators based on graphene can be realized by constructing asymmetric thermomechanical responsive structures (Zhao et al., 2013). For instance, a temperature-responsive GO film was fabricated by controlling the asymmetric microporous structures in two sides of the film (Cheng et al., 2016). The GO film exhibits reversible and quick bending upon heat induced by intermittent irradiation of infrared light. To further demonstrate the potential functions, laser writing circuits were in situ inserted into the GO film to detect its actuation behavior in real time, leading to an integrated self-detecting sensor.</p><p>The greater the difference of deformation properties, the higher the sensitivity to the temperature stimulus. In order to enhance deformation ability, Xu et al. applied small GO sheets (the size is ~1 μm) with rich edge functional oxygen-containing groups to obtain a maximum negative coefficient of thermal expansion of the GO sheets (Xu et al., 2017). After combining with a thermoplastic poly(vinylidene fluoride) (PVDF) film, the developed bilayer actuator possessed rapid and sensitive responses with excellent stability and repeatability. In addition, the photo-/electro-thermal conversion effects of the GO were applied to various mechanical responsive actuators. Zhu et al. (2011) demonstrated a sensitive heat-induced bending cantilever by coating the CVD grown graphene on a thin epoxy cantilever. The deflection of the graphene–epoxy hybrid cantilever could increase linearly with effective conversion factors of 0.17 μm °C−1 and 2.58 μm mW−1. A large displacement of 15 mm and high displacement-to-length ratio of ca. 0.79 could be achieved by constructing a bimorph actuator with PDMS with graphene as the raw material (Chen et al., 2011), in which a curvature was about 1.2 cm−1, with a strain of 0.41% at 10V for 3 s.</p><!><p>The hydration actuation behavior of GO materials induced by the strong hydrogen bond interaction with water molecules provides processing opportunities for fabricating new types of GO actuators responsive to changes in environmental water amounts and/or relative humidity (Zhao et al., 2013). The energy sources that triggers this behavior can be water vapor or thermal stimuli in an open system. In this regard, various humidity-based actuation micro-devices including robot, gripper, and electric generators have been developed recently. Take the GO sheets as an example, a kind of designed moisture-driven rotational motor was achieved by simply rotating the freshly spun GO fiber to form the twisted GO fiber (Cheng et al., 2014). This twisted GO exhibited a remarkable rotary speed up to 5,190 revolutions per min and a tensile expansion of 4.7% under humidity alternation (Figures 5A,B), which provides a way of building the moisture switches and moisture-triggered devices that convert mechanical-to-electric energy. By elaborately constructing an asymmetric structure, the actuation behaviors will be transmitted into various GO assemblies from 1D to 3D architectures. A rGO/GO asymmetric fiber actuator was fabricated in virtue of the laser positioning reduction along the one side of GO fiber, to perform bending/unbending actuation once it is exposed to humidity (Cheng et al., 2013, Figure 5C). To demonstrate the functions of the actuator, the asymmetric rGO/GO structure on the GO fiber was further regional-designed processed by a laser direct writing strategy. Consequently, the developed rGO/GO fiber actuator displayed sophisticated, programmed motions such as reversible multi-folding, S-shaped torsion, and spiral deformations (Figures 5D,E), which plays a vital role in single-fiber walking robots and shape-memory devices.</p><!><p>(A) The scheme of the designed humidity switch based on the humidity-responsive the twisted GO fiber in response to moisture (e.g., RH = 85%). (B) The alternating current generator. contains four copper coils around the TGF with a magnet. When the environment humidity changes, TGF can reversibly rotate the magnet within the surrounding copper coils to generate electricity (reprinted with permission from Cheng et al., 2014. Copyright 2014 Wiley-VCH). (C) Left: The scheme of laser positioning reduction on one side of a GO fiber. The black region corresponded to laser induced graphene region along the brown GO fiber. Right: optical image of the top surface for the as-prepared asymmetric G/GO fiber. (D) Photographs of a graphene/GO fiber (2 cm in length) under different RH values. (Reprinted with permission from Cheng et al., 2013. Copyright 2013 Wiley-VCH). (E) Photographs of three-wire moisture tentacles of the graphene/GO fibers at the sunny and rainy days, respectively. Scale bars: 5 mm. (F) Actuation of the bilayer paper sample as a function of RH. From left to right, RH was 12, 25, 49, 61, 70, and 90%, respectively. White-arrowed side: surface of the graphene oxide layer. (Reprinted with permission from Park et al., 2010. Copyright 2010 Wiley-VCH).</p><!><p>Along with this asymmetric moisture inserting strategy, more stimulus-induced deformation systems based on bimorph actuators have recently been reported (Sun G. et al., 2011; Sun Y. et al., 2011; Ji et al., 2014; Han et al., 2015; Kim et al., 2015). Park et al. (2010) demonstrated a bilayer actuator based on multi-walled carbon nanotubes (MWCNTs) and GO paper through sequential filtration method. At the initial state, the amount of water in the GO layer and MWCNT layer in the ambient is 17 and 0%, respectively. Differences in water adsorption capacity of MWCNTs and GO, leads to an interesting phenomenon: the bilayer paper will be rolled up at RH 12% of the room temperature because of the faster water absorption of MWCNTs compared to the GO side. In the meantime, it gradually becomes flat as the RH increases by 55%, due to the balance of the water absorption achieved by two sides of the film. This asymmetric paper will roll up again with the RH at 60–85% because of the large water absorption for the GO side (Figure 5F). It is noted that the differences in water molecular adsorptive capability in an asymmetric structure is essential in highly sensitive and advanced humidity-driven actuators.</p><!><p>Owing to the excellent electrostriction and electro-thermal features of graphene, the responsive to electrical or electrochemical stimulations are most widely researched among other types of graphene-related stimulus-sensitive actuators. Moreover, these remarkable microscopic characteristics of graphene sheets can be transmitted into macroscopic properties of rational assembled architectures, which leads to achievements of 1D to 3D electrical/electrochemical graphene actuators for different applications. A novel 3D graphene-based actuator based on the graphene framework coated with conductive PPy was successfully fabricated by the hydrothermal method (Liu et al., 2012; Figure 6A). It exhibited a maximum strain of up to 2.5%, superior to carbon nanotube film (Xu et al., 2008), graphene film (Xie et al., 2011), and the calculated strain induced in monolayer graphene by the formation of an electrostatic double layer (Rogers and Liu, 2011). The 3D prominent composite can be used as a smart filler for a controlled laser switch by adjusting the applied voltage (Figure 6B), which was filled into a plastic pipeline with a hole left in the center of the filler in order to let the red laser spots pass through. When applying a positive potential of 0.8 V, the initial red spot is blocked by the hole contraction induced by the expansion of the composite. This on/off switch process is repeatable and reliable, and can also be durably run for several months.</p><!><p>(A) Photograph of a designed device in which G–PPy was filled into a plastic pipeline with a microsized hole in the center of G–PPy. The G–PPy was partially immersed in 1 M NaClO4 solution and the hole was exposed to a laser beam for visibility. A Pt wire in contact with the sample was used as working electrode. (B) The open (on) and closed (off) state of the hole under the applied potential of +0.8 V and −0.8 V, respectively (reproduced from Liu et al., 2012 with permission from The Royal Society of Chemistry). The corresponding overlaid digital images captured at the starting point and the end point for the actuators (C1) IP -PPy, (C2) IP-PPy/rGO-1, (C3) IP-PPy/rGO-2, (C4) IP-PPy/rGO-3, and (C5) IP-PPy/rGO-4 in response to (A,D). (C) Voltage of 6 V in amplitude (reproduced from Rasouli et al., 2018 with permission from The Royal Society of Chemistry). (D,E) Snapshots of the tri-armed tweezers driven by an applied electric potential of +0.8 V and −0.8 V, respectively. (Reproduced with permission. Copyright Wang Y. et al., 2013, Elsevier Ltd).</p><!><p>Rasouli reported a PPy/rGO-based ionic actuator by simple electropolymerization of PPy/rGO nanocomposites on both faces of ink-coated Nafion membranes (Rasouli et al., 2018). When applying voltage on the composite film, the solvated ions would accumulate in a high concentration near the electrode regions, leading to bending deformations (MacDiarmid, 2001; Terasawa and Asaka, 2016). The maximum bending deformation of the actuators under the voltage of 6 V was about 25 cm compared to that of conventional ionic polymer (IP)–metal composites actuators (~23 cm, Shown et al., 2015). Except for the enormous deformation, the PPy/rGO-based ionic actuators also possessed excellent ion conductivity, capacitive characteristics, and large charge storage capacity (Figures 5C1–C5). Moreover, the electrochemical actuation behaviors are also reflected on 1D fiber actuators (Wang E. et al., 2013). A 1D fibrillar actuator based on PPy/GF was constructed by partially electropolymerizing of the PPy on the graphene fibers (GF). With a high strength of 230 MPa, a good maximum bending angle, and reasonable durability, this actuator can work as a micro tweezers (Figures 6D,E), which makes it possible to be the fiber-robot to operate multiple surgery or cell operation in the future.</p><p>Since the discovery that the single-walled carbon-nanotube incorporated into chitosan could generate electromechanical actuation properties at low alternating voltage (AC) stimuli (Hu et al., 2010), the electro-thermal graphene based actuators in the ambient solution, except for the electrical/electrochemical actuation in the solution, have also recently been widely investigated. Usually, the actuation ability is determined by Joule heating which is generated by the electric current passed through the graphene-based film. The higher the electric power input, the more thermal energy is converted. To this end, Xiao et al. reported an electromechanical actuator constituted by a porous graphene paper and PVDF layer (Xiao et al., 2016; Figure 7A). The hybrid film exhibited a large actuation motion with a maximum deflection of about 14.0 mm within 0.262 s and generated high actuation stress (>312.7 MPa g−1). This phenomenon is the same as that of the bilayer responsive structure of graphene and organic glass substrate which exhibited a reversible and large bending angle of 270° with a fast response of 8 s and recovery period of 19 s under the driving voltage of 65 V (Zhang et al., 2017). The difference in thermal expansion coefficient values between the thin graphene film and the organic glass substrate gives rise to an expanded volume of the two layers in different amounts, thus resulting in the deformation of the actuator (Figures 7B–E). In addition, Chen's group demonstrated an SG/PDMS bimorph actuator by combining spongy graphene (sG) paper with PDMS, which showed an ultra-large bending displacement of 15 mm with a curvature of about 1.2 cm−1 at 10 V for 3 s, a high displacement-to-length ratio of ~0.79, and vibration motion at AC voltage up to 10 Hz (Hu et al., 2014; Figures 7F,G), superior to those similar bimorph actuators reported previously (LeMieux et al., 2006; Sul and Yang, 2009; Hu et al., 2010; Jeon et al., 2012; Bi et al., 2013). More importantly, this bimorph actuator could mimic the fingers to fast grab, move and put down the objects, providing a basis for various device designs in the fields of artificial muscles, robotics, sensors, medicines and so on.</p><!><p>(A) Diagram to demonstrate the fish-like robot swimming, when the power is on or off, the "tail" bends down or up, then the fish-like robot will swim forward. The dimensions of the fish tail (the graphene-PVDF bimorph robot and the fish body (Expandable PS) are 14 × 3 mm2 and 30 × 8 mm2, respectively (reprinted with permission from Xiao et al., 2016. Copyright 2016 Wiley-VCH). (B) Laser reduced graphene oxide-based ETA without driving voltage. (C) The laser reduced graphene oxide-based electrothermal actuator is driven by the voltage of 65 V. (D) The LRGO-based ETA is hooking the plastic foam for moving a distance. (E) The driving voltage is switched off and the plastic foam is placed on the test bed (reprinted with permission from Zhang et al., 2017. Copyright 2017 Wiley-VCH). (F) A "tri-finger" mechanical gripper with 10 V turned off (left part) and turned on (right part). (G) A larger object is grabbed by the "tri-finger" gripper at applied 10 V (reproduced from Hu et al., 2014 with permission from The Royal Society of Chemistry).</p><!><p>As actuation active components, graphene and its derivatives with the extraordinary conversion of kinetic vibration energy to heat energy of graphene sheets, induced by the infrared phonon strong interactions during the resonance vibration process, have been applied to infrared light-driving actuators (Zhao et al., 2013). Lin and co-workers developed multi-responsive soft actuators with laser induced graphene (LIG) patterns as geometrically constraining elements coupled with PVDF and polyimide (PI) using a direct laser writing method (Deng et al., 2018). This PVDF/LIG/PI (PLP) sandwich actuator was a programmable shape transformation, in which responsive flowers with designed shapes were realized, in order to perform different bending behaviors under the controlled irradiation of the lamp (Figures 8a–e), imitating bionic robots. Another example is the combination of rGO and elastin-like polypeptides (ELPs) to simulate human finger actions (Wang E. et al., 2013). To demonstrate their idea, a hand-shaped rGO–ELP gel was fabricated, in which these "fingers" performed the given bending movement by controlling the irradiation position (Figure 8f). The bending rate and angle of the finger increased as enhancing the infrared radiation laser intensity and rGO concentration. Moreover, the rGO–ELP actuator can also be made into a light-driven crawler to "walk" on glass slides under the light radiation (Figure 8g). When applying infrared radiation on the one side of the crawler, it folded immediately. Once the light is off, the front rose and the back of the crawler produced a forward-directed force by pushing against the glass as it uncurled, which moved the entire gel forward 3 mm. These light-stimulated actuation strategies provide material platforms for the advanced smart muscles, robotics, and intelligent sensors. Recently, Wang et al., successfully prepared PDMS/graphene composite bilayer film, which could bend 7.9 mm in the horizontal direction under the light radiation. Moreover, this bilayer actuator can be further constructed into a beluga whale soft robot to swim in the pool at the speed of 6 mm/s deriving from its superhydrophobicity (Wang et al., 2019).</p><!><p>(a) Photographs of a PLP actuator upon exposure to light with different distances. (b,c) Photographs of a flower-shaped PLP actuator with (b) y = 901 and (c) y = +451 when exposed to light. (d) Photographs of a PLP robot crawling by switching the light on and off. (e) Schematic and photographs of a PLP robot with multi-way gaits under light stimulus (reproduced from Deng et al., 2018 with permission from The Royal Society of Chemistry). (f) Images of the fingers of a hand-shaped rGO-ELP hydrogel controllably bending and unbending in response to the location of an IR laser spot. (g) Schematic and images of a light-driven crawler. A rGO-ELP hydrogel molded with a slight curvature was placed with the porous side facing down. The laser was applied so as to induce gel curling. Subsequent uncurling during recovery after the laser was removed pushed the gel forward (1 mm tick marked) (reprinted with permission from Wang E. et al., 2013. Copyright 2013 American Chemical Society).</p><!><p>The actuators with the graphene as active components show great potential in the various intelligent bionic devices. Particularly, the actuating behaviors of graphene regulated by chemical bonds or intermolecular forces under multi-stimulus are discussed, which endow graphene-based responsive units with response capability to single and/or multiple environment stimulus, including electro-thermal stimuli, humidity stimuli, temperature stimuli and so on. For instance, one way is to selectively decorate the graphene with heteroatoms, such as oxygen, in which graphene oxide is fast and sensitive to water molecules, showing excellent responsive behavior to humidity. The other way is to specifically modify the graphene with conducting polymers or thermoplastic polymer, making it a crucial part of smart switch system or intelligent robotics. These prominent features present promising applications in smart systems with complex functions.</p><p>However, there are still some challenges that remain to be solved to meet future requests of graphene-based intelligent materials. The high manufacturing cost and delayed response time of graphene-based actuator have always been restricting the practice of advanced application in industry and intelligent home system. It is still an efficient way to introduce highly sensitive responsive polymers or gels into graphene systems to construct high performance bionic intelligent systems, such as modified PDMS, PMMA and other new designed organic polymers. Moreover, the mechanical properties and durability of the current graphene-based actuation system needs to be promoted, which involves preparation of high-quality graphene, matching of graphene with functional materials, and the design of specific graphene microstructures or assemblies. In addition, it is highly desirable to constitute smart graphene systems that handle multi-stimuli, have good sensitivity and high efficiency. Considering the promising potential application of bionic intelligent systems in the future, low cost biocompatible graphene-based actuators are also in great need of development, which remains a big challenge to overcome in the production process. There is no doubt that graphene-based smart devices will soon be playing an important role in modern intelligent systems, with various possibilities and continuous research efforts.</p><!><p>JW and YX are responsible for the main text. VC provided the adjustment suggestions. CS was responsible for drawing some figures. LQ and YZ hold the overall ideas and corrections.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p><!><p>Funding. We acknowledge the financial support from NSFC (No. 21604003, 51673026, 51433005), the National Key R&D Program of China (2017YFB1104300, 2016YFA0200200), NSFC-MAECI (51861135202).</p>

PubMed Open Access

Reversible uptake of molecular oxygen by heteroligand Co(II)–l-α-amino acid–imidazole systems: equilibrium models at full mass balance

BackgroundThe paper examines Co(II)–amino acid–imidazole systems (where amino acid = l-α-amino acid: alanine, asparagine, histidine) which, when in aqueous solutions, activate and reversibly take up dioxygen, while maintaining the structural scheme of the heme group (imidazole as axial ligand and O2 uptake at the sixth, trans position) thus imitating natural respiratory pigments such as myoglobin and hemoglobin. The oxygenated reaction shows higher reversibility than for Co(II)–amac systems with analogous amino acids without imidazole. Unlike previous investigations of the heteroligand Co(II)–amino acid–imidazole systems, the present study accurately calculates all equilibrium forms present in solution and determines the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{{{\text{O}}_{2} }}$$\end{document}KO2equilibrium constants without using any simplified approximations. The equilibrium concentrations of Co(II), amino acid, imidazole and the formed complex species were calculated using constant data obtained for analogous systems under oxygen-free conditions. Pehametric and volumetric (oxygenation) studies allowed the stoichiometry of O2 uptake reaction and coordination mode of the central ion in the forming oxygen adduct to be determined. The values of dioxygen uptake equilibrium constants \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{{{\text{O}}_{2} }}$$\end{document}KO2 were evaluated by applying the full mass balance equations.ResultsInvestigations of oxygenation of the Co(II)–amino acid–imidazole systems indicated that dioxygen uptake proceeds along with a rise in pH to 9–10. The percentage of reversibility noted after acidification of the solution to the initial pH ranged within ca 30–60% for alanine, 40–70% for asparagine and 50–90% for histidine, with a rising tendency along with the increasing share of amino acid in the Co(II): amino acid: imidazole ratio. Calculations of the share of the free Co(II) ion as well as of the particular complex species existing in solution beside the oxygen adduct (regarding dioxygen bound both reversibly and irreversibly) indicated quite significant values for the systems with alanine and asparagine—in those cases the of oxygenation reaction is right shifted to a relatively lower extent. The experimental results indicate that the “active” complex, able to take up dioxygen, is a heteroligand CoL2L′complex, where L = amac (an amino acid with a non-protonated amine group) while L′ = Himid, with the N1 nitrogen protonated within the entire pH range under study. Moreover, the corresponding log \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{{{\text{O}}_{2} }}$$\end{document}KO2 value at various initial total Co(II), amino acid and imidazole concentrations was found to be constant within the limits of error, which confirms those results. The highest log \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{{{\text{O}}_{2} }}$$\end{document}KO2 value, 14.9, occurs for the histidine system; in comparison, asparagine is 7.8 and alanine is 9.7. This high value is most likely due to the participation of the additional effective N3 donor of the imidazole side group of histidine.ConclusionsThe Co(II)–amac–Himid systems formed by using a [Co(imid)2]n polymer as starting material demonstrate that the reversible uptake of molecular oxygen occurs by forming dimeric μ-peroxy adducts. The essential impact on the electron structure of the dioxygen bridge, and therefore, on the reversibility of O2 uptake, is due to the imidazole group at axial position (trans towards O2). However, the results of reversibility measurements of O2 uptake, unequivocally indicate a much higher effectiveness of dioxygenation than in systems in which the oxygen adducts are formed in equilibrium mixtures during titration of solutions containing Co(II) ions, the amino acid and imidazole, separately.Electronic supplementary materialThe online version of this article (doi:10.1186/s13065-017-0319-8) contains supplementary material, which is available to authorized users.

reversible_uptake_of_molecular_oxygen_by_heteroligand_co(ii)–l-α-amino_acid–imidazole_systems:_equil

Background<!><!>Background<!>Results and discussion<!><!>Results and discussion<!>Conclusions<!>Reagents<!>Apparatus<!>Oxygenation reaction of the Co(II)–l-α-amino acid–imidazole systems<!>Determination of reaction stoichiometry of dioxygen uptake in the Co(II)–l-α-amino acid–imidazole systems by the molar ratio method<!>Confirmation of the coordination mode of the central ion by determination of the number of imidazole released from the coordination sphere of the Co(II)–l-α-amac–imidazole–O2 system<!>Calculations of equilibrium concentrations of Co(II), amac and Himid as well as evaluation of the equilibrium \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{{{\text{O}}_{2} }}$$\end{document}KO2 constants<!>

<p>The capability of compounds called natural respiratory pigments to reversibly absorb molecular oxygen has been the subject of intensive research since the end of the 19th Century and has been inspiring the creation of artificial systems to imitate their activity [1–14]. Example models of synthetic oxygen carriers include mixed complexes of the type Co(II)–auxiliary ligand–imidazole, in which imidazole coordinates in trans position against the bound O2 molecule, alike imidazole of the proximal histidine in myoglobin and hemoglobin [15]. In contrast to classical methods of preparing such compounds by mixing separate solutions of Co(II) salts, appropriate amino acids and imidazole [16–18], an original method has been applied, in which cobalt(II) and imidazole were introduced in the form of a polymeric, pseudo-tetrahedral, semi-conductive complex [Co(imid)2]n. This results in the formation of definite, unique structures with an imidazole molecule in an axial position opposite the O2 molecule [19–26].</p><!><p>Schematic structure of the polymeric [Co(imid)2]n complex</p><!><p>The peculiar property of O2 transport in such Co(II)–amac–Himid systems, as with the natural dioxygen carriers, results from the rapidly stabilizing equilibrium present in solution between the "active" form and the dioxygen-containing form. The "active" form, responsible for the dioxygen transport, is usually a paramagnetic, high-spin, hexacoordinate Co(II) complex of CoII(amac)2(Himid)(H2O) composition, containing two chelate–like connected amino acid molecules forming an equatorial plane, as well as two axial ligands–imidazole and water. After substitution of the dioxygen molecule for water, a dimeric, diamagnetic [CoIII(amac)2(Himid)]2O2 2− complex is formed with the O2 molecule coordinated in peroxide order i.e. with a O2 2− (μ-peroxy) bridge between two cobalt ions formally oxygenated to Co(III). This complex, because of the eventual partial irreversible oxidation of Co(II) to mononuclear Co(III) products, is frequently denoted as an intermediate oxygen adduct. Owing to the elongation of the dioxygen bond from 120.7 pm for the triplet O2 to 149.0 pm for the peroxide O2 2− anion, the oxygen adducts may be used as intermediate complexes in catalytic processes [29–34].</p><p>The O2 2− bridge (μ-peroxy) exists within pH = 3–9, but upon a rise in basicity above pH 10, this is transformed into a poorly reversible dibridged Co(III)O2 2−OH−Co(III) (μ-peroxy–μ-hydroxy) form. This double-bridge appears in place of the two carboxyl groups, which easily undergo dissociation and which are found in cis position towards the coordinated dioxygen molecule. Such a complex is a much less effective O2 carrier due to its higher affinity for autoxidation. An alternative known description of the oxygen bridges is the form type η, corresponding to "side on" bridge μ-peroxy structures [35]. In turn, acidification of the solution at a low temperature (−3 to 0 °C) leads to protonation of the μ-peroxy bridge, whereas the forming intermediate Co(III)O2 2−H+Co(III) product undergoes a rapid decay accompanied by Co(III) ion formation. In addition, at a temperature around 0 °C and in acidic medium, the O2 2− (μ-peroxy) bridge may be subsequently oxidized by means of strong oxidizers, e.g. Ce4+, MnO4 − or Cl2 ions. As a result, a paramagnetic, stable {[CoIII(amac)2(Himid)]2O2 −}+ complex is formed, with an irreversibly bound dioxygen moiety in the Co(III)–O2 −–Co(III) (μ-superoxy) bridge.</p><p>All known O2 carriers (both natural and synthetic) form complexes of two types: monomeric, with an M:O2 stoichiometry of 1:1, and dimeric, with an M:O2 stoichiometry of 2:1. An analysis of the theoretically estimated values of the free standard Gibbs energy of the O2 reactions with metal ions and their complexes could be expected to favor the dimeric structures. In fact, the ΔG° value for the dimer formation reaction attains negative values for a much higher number of metals than is the case for monomer formation. This effect refers to the displacement of complex-formation decidedly to the right [36]. The data find a practical confirmation because among all the known dioxygen carriers, in aqueous solution we observe formation of stable dimeric complexes.</p><p>Previous investigations of the Co(II)–amac–Himid systems have not included the key aspect, i.e. accurate calculations of the Co(II), amac and Himid concentrations at equilibrium, by using the formation constants reported in our work for analogous oxygen-free systems [37]. These calculations may allow the equilibrium concentrations of all equilibrium forms present in solution to be determined, and for the \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$K_{{{ ext{O}}_{2} }}$$\end{document}KO2 equilibrium constants to be evaluated without using any simplified approximations, which for instance take into account only the "active" complex and the oxygen adduct within the mass balance system [19, 38]. Moreover, the advantage of the experimental methods used in the present work, i.e. a direct gas–volumetric experiment with simultaneous pH measurement, is that it allows the degree of reversibility of O2 uptake to be taken into account. As for many other complexes, including a majority of complexes with amino acids and peptides, the irreversible part of the reaction is quite rapid (e.g. t1/2 <5 min for glygly), which excludes the use of the most commonly applied method based only on potentiometric titration [39–41].</p><!><p>The optimum amac to Co(II) ratio equaled 2:1. Above this value, the amount of dioxygen taken up did not change (see Additional file 1: Figure S1). The amount of imidazole released from the [Co(imid)2]n moiety as a result of the mixed Co(II)–amac–Himid–O2 complex formation (0.3 mol Himid per 0.3 mol Co) indicates that the stoichiometric Co(II): imidazole ratio was 1:1, which confirms that one of the two [Co(imid)2]n imidazole moieties remains in the coordination sphere of cobalt(II) of the final complex (see Additional file 2: Figure S2). In other words the structure of the forming dioxygen adducts are unified by the presence of one imidazole in the coordination sphere.</p><!><p>The Co(II)–l-α-histidine–Himid system at molar ratio 0.3:0.75:0.3 (mmol). Dependence of pH and number of mmole O2 bound on duration of the oxygenation reaction at a temperature of ~0°C (vertical segment corresponds to reversibility of O2 uptake after saturation = 86.69%)</p><p>Uptake of O2 by the Co(II)–l-α-amino acid–imidazole systems (duration time of uptake, final value of pH, number of mmoles of O2 bound, percentage of reversibility)</p><p>Percentage share of free Co(II) ion and complex forms at fixed equilibrium of O2 uptake reaction in the Co(II)–amac–Himid–O2 systems. l-α-amino acid (amac) = a alanine, b asparagine, c histidine. Internal diagrams show the equilibrium share of species other than the O2 adduct in an extended scale. Cadd O2 denotes the total concentration of the dioxygen adduct(with dioxygen bound both reversibly and irreversibly). L amac, L′ Himid. Molar ratio Co(II): amac: Himid = 0.3: 0.6:0.3 (mmol)</p><p>Equilibrium constants \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$K_{{{ ext{O}}_{2} }}$$\end{document}KO2 of dioxygen uptake in the Co(II)–l-α-amino acid–imidazole–O2 systems</p><p>The mean values are followed by standard deviations</p><p>Coordination modes in the Co(II)–amac–Himid system with amac = Ala (R = CH3) and Asn (R = CH2–CO–NH2). a "active" heteroligand complex CoL2L′, where L amac, L′ Himid, b dioxygen adduct</p><p>Coordination modes in the Co(II)–amac–Himid system with amac = His. a "active" heteroligand complex CoL2L′, where L amac, L′ Himid, b dioxygen adduct</p><p>UV/Vis spectra in the Co(II)–amac–Himid system at temperature ~0 °C, where amac = l-α-histidine. Right Y-axis: molar absorption coefficients of (a) Co(II) and (b) "active" heteroligand complex CoL2L′, where L amac, L′ Himid; left Y-axis: (c) molar absorption coefficients of the dioxygen adduct</p><!><p>As can be seen in Table 2, the highest value of log \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$K_{{{ ext{O}}_{2} }}$$\end{document}KO2 occurs for the histidine system, most likely due to participation of the aforementioned additional effective N3 donor of the histidine imidazole side group. This is not surprising as it is already known that for histidine, the "active" complex is the most thermodynamically stable complex also under oxygen-free conditions [37]. On the other hand, the lower value of log  \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$K_{{{ ext{O}}_{2} }}$$\end{document}KO2 for the asparagine system in comparison with the alanine system is most likely due to steric hindrance, which arises from one of the asparagine amide side groups during formation of the dimer. In this case, a greater share of the amino acid in the Co(II): amac: Himid molar ratio favorably displaces the equilibrium towards oxygen adduct formation. This also makes it possible to obtain chemically reasonable (positive) solutions of equation system (1) at higher excesses of the amino acid (cf. Table 2). For the two remaining amino acids alanine and histidine, particularly histidine, the oxygen adduct (for both the reversible and irreversible parts together) almost entirely uses up the accessible cobalt when the share of the amino acid greatly exceeds the stoichiometric ratio Co(II): amac:Himid = 1:2:1; the concentrations of the other complex species, including the "active" complex, fall to such low levels that it is impossible for the equation system (1) to converge in the form of three positive solutions.</p><!><p>At a decreased temperature close to 0 °C, the Co(II)–amac–Himid systems demonstrate enhanced reversible uptake of molecular oxygen. Coordination of the dioxygen molecule by the "active" complex occurs as exchange of the axial H2O or carboxyl oxygen to O2, occurring together with simultaneous formal intramolecular redox oxidation of Co(II) to Co(III) and the reduction of the charge of the dioxygen molecule to a bridging peroxide O2 2− ion.</p><p>The log  \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$K_{{{ ext{O}}_{2} }}$$\end{document}KO2 values are highest for the oxygenated forms of the heteroligand complexes with histidine, as their coordination sphere is formed by a chelating tridentate ligand (with imidazole, NH2, COO− donors). The essential impact on the electron structure of the dioxygen bridge, and by that on reversibility of O2 uptake, is due to the first of the groups mentioned above. The two remaining amac ligands engaged in the mixed complexes (i.e. alanine and asparagine) were bidentate ligands. Even the potentially tridentate l-α-asparagine behaves as a bidentate ligand in the attainable pH range of around 9–10, illustrated in Table 1, which follows also from the previous reports concerning oxygen–free conditions. However, the reversibility of O2 uptake in the latter systems containing an axial imidazole, unequivocally indicates a much higher reversibility than that previously reported for Co(II)–amac systems in the absence of imidazole.</p><p>Chemically reasonable (positive) values of both [Co(II)], [amac], [Himid] equilibrium concentrations and hence, appropriate log \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$K_{{{ ext{O}}_{2} }}$$\end{document}KO2 values, could be attained only for limited Co(II):amac:Himid molar ratios, irrespective of the degree of equilibrium displacement towards oxygen adduct formation.</p><!><p>l-α-amino acids: asparagine, pure, Sigma Chemical Co., histidine, pure (≥99.0%), Fluka Chemie GmbH, alanine, pure, International Enzymes Limited; polymeric [Co(imid)2]n complex prepared by A. Vogt, Faculty of Chemistry, University of Wrocław [27, 38, 45]; potassium nitrate (V), p.a., P.O.Ch. Gliwice; nitric (V) acid, p.a., P.O.Ch. Lublin; sodium hydroxide—0.5021 M solution determined by potassium hydrogen phthalate; acetone, p.a., P.O.Ch. Gliwice; oxygen pure medical (99.7–99.8%); argon, p.a. (99.999%) from Linde Gas (Poland).</p><!><p>An isobaric laboratory set for volumetric and pH-metric measurements (see Additional file 3: Figure S3), composed of the following elements: a double-walled thermostated glass vessel of volume ca 80 mL, tightly closed with a silicon stopper and equipped with a burette nozzle supplying the 4 M HNO3; a combination pH glass electrode C2401, Radiometer (Copenhagen); a Radiometer Analytical 101 temperature sensor; a gas inlet tube (dioxygen) connected with the gas burette; outlet tube; a glass rode to hang a small glass vessel with the [Co(imid)2]n polymer. A PHM 85 Precision pH Meter Radiometer (Copenhagen), a Fisherbrand FBC 620 cryostat, Fisher Scientific, an Electromagnetic Stirrer ES 21H (Piastów, Poland), an oxygen tank with reducing valve and a CO-501 Oxygen Meter, Elmetron (Zabrze, Poland) were also used. The following glass set was used to determine the imidazole released from the coordination sphere of the mixed complexes: suction flask, water suction pump, washer, Schott funnel POR 40 (see Additional file 4: Figure S4).</p><!><p>The thermostated vessel was filled with a solution containing an exactly weighted sample of chosen amino acid, so as to obtain a predicted Co(II)–amac–Himid ratio when adding the [Co(imid)2]n polymer. Adjustment of the solution to constant ionic strength I = 0.5 M was achieved by means of potassium nitrate. The solution was topped up with water to 30 mL. A small glass vessel with 0.3 mmol of the [Co(imid)2]n polymer (hence the same 0.6 mmol of imid) was hung from a glass rod over the solution surface. After the entire vessel reached a temperature close to 0 °C [decrease of temperature inhibits the irreversible oxidation of Co(II)], the initial pH and the initial volume level in the gas burette was read and the main experiment started by inserting the polymer into the sample. The current values of pH and dioxygen volume were noted in definite time intervals up to saturation. A rise in pH was observed along with a change in color from entirely colorless to brown or even dark-brown. At the end of oxygenation, which occurred when reaching pH ≈9 to 10, the solution was acidified to the initial pH with a small aliquot of 4 M nitric acid solution. This caused a partial discoloration of the solution and evolution of dioxygen. The volume of dioxygen evolved against the total volume of dioxygen bound served as a measure of reversibility of oxygenation.</p><!><p>For each system under study, a dependence plot of the number of bound O2 (mmol) against the C L/C M ratio was prepared, where C L—total amac concentration, C M—total Co(II) concentration, which enabled the determination of stoichiometry of dioxygen uptake.</p><!><p>Exactly weighed samples of amino acid and the [Co(imid)2]n polymer were placed into a washer so as to attain a molar ratio of Co(II):l-α-amac: imidazole = 0.3: 0.9: 0.3 (mmol). The washer immersed in ice was filled with 2 mL of argonated water and then, during 15 min, the forming "active" complex was argonated continuously. After 10 min, argonation was changed to oxygenation. The final content of the washer, the freshly formed dioxygen complex, was quantitatively added to a Schott funnel previously filled with oxygenated acetone. The oxygen complex, insoluble in water, precipitated as a dark brown solid. At the moment a water suction pump was connected to the Schott funnel. Acetone was filtered off together with the water containing the imidazole released along with oxygen complex formation. The filtrate obtained was titrated potentiometrically with nitric acid. All the steps of experiment were carried out at temperature close to 0 °C.</p><!><p>The calculations were performed by means of a Mathcad 13 computer program [46]. The mass balance non–linear equation system was solved by the Levenberg–Marquardt method [47, 48], which enables a faster convergence of the solutions than the Gauss–Newton iteration. Such effect is due to the introduction of an additional λ parameter to the Gauss–Newton iteration formula, which corrects the appropriate direction of the procedure depending on whether the solutions go close to or far from the convergence series.</p><p>The procedure used the corresponding equilibrium concentrations [M], [L], [L′] (where: [M] = [Co(II)]), which were the searched unknown quantities x 1, x 2, x 3 of the following system:1\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$egin{aligned} f_{ 1} (x_{ 1} ,x_{ 2} ,x_{ 3} ) = \, 0 \hfill \ f_{ 2} (x_{ 1} ,x_{ 2} ,x_{ 3} ) = \, 0 \hfill \ f_{ 3} (x_{ 1} ,x_{ 2} ,x_{ 3} ) = \, 0 \hfill \ \end{aligned}$$\end{document}f1(x1,x2,x3)=0f2(x1,x2,x3)=0f3(x1,x2,x3)=0</p><p>The solution vector of the system:2\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$X = \left[ {egin{array}{*{20}c} {x_{1} } \ {x_{2} } \ {x_{3} } \ \end{array} } ight]$$\end{document}X=x1x2x3follows Newton's formula:3\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$X_{{{ ext{i}} + 1}} = X_{ ext{i}} - \, \left( {F^{\prime} \, \left( {X_{ ext{i}} } ight)^{ - 1} \cdot F\left( {X_{ ext{i}} } ight)} ight)$$\end{document}Xi+1=Xi-F′Xi-1·FXiafter an appropriate initial estimation of the X 0 vector. The function vector is:4\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$F (X) = \left[ {egin{array}{*{20}c} {f_{1} (x_{1} ,x_{2} ,x_{3} )} \ {f_{2} (x_{1} ,x_{2} ,x_{3} )} \ {f_{3} (x_{1} ,x_{2} ,x_{3} )} \ \end{array} } ight]$$\end{document}F(X)=f1(x1,x2,x3)f2(x1,x2,x3)f3(x1,x2,x3)whereas the matrix of derivatives, i.e. Jacobi matrix, is:5\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$F^{\prime}(X) = \left[ {egin{array}{*{20}c} {rac{{\partial f_{1} }}{{\partial x_{1} }}\quad rac{{\partial f_{1} }}{{\partial x_{2} }}\quad rac{{\partial f_{1} }}{{\partial x_{3} }}} \ {rac{{\partial f_{2} }}{{\partial x_{1} }}\quad rac{{\partial f_{2} }}{{\partial x_{2} }}\quad rac{{\partial f_{2} }}{{\partial x_{3} }}} \ {rac{{\partial f_{3} }}{{\partial x_{1} }}\quad rac{{\partial f_{3} }}{{\partial x_{2} }}\quad rac{{\partial f_{3} }}{{\partial x_{3} }}} \ \end{array} } ight]$$\end{document}F′(X)=∂f1∂x1∂f1∂x2∂f1∂x3∂f2∂x1∂f2∂x2∂f2∂x3∂f3∂x1∂f3∂x2∂f3∂x3(F'(X))−1 in Eq. (3) denotes the inverted Jacobi matrix.</p><p>In the mass balance system all the ligand (both amac and Himid) protonation constants as well as the complex–formation constants with Co(II) were known from the previous reports [37, 45]. In cumulative form the formation constants may be written as:6\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$eta_{mll^\prime h} = { aise0.7ex\hbox{${[{ ext{M}}_{m} { ext{L}}_{l} L^\prime_{l\prime } H_{h} ]}$} \!\mathord{\left/ {phantom {{[{ ext{M}}_{m} { ext{L}}_{l} L\prime_{l\prime } H_{h} ]} {[{ ext{M}}]^{m} [{ ext{L}}]^{l} [{ ext{L}}^\prime ]^{l^\prime } [{ ext{H}}]^{h} }}} ight.\kern-0pt} \!\lower0.7ex\hbox{${[{ ext{M}}]^{m} [{ ext{L}}]^{l} [{ ext{L}}^\prime ]^{l^\prime } [{ ext{H}}]^{h} }$}}$$\end{document}βmll′h=[MmLlLl′′Hh][M]m[L]l[L′]l′[H]h</p><p>The functions used for the equation systems of l-α-alanine and l-α-asparagine were due to the fact that the mixed ML2L′ complex capable of dioxygen uptake (existing outside of the non-active mixed MLL′ complex) contains the sufficient three nitrogen donors in the coordination sphere, in accordance with Fallab's "3 N" rule [49]:7\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$f_{1} = C_{ ext{M}} - Y[{ ext{M]}} - \sum\limits_{l = 1}^{3} {eta_{ml} [{ ext{M]}}} \,[{ ext{L}}]{\kern 1pt}^{l} - \sum\limits_{l' = 1}^{5} {eta_{ml'} [{ ext{M]}}} \,[{ ext{L}}^{\prime}]{\kern 1pt}^{l'} - \sum\limits_{l = 1}^{2} {eta_{mll'} [{ ext{M]}}} \,[{ ext{L}}]{\kern 1pt}^{l} [{ ext{L}}^{\prime}] - 2C_{{{ ext{O}}_{2} }}$$\end{document}f1=CM-Y[M]-∑l=13βml[M][L]l-∑l′=15βml′[M][L′]l′-∑l=12βmll′[M][L]l[L′]-2CO2 8\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$f_{2} = C_{ ext{L}} - Y_{1} [{ ext{L]}} - l\sum\limits_{l = 1}^{3} {eta_{ml} [{ ext{M]}}} \,[{ ext{L}}]{\kern 1pt}^{l} - l\sum\limits_{l = 1}^{2} {eta_{mll'} [{ ext{M]}}} \,[{ ext{L]}}{\kern 1pt}^{l} [{ ext{L}}^{\prime}] - 4C_{{{ ext{O}}_{2} }}$$\end{document}f2=CL-Y1[L]-l∑l=13βml[M][L]l-l∑l=12βmll′[M][L]l[L′]-4CO2 9\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$f_{3} = C_{ ext{L}^{\prime}} - Y_{2} [{ ext{L}}^{\prime}] - l'\sum\limits_{l' = 1}^{5} {eta_{ml'} [{ ext{M]}}} \,[{ ext{L}}^{\prime}]{\kern 1pt}^{l'} - \sum\limits_{l = 1}^{2} {eta_{mll'} [{ ext{M]}}} \,[{ ext{L]}}{\kern 1pt}^{l} [{ ext{L}}^{\prime}] - 2C_{{{ ext{O}}_{2} }}$$\end{document}f3=CL′-Y2[L′]-l′∑l′=15βml′[M][L′]l′-∑l=12βmll′[M][L]l[L′]-2CO2</p><p>For l-α-histidine, the mixed not oxygen binding complex was a MLL′H species, in which the side group imidazole was protonated at the N3 nitrogen, thus the number of nitrogen atoms in the coordination sphere of the central ion was two, i.e. less than the minimum suggested by Fallab's rule. However, as the number of nitrogen atoms was sufficient in the "active" complex ML2L′, capable of O2 was as follows:10\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$f_{1} = C_{ ext{M}} - Y[{ ext{M]}} - \sum\limits_{l = 1}^{ 2} {\sum\limits_{h = 1}^{1} {eta_{mlh} } } \,[{ ext{M}}][{ ext{L}}]{\kern 1pt}^{l} [{ ext{H}}]{\kern 1pt}^{h} - \sum\limits_{l' = 1}^{5} {eta_{ml'} [{ ext{M]}}} \,[{ ext{L}}^{\prime}]{\kern 1pt}^{l'} - eta_{1210} [{ ext{M}}][{ ext{L}}]{\kern 1pt}^{2} [{ ext{L}}^{\prime}] - eta_{1111} [{ ext{M}}][{ ext{L}}]{\kern 1pt} [{ ext{L}}^{\prime}][{ ext{H}}]\, - 2C_{{{ ext{O}}_{2} }}$$\end{document}f1=CM-Y[M]-∑l=12∑h=11βmlh[M][L]l[H]h-∑l′=15βml′[M][L′]l′-β1210[M][L]2[L′]-β1111[M][L][L′][H]-2CO2 11\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$f_{2} = C_{ ext{L}} - Y_{1} [{ ext{L]}} - l\sum\limits_{l = 1}^{ 2} {\sum\limits_{h = 0}^{1} {eta_{mlh} } } \,[{ ext{M}}][{ ext{L}}]{\kern 1pt}^{l} [{ ext{H}}]{\kern 1pt}^{h} - 2eta_{1210} [{ ext{M}}][{ ext{L}}]{\kern 1pt}^{2} [{ ext{L}}^{\prime}] - eta_{1111} [{ ext{M}}][{ ext{L}}]{\kern 1pt} [{ ext{L}}^{\prime}][{ ext{H}}] - 4C_{{{ ext{O}}_{2} }}$$\end{document}f2=CL-Y1[L]-l∑l=12∑h=01βmlh[M][L]l[H]h-2β1210[M][L]2[L′]-β1111[M][L][L′][H]-4CO2 12\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$f_{3} = C_{ ext{L}^{\prime}} - Y_{2} [{ ext{L}^{\prime}]} - l'\sum\limits_{l' = 1}^{5} {eta_{ml'} [{ ext{M]}}} \,[{ ext{L}^{\prime}]}{\kern 1pt}^{l'} - eta_{1210} [{ ext{M}}][{ ext{L}}]{\kern 1pt}^{2} [{ ext{L}^{\prime}]} - eta_{1111} [{ ext{M}}][{ ext{L}}]{\kern 1pt} [{ ext{L}^{\prime}][H]} - 2C_{{{ ext{O}}_{2} }}$$\end{document}f3=CL′-Y2[L′]-l′∑l′=15βml′[M][L′]l′-β1210[M][L]2[L′]-β1111[M][L][L′][H]-2CO2where: C M—total concentration of the metal: Co(II), C L—total concentration of the l-α-amino acid, \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$C_{{L^{\prime}}}$$\end{document}CL′—total concentration of imidazole, \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$C_{{{ ext{O}}_{2} }}$$\end{document}CO2—concentration of the oxygen adduct, β ml—summary stability constants of the Co(II)–l-α-amino acid complexes, β ml'—summary stability constants of the Co(II)–imidazole complexes, β mll'—summary stability constants of the mixed Co(II)– l-α-alanine/asparagine–imidazole complexes, β 1210, β 1111—summary stability constants of the mixed Co(II)–l-α-histidine–imidazole complexes.</p><p>The hydrolyzed Co(II) aqua-ion and the protonated (not complexed) ligand forms were considered in expressions:\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$egin{aligned} Y &= 1 + \left( {{ ext{1}}/K_{{{ ext{OH}}}} [{ ext{H}}]} ight) \Y_{1} &= 1 + eta _{{{ ext{LH}}}} [{ ext{H}}] + eta _{{{ ext{LH2}}}} [{ ext{H}}]^{{ ext{2}}} \,&{ ext{for}} \,{ extsc{{l}}} ext{-}lpha{ ext{-alanine and}}\,{ extsc{{l}}} ext{-}lpha { ext{-asparagine}};\hfill \ { ext{Y}}_{{ ext{1}}}&= { ext{ 1 }} + eta _{{{ ext{LH}}}} [{ ext{H}}] + eta _{{{ ext{LH2}}}} [{ ext{H}}]^{{ ext{2}}} + eta _{{{ ext{LH3}}}} [{ ext{H}}]^{{ ext{3}}}\;&{ ext{for}}\, { extsc{{l}}} ext{-}lpha ext{-histidine} \hfill\ Y_{2} &= 1 + eta _{{{ ext{L}}^{\prime } { ext{H}}}} \left[ { ext{H}} ight] \hfill\end{aligned}$$\end{document}Y=1+1/KOH[H]Y1=1+βLH[H]+βLH2[H]2forL-α-alanine andL-α-asparagine;Y1=1+βLH[H]+βLH2[H]2+βLH3[H]3forL-α-histidineY2=1+βL′HHwhere: K OH—hydrolysis constant of tshe Co(II) aqua-ion = 10−9.8 [50], β LH, β LH2, β LH3—summary (overall) protonation constants of the l-α-amino acid, β L'H—protonation constant of imidazole.</p><p>It is noteworthy that solving the nonlinear equation system at very erroneous initial estimations may lead to quite different results or lack of convergence. However, in the case of the systems under study, the solutions [M], [L] and [L′] are not allowed to be negative numbers and they should be found within the limits of zero and the total concentrations C M, C L, C L′. This makes it possible to reject the solutions without a chemical meaning.</p><p>The used summary protonation constants of l-α-amino acids and imidazole, the stability constants of the primary Co(II)–amac, Co(II)–Himid complexes, as well as the stability constants of the heteroligand Co(II)–l-α-amino acid–imidazole complexes have been determined previously in the same medium and the same ionic strength as in the present work (KNO3, I = 0.5) [37, 45]. The only different parameter was the temperature: 25.0 °C, instead of 0–1 °C. The lack of data due to the lower temperature is usually caused by lowered sensibility of the glass electrodes. Nevertheless, the systematic error of the stability constants recently used could be estimated on the basis of corresponding literature data as 0.1–0.2 in logarithm [51].</p><p>The obtained equilibrium concentrations [M], [L], [L′] were needed to calculate the \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$K_{{{ ext{O}}_{2} }}$$\end{document}KO2 constant. In the present reaction scheme, the first step corresponded to formation of the "active" complexes:13\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ ext{Co}}\left( { ext{imid}} ight)_{ 2} + { ext{ 2 Hamac }} + { ext{ H}}_{ 2} { ext{O}} o { ext{Co}}\left( { ext{amac}} ight)_{ 2} \left( { ext{Himid}} ight)\left( {{ ext{H}}_{ 2} { ext{O}}} ight) \, + { ext{ Himid}}$$\end{document}Coimid2+2 Hamac+H2O→Coamac2HimidH2O+Himid</p><p>Consecutively the "active" complex takes up dioxygen by forming the dimeric oxygen adduct:14\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$2 { ext{ Co}}\left( { ext{amac}} ight)_{ 2} \left( { ext{Himid}} ight)\left( {{ ext{H}}_{ 2} { ext{O}}} ight) \, + { ext{ O}}_{ 2} o \, \left[ {{ ext{Co}}\left( { ext{amac}} ight)_{ 2} \left( { ext{Himid}} ight)} ight]_{ 2} { ext{O}}_{ 2}^{ 2- } + { ext{ 2 H}}_{ 2} { ext{O}}$$\end{document}2Coamac2HimidH2O+O2→Coamac2Himid2O22-+2 H2O</p><p>By treating the O2 uptake as a reversible reaction:15the equilibrium constant may be calculated from the formula:16\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$K_{{{ ext{O}}_{2} }} = rac{{[{ ext{O}}_{2} \,{ ext{adduct}}]}}{{[ \, { ext{"active"}}{ ext{ complex}}]^{2} [{ ext{O}}_{2} ]}}$$\end{document}KO2=[O2adduct]["active"complex]2[O2]where [O2 adduct]—equilibrium concentration of this part of the oxygen adduct, in which dioxygen was bound reversibly. The value was found by using the percentage of reversibility of O2 uptake, that is to say by rejecting the part of O2 adduct, in which the metal undergoes irreversible oxidation to Co(III) during the experiment. The equilibrium [O2] concentration was calculated on the basis of table data of dioxygen solubility in water [52].</p><p>According to Henry's law, if the experiment proceeds at the same temperature but at decreased pressure, the volume of gas dissolved in water (or in a diluted solution) is proportionally lower. Under the experimental conditions we have:\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$V_{{{ ext{O}}_{ 2} }} = V_{ ext{g}} \cdot f = V_{ ext{g}} \cdot p_{{{ ext{O}}_{ 2} }} / 7 60$$\end{document}VO2=Vg·f=Vg·pO2/760where: V g = 0,04758 mL—table value of dioxygen solubility in 1 L of water, at temperature 1 °C under normal pressure 1013 × 105 Pa. \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$p_{{{ ext{O}}_{ 2} }}$$\end{document}pO2—partial pressure of dioxygen in the gas burette.</p><p>The \documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$$V_{{{ ext{O}}_{ 2} }}$$\end{document}VO2 value gives the [O2] concentration after adjustment to the number of mmoles of O2 dissolved in 1 L of the solution.</p><!><p>Additional file 1: Figure S1. Determination of stoichiometry of the O2 uptake by the molar ratio method in the Co(II) – amac – Himid – O2 systems. L-α-amino acid (amac) = (a) alanine, (b) asparagine, (c) histidine. C L – total concentration of amac, C Co – total concentration of Co(II). Mmole O2 – number of mmol of dioxygen taken up. All the samples contained 0.3 mmol of Co(imid)2 in 30 mL of solution.</p><p>Additional file 2: Figure S2. Titration curve of the water-acetone filtrate obtained when the dioxygen adduct formed in aqueous solution precipitated in acetone. Co(II) : amac : Himid at a molar ratio of 0.3 : 0.9 : 0.3 (mmol); L-α-amino acid (amac) = (a) alanine, (b) asparagine, (c) histidine.</p><p>Additional file 3: Figure S3. Laboratory set for pehametric – volumetric measurements.</p><p>Additional file 4: Figure S4. Laboratory set for determination of imidazole released from the coordination sphereof Co(II): (a) initial preparation, (b) collection of the filtrate.</p><p></p><p></p><p>amino acid (α-amine group non-protonated, carboxyl group deprotonated)</p><p>amino acid (α-amine group protonated, carboxyl group deprotonated)</p><p>Electronic supplementary material</p><p>The online version of this article (doi:10.1186/s13065-017-0319-8) contains supplementary material, which is available to authorized users.</p>

PubMed Open Access

A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase

The catalytic mechanism of the cyclic amidohydrolase isatin hydrolase depends on a catalytically active manganese in the substrate-binding pocket. The Mn 2+ ion is bound by a motif also present in other metal dependent hydrolases like the bacterial kynurenine formamidase. The crystal structures of the isatin hydrolases from Labrenzia aggregata and Ralstonia solanacearum combined with activity assays allow for the identification of key determinants specific for the reaction mechanism. Active site residues central to the hydrolytic mechanism include a novel catalytic triad Asp-His-His supported by structural comparison and hybrid quantum mechanics/classical mechanics simulations. A hydrolytic mechanism for a Mn 2+ dependent amidohydrolases that disfavour Zn 2+ as the primary catalytically active site metal proposed here is supported by these likely cases of convergent evolution. The work illustrates a fundamental difference in the substrate-binding mode between Mn 2+ dependent isatin hydrolase like enzymes in comparison with the vast number of Zn 2+ dependent enzymes.The catalytic activity of metallohydrolase enzymes strongly depends on the identity of the metal and on the nature of its binding site. Zn 2+ bound hydrolytic enzymes, in particular metallo-β-lactamases 1 , probably constitute the most extensively studied cases for such a group. Depending on the enzymatic class, hydrolytic enzymes with both a mononuclear or dinuclear Zn 2+ sites have been identified as widely distributed throughout all kingdoms of life 2 . The catalytic mechanism for the mononuclear and dinuclear Zn 2+ sites have been debated for years [3][4][5] , and only recently the collection of accumulated knowledge allowed the proposition of a plausible mechanism [6][7][8][9] . Despite Zn 2+ being the most widely distributed hydrolytic cation, it has been shown that Zn 2+ also possesses an inhibitory potential for certain metallohydrolases as, for example, in carboxypeptidase A 10 .The specialized metallo-enzyme amidohydrolase superfamily (AHS) is able to carry out a diverse range of chemical reactions including the degradation of metabolic precursors like pyrimidine (dihydropyrimidinases) and hydantoin, (hydantoinases) 11 . The AHS members are often categorised as both Zn 2+ and Mn 2+ dependent hydrolases. However, amidohydrolases involved with purines degradation in both bacteria and plants are mainly Mn 2+ dependent allantoinases 12,13 , even though Zn 2+ dependent allantoinase activity has been reported 14 .The compound isatin is a metabolic precursors and was originally identified as an intermediate product of the indole-3-acetic acid (IAA) degradation pathway in Bradyrhizobium diazoefficiens 15 . Hydrolytic activity of isatin hydrolase (IH) was first demonstrated in various symbiotic rhizobial species 16 . Recent bioinformatics studies

a_fundamental_catalytic_difference_between_zinc_and_manganese_dependent_enzymes_revealed_in_a_bacter

<!>Materials and Methods<!>25<!>Enzymatic assay.<!>Sequence analysis.<!>Molecular modelling.<!>Results<!>QM/MM calculations reveal the importance of an Asp-His dyad in the proton abstraction. In<!>Figure 3.<!>Discussion

<p>indicates that IH are found widespread in bacteria including pathogens specific to the human gut and plants, e.g., B. enterica and R. solanacearum 17 . Isatin hydrolase A from Labrenzia aggregata (LaIHA) and R. solanacearum (RsIHA) share 59% sequence identity while the two putative orthologues LaIHA and LaIHB in L. aggregata share 51% sequence identity. Both LaIHA and LaIHB contain the central metal binding motif HxG[T/A]HxDxPxH in each protomer of the catalytically active dimer. Structural characterization of LaIHB revealed a novel fold, which later was also attributed to other amidohydrolases such as bacterial kynurenine formamidase B (KynB) (EC 3.5.1.9) 17 . The fold is generally described as an α/β hydrolase with a central scaffold resembling the swivelling β/α/β fold 18 , while the majority of AHS members contains a monomeric (β/α) 8 -TIM like-barrel structural fold 19 . A notable difference between LaIHB and kynurenine formamidase from Bacillus anthracis (BaKynB) is the presence of a mononuclear manganese-binding site in LaIHB, while the structure of KynB accommodates a bi-nuclear zinc site 20 . LaIHB shows a strict specificity for Mn 2+ over Zn 2+ , as reported by kinetic measurements 17 , and rationalised by computer simulations 21 .</p><p>Here we present two crystal structures of LaIHA, one bound to the hydrolytic product isatinate, and one bound to benzyl benzoate, as well as the apo structure of homologous RsIHA. Together with results from enzyme kinetic analysis, the resolved structures are used to describe the molecular basis for the Mn 2+ -dependent mechanism of hydrolysis, showing that IHA is a true orthologue of IHB. For the first time, the mechanism of a Mn 2+ dependent hydrolysis reaction is characterised in detail by computer simulations using multiscale quantum mechanics/molecular mechanics (QM/MM) 22 and ab initio molecular dynamics (MD) simulations 23 . The simulations were crucial to unravelling the essential catalytic role of the conserved residues His79 and His207 for the formation of the product. Based on the identification of mechanistically important residues presented in this work, including the Mn 2+ binding residues in the LaIHA active site, we further present evidence that the Mn 2+ dependent allantoate amidohydrolase (AAH) (E.C. 3.5.3.9) displays a striking similarity in the active site geometry despite the differences in the overall protein fold. As this likely represents a case of convergent evolution, we propose that the generalised catalytic mechanism described here for the IHs may also apply to the AAH family and other similar Mn 2+ dependent hydrolases.</p><!><p>Cloning, expression and purification of LaIHA and RsIHA. Chemicals were purchased from Sigma-Aldrich (Norway) unless stated otherwise. The open reading frame encoding LaIHA (UniProtKB: A0P0F0) was amplified by PCR from a boiled colony of L. aggregata IAM12614. DNA fragments were isolated and digested with BamHI and EcoRI and cloned into the T7-RNA polymerase dependent E. coli expression plasmid pT7H6 24 . The 6xHis containing LaIHA was expressed in E. coli BL21 AI cells (Invitrogen) in 2xTYE-medium for 4 hours at 37 °C. Cells were harvested by centrifugation, re-suspended, and lysed by sonication in a buffer containing 50 mM Tris-HCl pH 8.0 and 0.5 M NaCl supplemented with a protease inhibitor tablet (Roche cOmplete, EDTA-free). Insoluble material was removed by centrifugation (4000 g, 10 min.). The soluble protein extract was batch-adsorbed onto 25 mL Ni-NTA agarose resin (Qiagen) per litre of original culture and loaded into liquid chromatography columns. The protein loaded Ni-NTA columns were washed with >20 column volumes (CV) of equilibration buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 50 mM imidazole). The protein was eluted from the resin with elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM Na 2 EDTA). Fractions containing the recombinant proteins were pooled, and the buffer changed into a low salt buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, and 1 mM Na 2 EDTA) on a Sephadex G-25 column (GE Healthcare). Before crystallisation and kinetic experiments, LaIHA was further treated with 10 mM EDTA and dialysed (500 fold dilution) into a stability buffer (5 mM bis-tris pH 7.0, 100 mM NaCl and 1 mM DTT). The protein solutions were centrifuged at 180,000 g for 10 minutes at 4 °C, and the supernatant could hereafter be stored in a stable condition at 4 °C for at least 12 months.</p><p>The RsIHA (UniProtKB: Q8XYC3) was ordered from Genscript and inserted into the expression vector pET-M11. The six-histidine containing construct was expressed in E. coli BL21 gold cells, in LB-medium (containing 50 µg/mL kanamycin) overnight at 18 °C. Cells were harvested by centrifugation, re-suspended, and lysed by a bead-beater (Biospec) in a lysis buffer (50 mM Tris-HCl pH 8.0 and 0.1 M NaCl), protease inhibitor tablet (Roche cOmplete, EDTA-free). Insoluble material was removed by centrifugation, and the lysate was supplemented with 20 mM imidazole before loaded onto a pre-equilibrated HisTrap HP (GE). The column was washed with 10 CV with equilibration buffer (50 mM Tris-HCl pH 8.0, 0.1 M NaCl, and 20 mM imidazole) and eluted with elution buffer (50 mM Tris-HCl pH 8.0, 0.1 M NaCl, and 300 mM imidazole). The pooled fractions were supplemented with a molar 1:50 ratio of tobacco etch virus (TEV) protease and dialysed overnight against dialysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl). The RsIHA was again loaded to the HisTrap HP column and the flow-through was collected. RsIHA was concentrated to 20 mg/mL and loaded onto a Superdex 200 10/300 GL size exclusion column (GE) pre-equilibrated in SEC buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl). Pure RsIHA were pooled, concentrated, and stored at −80 °C. RsIHA was treated like LaIHA before crystallisation and kinetic experiments.</p><p>Crystallization of LaIHA and RsIHA ligand complexes. The pre-treatment of LaIHA and RsIHA with an excess of EDTA and dialysis was mainly carried out to remove any potential trace of metals, leaving an estimated EDTA concentration at 0.02 mM. Prior to crystallization 1 mM MnCl 2 was added. LaIHA and RsIHA were crystallized at 22 °C using a protein concentration of 15 mg/mL in 5 mM Bis-Tris pH 7.0, 100 mM NaCl, and 1.0 mM MnCl 2 . LaIHA was crystallised by vapour diffusion using sitting drops 1 + 1 μL (LaIHA: Reservoir) over 100 μL reservoir solution. The best diffracting crystals were obtained in 20% glycerol and 20% PEG 1500. The LaIHA:isatinate structure was obtained by soaking the crystals for approximately 1 min with 500 µM isatin before flash-freezing the crystals in liquid nitrogen (LN 2 ). This condition also served as cryo-protection. RsIHA was also crystallised with vapour diffusion using hanging drops, 1 + 1 μL (RsIHA: Reservoir) over 400 μL reservoir solution sealed with immersion oil (56822, Sigma). The best diffracting crystals of RsIHA grew in 1 M succinate, 100 mM HEPES pH 6.5. Cryo-protection was achieved by adding 1 μL of 60% w/v PEG400 suspended in reservoir solution to the drop. The crystals were mounted and flash-cooled immediately after. Crystals of LaIHA:benzyl benzoate was obtained in 28% PEG 1500 and 1 mM MnCl 2 with a hanging drop setup using 250 μL reservoir and 1 + 1 μL drops. With a protein concentration of 20 mg/mL used. The hanging drop wells were sealed with immersion oil. Crystals appeared within one week. Cryo-protection was obtained by adding glycerol to the reservoir solution to a final concentration of ~12%, which after 12 hours would allow additional vapor diffusion to take place before mounting and flash-cooling in LN 2</p><!><p>.</p><p>Data collection and structure determination. All datasets were collected at 100 K. For LaIHA:Benzyl benzoate a dataset extending to 1.50 Å resolution was collected at a wavelength of 1.0004 Å. The space group was determined to be P1, and a collection strategy was calculated by iMOSFLM 26 . The data were processed with XDS 27 . A test set of 5% was used for R free calculation 28 . Phasing was performed by molecular replacement (MR) using LaIHB (PDB ID: 4J0N) as a search model for Phaser 29 . Initial model building and refinement were performed in Phenix 30 . The final model was built using Coot 31 with small ligand models and restraints produced using eLBOW 30 . For LaIHA:Isatinate, a diffraction dataset was collected at beamline P13 at PETRA III (Hamburg, Germany) 32 . The space group was determined and would only allow a P1 lattice. A complete dataset was collected at 1.79 Å resolution with and oscillation range of Ω = 0.01° and processed with XDS. The structure was solved with MR using a LaIHA: Benzyl benzoate monomer as the search model and performed with Phaser in Phenix. Diffraction data on RsIHA was collected at beamline ID30B at ESRF (Grenoble, France). The space group was determined and allowed P 3 2 2 1, a complete dataset with at Ω = 0.05° over 126° was collected. The data extended to 2.65 Å and was processed with XDS. The structure was solved with MR using LaIHA:Benzyl benzoate monomer as the search model in Phenix. Model building and refinement was performed using coot 33 and phenix. refine within the phenix package 30 . NCS restraints were applied throughout the refinement, however released in the final round (Number of molecules in the asymmetric of each crystals structure have been added in the Supplementary Table S1) 34 .</p><p>All structure validations were performed using MolProbity 30 . Structure analysis and figure preparation were done using PyMOL Molecular Graphics System, Version 1.5.0.3, Schrödinger, LLC. The finalised model and structure factors were deposited to Protein Data Bank (PDB) and given the PDB accession codes (LaIHA:Benzyl benzoate PDB ID 5NNA; RsIHA PDB ID 5NMP; LaIHA:isatinate PDB ID 5NNB).</p><!><p>The measurement of the activity, and its metal dependence, were performed as described by Bjerregaard-Andersen et al. 17 . The method is based on an increased absorption of the product isatinate at 368 nm. This can be followed using a standard spectrophotometer. Measurements were performed using a JASCO V-630 spectrophotometer with a cuvette light path of 1.0 cm. An extinction coefficient of 4.5 × 10 3 cm −1 mol −1 L was used for isatinate. The data were fitted with the Michaelis-Menten equation assuming one binding site using the software GraphPad Prism 6.</p><!><p>Structural homologues of LaIHA and E. coli allantoate amidohydrolase (EcAAH) were identified using the DaliLite v. 3 35 , followed by inspection in PyMoL. The sequences of the identified homologues were then imported in Jalview 36 using the fetch from PDB option and combined with sequences of LaIHA, RsIHA, and Rhodococcus rhodochrous HpoH (RrHpoH). Sequences with the following UniProt accession codes (in parentheses) were used: LaIHA (A0P0F0), LaIHB (A0NLY7), RsIHA (Q8XYC3), EcKynB (B4E9I9), PaKynB (Q9I234), BaKynB (Q81PP9), BsAHD (P84132), MjAHD (Q58193), RrHpoH (B5MAD9). For AAH alignment, the following accession codes were used: EcAAH (P77425), AtAAH (Q8VXY9), BvHYD (A4JQA0), BmHYD (A0A0H3KRF1), BcHYD (B4EHA1), GeRAC (Q53389), SkSYN (Q96W94). Sequence alignments were performed using the Clustal algorithm with default settings in Jalview. Phylogenetic trees were calculated in Jalview as average distance using identity percentage and exported to TreeDyn 198.3 37 for visualisation. The sequences were included in alignment as supplied by the PDB.</p><!><p>The system consisting of LaIHA, isatin, 47331 water molecules, and 19 Na + ions was placed in a 105 × 126 × 118 Å 3 simulation box. The initial geometry of the protein was taken from the X-ray structure of LaIHA:isatinate resolved by us and described in this text. Isatin was introduced in the system by molecular replacement, fitting the aromatic moiety of isatinate. The Mn 2+ ion, isatin, the catalytic water, the side chains of the ligating amino acids (His69, His73, Asp75, Gln219), and of the neighbouring His79, His207, Asp193 residues were treated at a quantum-mechanical level (QM) using density functional theory with the Becke and Lee-Yang-Parr (BLYP) approximations for the exchange-correlation functional 38,39 . The Kohn-Sham orbitals for the valence electrons were expanded over a DZVP Gaussian basis set 40 and an auxiliary plane-wave basis set with a cut-off of 240 Ry. The core electrons were integrated out using Goedecker-Teter-Hutter pseudopotentials 41 . The remainder of the system was described at the molecular-mechanics (MM) level using the Amberff14SB force field 42 .</p><p>In a first step, the system was equilibrated by full classical molecular dynamics (MD) simulations in the NpT ensemble (T = 300 K, p = 1 atm), constraining the QM region to its initial positions, and using a time-step of 1.5 fs. Thereafter, QM/MM Born-Oppenheimer MD simulations were performed at 300 K within the NVT ensemble using the Nosé-Hoover thermostat [43][44][45] and a time-step of 0.25 fs. The simulations were run using the CP2K package (https://www.cp2k.org/) [46][47][48] . The mechanism of hydrolysis was sampled by coupling QM/MM MD to metadynamics simulations 49,50 using collective variables defined as differences between coordination numbers (Visualized in Fig. S1, in the supporting information): where CN is the coordination number between two atoms. Each CN is defined as:</p><p>where d ij is the distance between atoms i, j, and d 0 , p, and q are free parameters. In our simulations, d 0 was set to 1.6 Å for CV1, and 1.1 Å for CV2 and CV3. Values of p = 12 and q = 14 were used for all the three CVs. The d0, p, q values where chosen so that the profile of the sigmoidal function (equation 1) did not change significantly during a standard thermal oscillation of the bond, while it signalled the formation or breaking of a chemical bond. Gaussian hills with a height of 2 kcal/mol were spanned every 100 step (i.e., every 25 fs). A Gaussian spread of 0.15 was used for all three collective variables. The free energy profile for the reaction was obtained as the sum of the Gaussians added during the whole metadynamics simulation. The three CVs used in this study were chosen to take into account both the nucleophilic addition/elimination at the carbonyl group and the proton rearrangements necessary to yield the product. Runs using any combination of only two of these CVs did not produce any reactive profile, indicating that all the three CVs are necessary for the determination of the reaction coordinate. With this setup, the system reacted after 1.1 ps of metadynamics simulations, the run was continued until a total time of 1.85 ps to reach convergence in the free energy profile. We assumed convergence of the profile after observation of a second re-crossing of the barrier (Supplementary Fig. S2).</p><!><p>IHA contains a mononuclear manganese binding site involving an additionally conserved glutamine. The structures of LaIHA:benzyl benzoate and LaIHA:isatinate were determined at 1.50 Å and 1.79 Å resolution, respectively, while the one of RsIHA was determined at 2.65 Å resolution. Data collection and model refinement statistics are collected as described in Supplementary Table S1. Both LaIHA structures are highly similar to the one of LaIHB with a root mean square deviation (rmsd) value of ~0.7 Å computed on all main chain atoms. Both LaIHB and LaIHA are dimers with two exchanged regions -a small N-terminal α-helical exchange and a larger β-hairpin exchange (Fig. 1a). A hallmark of this fold is the contribution of two conserved residues of one monomer in the formation of the substrate-binding pocket located in the other monomer. For the IH's these two residues are two tryptophans (Fig. 1b).</p><p>The highly conserved Mn 2+ binding site is located at the bottom of the substrate-binding pocket. The Mn 2+ is found in an octahedral complex coordinated by His69, His73, Asp75, and two water molecules (Fig. 1c). Asp75 forms a bidentate contact in LaIHA:Benzyl benzoate and a mono-dentate electrostatic contact in the LaIHA:isatinate structures. The mono or bi-dentate coordination results in two or one water molecules as additional ligands, respectively, preserving the octahedral geometry. The side chain carbonyl of Gln219 in LaIHA (Ala224 in LaIHB) is found in alternative conformations, one of which coordinates Mn 2+ . The non-binding conformation allows additional water to approach to Mn 2+ (W1025 A/B , Fig. 1c). Sequence analysis reveals that Gln219 is part of a motif, conserved as either GLAS (e.g. in LaIHB) or GLQC (e.g. in LaIHA) (Figs 1 and 2a). The short residues motifs AS and QC are conserved as sequence pairs, which could indicate a functional dependency. Interestingly, the residues S or C are found to be the key residue involved in regulating the proton flow through the water channel. A mutation of the serine residue to a cysteine to form a GLAC motif caused a gain in activity in LaIHB 17 . The GLQC/GLAS sequence, combined with the conserved manganese binding site appears to be a signature motif for isatin hydrolase activity. In KynB, the comparable position holds a conserved I(L/I)E motif, which is also found in the two uncharacterized structures of amidohydrolases (AHD) from Bacillus stearothermophilus (BsAHD) and Methanococcus jannaschii (MjAHD) (Fig. 2a, Full sequence alignment shown in Supplementary Fig. S3). The motif is absent in RrHpoH, which has confirmed cyclase activity. Whether the I(L/I) E motif and perhaps, in particular, the glutamate is indicative of activity on linear amides or a binuclear metal site remains uncertain.</p><p>Activity measurements confirm that both LaIHA and RsIHA have isatin hydrolase activity (Fig. 1d,f). The determination of the catalytic parameters assuming Michaelis-Menten kinetics yields K m values for LaIHA and RsIHA of 16 μM and 10 μM, respectively. Structural superposition revealed identical metal binding sites in the two homologues. Thus, a metal dependence analysis was only performed on LaIHA. LaIHA shows the highest activity in the presence of Mn 2+ confirming that this enzyme is Mn 2+ dependent. However, the relative activity of 15.1 ± 2.2% is also observed in the presence of Cd 2+ . Notably, LaIHA could not be activated in the presence of both Zn 2+ and Cu 2+ (Fig. 1e).</p><!><p>LaIHA, both the carbonyl group of the substrate and the catalytic water coordinate to the Mn 2+ ion. Like in other hydrolytic enzymes, the nucleophilic water is activated by a pre-organized base, which can efficiently extract a proton to form hydroxide (OH − ). In LaIHA, His207 acts as such base. The positively charged protonated His207-H + is stabilised by the presence of a hydrogen-bonded partner, i.e., Asp193. The water-His207-Asp193 H-bond network closely resembles the Ser-His-Asp triad common to serine proteases. His79, also H-bonded to the catalytic water, completes a complex His79-water-His207-Asp193 proton-shuttle system (Fig. 2b,d). Water deprotonation in LaIHA is extremely efficient and occurs spontaneously during the QM/MM simulations at room temperature 21 . Activation of the catalytic water is favoured by its isolation from the water channel that The manganese is found in octahedral coordination similarly to that described in 17 . Gln219 resides in a double conformation and only partially coordinates to the manganese. Also, W1025 is found in a double conformation (denoted A and B in Fig. 1). Note that Asp75 is coordinating bidentate in LaIHA:isatinate while monodentate in LaIHA: benzyl benzoate. connects the substrate cavity to the exterior. Confinement of the catalytic water has been reported as an important structural requirement in other hydrolytic (metallo)-enzymes [51][52][53][54][55] . Figure 3 depicts the molecular details of the hydrolytic reaction. The nucleophilic attack of OH − to the carbonyl of isatin leads to the formation of a tetrahedral intermediate. Our model predicts an activation barrier of a few (i.e., less than 6) kcal mol −1 . Thus, as in other metallohydrolases (i.e., zinc-β-lactamases), this step is not rate-limiting for the reaction. The tetrahedral intermediate is only metastable and it can easily recombine into the reactant state. In fact, the following separation of the scissile C-N bond constitutes the rate-limiting step of the reaction with a barrier height of ~12 kcal mol −1 .</p><p>Our data report that C-N separation occurs through a dissociative pathway. The transition state is stabilised by formation of two hydrogen-bonds (H-bonds), the first between the nucleophilic OH − and the Nδ atom of His79, and the second between the isatin N atom and the Nε-H moiety of His207 (Fig. 3, Supplementary movie). The final product formation of isatinate is accompanied by synchronous proton exchange in both of these two H-bonds. Our simulations indicate that the proton transfers occur after the transition state has been reached and the C-N bond is broken. Kinetic measurements in a deuterated environment did not reveal any observable kinetic isotope effect on the reaction, providing an additional indication that proton transfers are not involved in the rate-limiting step of the enzymatic process (data not shown).</p><p>The preference of a dissociative pathway over an associative or concerted mechanism may be a consequence of the fact that the N-atom in isatin acts as a poor base. In general, the π conjugation between the lone pair of N and the aromatic ring present in aryl-amines decreases the pKa value by few pH units. As a result, we observe that formation of a stable H-bond with His207, and consequently the proton transfer, occurs only upon a major elongation of the isatin C-N bond, which is associated to increased localization of the electron density at the N atom. Additional stabilization of the moiety into the product is obtained by the deprotonation of the nucleophile OH − group by His79 to produce a carboxylate group chelating the Mn 2+ as observed in the crystal structure.</p><p>The isatinate conformer produced by hydrolysis is not the most stable one. In fact, the α-carbonyl can isomerise via a bicycle motion, similar to that described in excited state isomerisation of retinal in rhodopsin 56 . This isomerised product found in our simulations is in agreement with the binding geometry of the product, isatinate, described in the crystal structure (Fig. 1b). In particular, the final binding state of isatinate shows bidentate coordination to the manganese ion, and similar stacking interactions with the indole rings of Trp59, Trp61, Trp80 and Tyr204 (Fig. 1b).</p><p>The strong exothermic isomerisation step (−10 kcal mol −1 ) yields a structure of isatinate with a strong intra-molecular H-bond between the amino and the α-carbonyl groups. This possible intermediate most likely constitutes the driving force of the reaction and traps the isatinate as a ligand to Mn 2+ in its product state before it is replaced by water and released from the binding pocket (Fig. 3).</p><!><p>Reaction mechanism describing the hydrolysis of isatin. The reaction is visualised from the initial nucleophilic attack of OH − to the carbonyl of isatin until the final substrate isatinate is formed. The final step involves isatinate isomerization, promoted by the strong intra-molecular H-bond between its amino and α-carbonyl groups, as well as the reformation of the hydrogen bond between Asp193 and His207. Free energy differences were estimated from metadynamics simulations.</p><p>Isatin hydrolase and kynurenine formamidase active-site conservation indicates a similar mechanism. The recently determined crystal structure of kynurenine formamidase from Bacillus anthracis (BaKynB) contains a binuclear Zn 2+ site and thus is part of the EC 3.5 class that has a similar overall protein fold when compared with LaIHA (sequence identity 24%). Figure 2d shows the superposition of the metal binding sites for LaIHA:isatinate and BaKynB (PDB ID: 4COG). The structural architecture of the metal binding sites is highly conserved between both enzymes (Fig. 2d). The most striking difference is the position of Gln219 in LaIHA, which is occupied by glutamate (Glu172) in BaKynB. This residue is conserved for all verified KynB enzymes. In BaKynB, the His60 (equivalent to His79 in LaIHA) is proposed to be necessary for the hydrolysis reaction to take place, by accepting a proton from the bi-coordinated activated water. In the mechanism for the isatin hydrolysis presented here, both His79 and His207 are involved in the catalytic mechanism (Supplementary Movie M1). In BaKynB, the Zn 2+ in position (I) Zn I 2+ is octahedrally coordinated and bound in an equivalent position as Mn 2+ in LaIHA. In a homologous structure of KynB from Burkholderia cenopacia (BcKynB), this position is occupied by Cd 2+ . The Zn 2+ in position (II), Zn II 2+ is also found in an octahedral conformation.</p><p>Convergent evolution of the active-site geometry in LaIHA and EcAAH. Amidohydrolases with known manganese dependency where identified through literature searches and examined manually for structural similarity. This approach revealed two candidates that, upon close inspection, shared metal binding sites residues and residues identified as catalytically important. The allantoate amidohydrolases (AAHs) are part of the purine degradation pathway and hydrolyse the allantoate to ureidoglycolate, carbon dioxide, and ammonium in the process, the allantoin pathway is found in both plants 13 and bacteria 57 (Fig. 2a,c). The identified structures were manually superposed with the active site of representatives from the AAH's, (Fig. 2e). The structure for E. coli AAH (EcAAH, PDB ID: 4PXD) was used as input for the DALI server 35 . The found hits were visually inspected for structural homology and included in alignment to confirm residue conservation (Fig. 2a).</p><!><p>The Baltic sea bacteria Labrenzia aggregata genome includes two homologous open reading frames for isatin hydrolases A and B that share 51% sequence identity (SI). The more distant homolog from Ralstonia solanacearum (RsIHA) shares 59% SI with LaIHA and 51% SI with LaIHB. Unlike the whole sequence, the active site and substrate binding pocket are highly conserved among these proteins. Even though the key substrate binding pocket residues originally identified in the structure of LaIHB -Phe63, Trp65 and Trp84 and Phe209 17 do not share equivalent positions in the sequences of LaIHA or RsIHA, their aromatic side chains do adopt an identical structural conformation in the respective binding pockets, thus indicating functional conservation (Fig. 1b). Compared to LaIHB, the Mn 2+ binding sites in both RsIHA and LaIHA include Gln219, also responsible for increased metal specificity 21 . The crystal structure of BaKynB from Bacillus anthracis (PDB ID: 4COG) presents a glutamate (Glu173, Fig. 2d) adopting an equivalent conformation as that of Gln219 in LaIHA. In conjunction with the conserved His161 and Asp56 (BaKynB numbering), it forms a secondary Zn 2+ site (Zn II ) binding site in the presented binuclear metal binding (Fig. 2d). The remaining metal-coordinating residues are identical to both RsIHA and LaIHA. It cannot be completely ruled out that, in certain conditions, Gln219 would allow the formation of a secondary metal-binding site in LaIHA. This is however unlikely, due to the different Lewis-acid/ base properties of an amide group, compared to those of a carboxylate. The structure of LaIHB, which features an alanine residue in the position of Gln219, does not have this option and thus is expected to be strictly mononuclear, with the highest hydrolytic activity measured in the presence of Mn 2+ , as also observed for LaIHA presented in this manuscript.</p><p>In the case of the amidohydrolase diaminopimelate desuccinylase (dapE), it was found that the compound L-captopril only binds to the Zn 2+ bound form of the enzyme and not to the physiologically relevant Mn 2+ bound form, thus stressing the importance of identifying the relevant metal 58 .</p><p>Different metallo-β-lactamases can bind and be catalytically active in the presence of both one and two Zn 2+ metals in the binding site. In both cases, the reaction mechanisms were characterized by computational modelling in the past (1 Zinc: B1 BcII from Bacillus cereus and B2 CphA from Aeromonas hydrophila, 2 Zinc: B1 CcrA from Bacteroides fragilis) [59][60][61] , the mono and binuclear reaction scheme for Zn 2+ binding beta-lactamases has recently been revised in Lisa MN et al. 6 . The mechanism of hydrolysis in LaIHA shares similarities with those found in single-Zn 2+ enzymes. In particular, the reaction proceeds through a two-step mechanism, where the nucleophilic attack on the amide carbonyl of isatin is followed by the breaking of the C-N bond. On the contrary, in CcrA from B. fragilis, the reaction follows a concerted mechanism where the nucleophilic attack of the hydroxyl takes place simultaneously with the opening of the β-lactam ring.</p><p>Common to all these enzymes is that the rate-limiting second step of the reaction is constituted by the dissociation of the C-N bond, and that the pathway to the product is facilitated by proton transfer on the N atom that usually occurs at the transition state or immediately after. The proton-shuttling pathways are nonetheless different in the Zn-bound enzymes and IH. The lone pair of the amidic nitrogen of isatin is π-conjugated with both the amidic carbonyl and the aromatic phenyl ring present in the molecular structure. As a consequence of the resulting electronic delocalisation, its basicity remains low even after the initial attack of the OH − and the subsequent formation of the tetrahedral intermediate. In fact, the evolution from the TS to the product requires both the pre-organised His207-Asp193 dyad shuttling one proton on the N atom and the stabilisation of C-N heterolytic breakage by a secondary proton transfer from the carboxylic group to His79. As a consequence, again differently from metallo-β-lactamases, the product formation in LaIHA leaves the protein in a higher protonation state than the initial complex. The deprotonation of the active site after the release of the product likely occurs through the water channel originally described for LaIHB 17 . Our in silico studies yield further support to the necessity for proton removal, in order to recover the catalyst, and allow further enzymatic cycles.</p><p>As the calculations produced a slightly endothermic profile (+2 kcal), the excess energy released by deprotonation of the protein may also constitute the driving force for the enzymatic process. Though, we stress that due to high computational costs, the free energy profile was obtained with a resolution that is of the same order as the reaction free energy balance.</p><p>The sequence alignment and structural superposition confirm that not only the metal coordinating residues (Fig. 2a-c, dots), but also the residues involved in the proton transfer (Fig. 2a-c, asterisk) are highly conserved. Only the position corresponding to LaIHA residue Asp193 (Fig. 2a) alternates between aspartate and glutamate, thus showing anyway functional conservation. The conservation suggests functional importance and, supported by the structural superposition and mechanistic elucidation, points to a common catalytic role. This is particularly interesting in the light of the sequence variation within the IH-like and AAH-like folds as illustrated in Fig. 2b,c,e. Here, the phylogenetic tree of proteins belonging to the respective folds indicates a large sequence variation, perhaps best exemplified by the significant divergence of EcAAH compared to Arabidopsis thaliana AAH (AtAAH), i.e., prokaryotic and eukaryotic. However, both the overall fold, displaying domain-swapped dimers, and the active site geometry are highly conserved (Fig. 2c-e).</p><p>It remains to be established whether the catalytic mechanism exemplified in LaIHA by our structural and in silico studies is specific to the IH-like enzymes, or could represent a broader case of convergent evolution across a range of AHS metallo-hydrolases. In the case of the AAH homologues, the Mn 2+ binding site is structurally completely conserved, including the binding residues in the Mn I site (His69, His73 and Asp75 for LaIHA), and the proton transfers triad (His79, His203 and Asp193) in LaIHA compared to Glu127, His382 and His226 in EcAAH, with the peculiar difference that in EcAAH the Mn 2+ occupies site II (See comparison in Fig. 2c,e). The IH, KynB and AAH families are not the only ones to have divergent metal site constellation, despite similar metal binding residues, the ureidoglycolate amidohydrolase from Arabidopsis thaliana (AtUAH) has a binuclear Mn 2+ site 62 (PDB ID 4PXB), while the putative hydantoinases (PDB ID 5I4M, 5THW and 4WJB) and β-alanine synthase from Saccharomyces kluyveri 63 (PDB ID 2VL1) have binuclear Zn 2+ sites, the L-N-carbamoylase from Geobacillus stearothermophilus has a mononuclear Co 2+ site 64 (PDB ID 3N5F). The model for hydrolysis presented here would only allow a Mn 2+ in site I, and a Zn 2+ would act as an inhibitor. In particular, according to previous simulations, Zn 2+ is not able to bind both isatin and the water, disrupting the preorganization of the active site and preventing the initial nucleophilic attack 21 . Thus, it remains an open question whether the Mn 2+ in site II also act as an inhibitor or merely adopt an alternative position in the structure of EcAAH due to the crystal conditions. It is a reoccurring problem for metal dependent hydrolases that the majority of the information is collected from in vitro experiments and that conflicting conclusions are reached when it comes to metal specificity. As in the case of KynB were a binuclear Zn 2+ is described in the structure 20 and Zn 2+ dependent activity is presented, however it still remains an open question whether this is the physiological metal, as in other systems such as Ralstonia metallidurans a 15 to 20-fold activation of kynurenine formamidase was detected after treatment with Mn 2+ 65 .</p><p>The major difference between the Michaelis complexes in LaIHA/B and zinc-β-lactamases is constituted by the binding mode of the substrate. β-lactam substrates, bind in the proximity of the metal(s), but their amide moiety do not participate directly to their coordination sphere(s) as is the case for the reacting cyclic amide in the IH. It is carboxylate groups of the carpapenem family, that later during the catalysis interact directly with the Zn 2+ ion as proposed is for several beta lactamases [6][7][8] including the more recent New Delhi metallo-β-lactamases 9,66,67 . In LaIHA it is the oxygen of the electrophilic amide carbonyl on isatin that is directly coordinated to the catalytic Mn 2+ ion (Fig. 3). The enzyme kinetics analyses, accompanying structural determination of LaIHA and RsIHA, present striking evidence that RsIHA, LaIHA, and LaIHB are true orthologues enzymes. These data support the claim in Bjerregaard-Andersen et al. 17 that isatin metabolism is present in bacteria native to the human gut and strengthen the hypothesis that the molecule isatin may be a signalling molecule that links the gut-brain axis under conditions such as stress 68,69 .</p>

Scientific Reports - Nature

Asymmetric Synthesis of All the Known Phlegmarine Alkaloids

The asymmetric synthesis of all four of the known natural phlegmarines and one synthetic derivative has been accomplished in 19 to 22 steps from 4-methoxy-3-(triisopropylsilyl)pyridine. Chiral N-acylpyridinium salt chemistry was used twice to set the stereocenters at the C-9 and C-2\xe2\x80\xb2 positions of the phlegmarine skeleton. Key reactions include the use of a mixed Grignard reagent for the second N-acylpyridinium salt addition, zinc/acetic acid reduction of a complex dihydropyridone, and a von Braun cyanogen bromide N-demethylation of a late intermediate. These syntheses confirmed the absolute stereochemistry of all the known phlegmarines.

asymmetric_synthesis_of_all_the_known_phlegmarine_alkaloids

Introduction<!>Results and Discussion<!>N\xce\xb2-Methylphlegmarine (1b) and N \xce\xb1-acetyl-N \xce\xb2-methylphlegmarine (1d)<!>Phlegmarine (1a), N \xce\xb1-methylphlegmarine (1c) and unnatural N\xce\xb1-methyl-N\xce\xb2-acetyl-phlegmarine (1e)<!>Experimental Section<!>2S-2-[(4aR,5S,7R,8aR)-1,2,3,4a,5,6,7,8,8a-Decahydroquinolin-1,7-dimethyl-5-ylmethyl)-1-[(benzyloxy)carbonyl]-2,3-dihydro-4-pyridone (4)<!>(2R)-2-[(R)-2-methyl-4-pentenyl]-1-(phenoxycarbonyl)-2,3,5,6-tetrahydro-4-pyridone (8)4c. Improved procedure<!>2S-2-[(4aR,5S,7R,8aR)-1,2,3,4a,5,6,7,8,8a-Decahydroquinolin-1,7-dimethyl-5-ylmethyl)-1-[(1R,2S]-2-((1-methyl-1-phenyl)ethyl)-cyclohexyloxyl-oxycarbonyl]-5-(triisopropylsilanyl)-2,3-dihydro-4-pyridone (19).4c Improved procedure<!>2S-2-[(4aR,5S,7R,8aR)-1,2,3,4a,5,6,7,8,8a-Decahydroquinolin-1,7-dimethyl-5-ylmethyl)-2,3-dihydro-4-pyridone (20).4c Improved procedure<!>2S-2-[(4aR,5S,7R,8aR)-1,2,3,4a,5,6,7,8,8a-Decahydroquinolin-1,7-dimethyl-5-ylmethyl)-1-[(benzyloxy)carbonyl]-4-piperidone (21)<!>Mixture of enol triflate isomers (22)<!>2S-2-[(4aR,5S,7R,8aR)-1,2,3,4a,5,6,7,8,8a-Decahydroquinolin-1,7-dimethyl-5-ylmethyl)-1-[(benzyloxy)carbonyl]piperidine (23)<!>2S-2-[(4aR,5S,7R,8aR)-1,2,3,4a,5,6,7,8,8a-Decahydroquinolin-1-cyano-7-methyl-5-ylmethyl)-1-[(benzyloxy)carbonyl]piperidine (26)<!>Phlegmarine (1a)<!>N\xce\xb2-Methylphlegmarine (1b)<!>N\xce\xb1-Methylphlegmarine (1c)<!>N\xce\xb1-Acetyl-N\xce\xb2-methylphlegmarine (1d)<!>N\xce\xb1-Methyl-N\xce\xb2-acetylphlegmarine (1e)<!>

<p>The lycopodiaceous plants have produced numerous and structurally interesting alkaloids which have proven to be challenging targets for total synthesis.1 One of the Lycopodium alkaloids, huperzine A, is a potential therapeutic agent for treatment of Alzheimer's disease.2 This medicinally important compound has spurred the isolation of several new Lycopodium alkaloids having various biological activities including cytotoxicity.3 The discovery of significant biological activities among the Lycopodium alkaloids has prompted renewed interest in the development of new synthetic strategies for their preparation.4 As part of our natural product synthesis program,5,6 we have examined approaches to the phlegmarine alkaloids.</p><p>The phlegmarines are a C16N2 skeletal group of Lycopodium alkaloids discovered by Braekman and coworkers in 1978.7a All four naturally occurring members of this group (1a–d) possess the same skeleton and differ only by their nitrogen atom substituents.7b Synthesis and spectroscopic structure studies carried out by Braekman's group7a determined the basic carbon skeleton, but it was not until the work of MacLean8 and co-workers that the relative stereochemistry of all five stereogenic centers of the phlegmarines was defined. The absolute stereochemistry of these alkaloids was established in our laboratories through the asymmetric total synthesis of (−)-Nα-acetyl-N-β-methylphlegmarine (1d).4c We report herein the total synthesis of all the known phlegmarine alkaloids 1a–d, and the Nβ-acetyl derivative 1e.</p><!><p>Our strategy for synthesizing the phlegmarines is depicted in Scheme 1. All of the alkaloid targets were to be prepared from the common dihydropyridone intermediate 4, which would arise from the key fragment 3 and chiral 1-acylpyridinium salt 2. Fragment 3 would also be prepared from the same antipode of 2 by a modification of our published procedure.4c</p><p>The Grignard of (R)-5-chloro-4-methylpentene9 was added to chiral N-acylpyridinium salt 2, prepared in situ from 4-methoxy-3-(triisopropylsilyl)pyridine10 and the chloroformate of (−)-trans-2-(α-cumyl)cyclohexanol (TCC),11 to give the crude N-acyldihydropyridone 5 in 90% yield and 88% de (Scheme 2). Purification by recrystallization from ethanol provided a 76% yield of the major diastereomer 5 as an isomerically pure white solid. A one-pot reaction of 5 with NaOMe/MeOH followed by aqueous 10% HCl furnished dihydropyridone 6 in 95% yield with 95% recovery of the chiral auxiliary, (−)-TCC. N-Acylation of 6 with n-BuLi and phenyl chloroformate gave a quantitative yield of enantiopure carbamate 7. Conjugate reduction of 7 can be effected with L-Selectride/BF3•OEt2 4c (86% yield) or more conveniently with Zn/AcOH12 to give 8 in 93% yield. Ozonolysis of the terminal alkene of 8 provided a high yield of aldehyde 9, which on acid-mediated cyclization13 was converted efficiently to bicyclic enone 10.</p><p>The next step required a facial selective conjugate addition of a nucleophile containing the latent functionality of a hydroxymethyl group. Based on our earlier model studies,14 (dimethylphenylsilyl)methylmagnesium chloride was chosen for use in a copper-mediated 1,4-addition to enone 10. The resulting facial selectivity was anticipated to be high based on conformational and stereoelectronic arguments. Due to A(1,3) strain,15 the reactive conformation is concave as shown in Figure 2. Stereoelectronically controlled axial attack of the nucleophile at C-5 would afford the desired stereochemical outcome. In the presence of copper iodide, addition of Grignard 11 to 10 and trapping of the resulting enolate with N-(5-(chloro-2-pyridyl)triflimide16 provided a 96% yield of vinyl triflate 12. Since protonation of the enolate leads to the cis-fused ring juncture,14 in situ vinyl triflate formation was necessary to set up the eventual incorporation of the required stereochemistry at C-10. The vinyl triflate function would not survive the subsequent hydrolysis of the carbamate group, so 12 was cleanly reduced to the more stable alkene 13 using Cacchi's conditions.17</p><p>Carbamate hydrolysis using KOH/2-propanol at reflux gave a high yield of the secondary amine 14. The use of 2-propanol was essential to obtaining a clean product, for the analogous reaction with KOH/ethanol gave a significant amount of the corresponding ethyl carbamate via carbamate exchange. Catalytic hydrogenation of 14 provided an 89/11 mixture of crude amines that were converted to Cbz carbamate 15 (78%) and the corresponding cis C-10 epimer. The trans selectivity can be attributed to significant shielding of the bottom face of the alkene in 14 by the axial (phenyldimethylsilyl)methyl group. Oxidation using Fleming's conditions18 and subsequent lithium aluminum hydride reduction gave amino alcohol 16 in high yield. Conversion to iodide 3 was effected using 1,2-bis-(triphenylphosphino)ethane (17) and I2.19 This method proved superior to the more common procedure using triphenylphosphine/I2 due to ease of product purification.</p><p>The key intermediate 4 was prepared as shown in Scheme 3. The mixed Grignard reagent 18 was prepared from iodide 3, by lithium-halogen exchange and addition of methylmagnesium iodide, and treated with 2 equiv of N-acylpyridinium salt 2 to give dihydropyridone 19 in 53% yield. The five stereocenters were correctly installed as determined by single-crystal X-ray analysis.4c The TIPS group and TCC auxiliary were removed from 19 in one step using our standard procedure to afford 20, which was converted to key intermediate 4 in high yield on lithiation and treatment with benzyl chloroformate. With the intermediate 4 in hand, the five target alkaloids were prepared as described below.</p><!><p>Conjugate reduction of 4 with zinc in acetic acid gave piperidone 21 in near quantitative yield (Scheme 4). Deprotonation with KHMDS and trapping with N-(5-chloro-2-pyridyl)triflimide provided the vinyl triflates 22 in a 3:1 ratio favoring olefin formation at the 4,′5′ position. Catalytic hydrogenation over Pearlman's catalyst afforded the natural product 1b, which exhibited spectral data and optical rotation in agreement with its assigned structure.</p><p>The alkaloid Nα-acetyl-Nβ-methylphlegmarine (1d) was prepared in one step from 1b by simple acetylation. Although the rotation of 1d was higher than that previously reported for the natural product, all spectral data were in agreement with literature values.7a</p><!><p>In order to prepare the remaining two natural phlegmarines (1a,c) and the known synthetic derivative 1e from intermediate 4, a demethylation of the beta nitrogen would be required toward the end of the synthesis. The vinyl triflate mixture 22 was reduced selectively via hydrogenation over platinum on carbon to afford the benzyl carbamate 23 (Scheme 5). Initial attempts to N-demethylate 23 with phenyl chloroformate proved problematic giving an inseparable mixture of dicarbamate 24 and the ring-opened product 25 as determined by 1H NMR and MS analysis. The use of other chloroformates20 led to similar results with a poor ratio of products regardless of temperature, solvent, or the addition of additives (LiBr, LiCl). The von Braun demethylation reaction using cyanogen bromide21 was examined next. To our delight, treatment of tertiary amine 23 with cyanogen bromide at rt gave a near quantitative conversion to cyanamide 26 (Scheme 6). When 26 was treated with dilute HCl at reflux, both the cyanamide and Cbz groups were hydrolyzed to the secondary amines providing natural phlegmarine (1a) in high yield. Nα-Methylphlegmarine (1c) was also prepared in excellent yield from 26 in one step by concomitant reduction of the cyanamide and carbamate groups with LAH. N-Acylation of 1c afforded Nα-methyl-Nβ-acetylphlegmarine (1e) in good yield. All three of our synthetic phlegmarines (1a,c,e) exhibited characterization data in agreement with literature values for the known compounds.</p><p>In summary, all four of the known phlegmarine alkaloids and one previoiusly reported synthetic derivative have been synthesized enantiopure from key dihydropyridone intermediate 4. These syntheses have confirmed the absolute stereochemistry of the phlegmarines as 2′S, 5S, 7R, 9R, 10R. Our chiral N-acylpyridinium salt chemistry was used twice during the synthetic route to set the stereocenter at C-9 and the remote center at C-2′ of the phlegmarines. The syntheses ranged from 19 to 22 steps and were accomplished with excellent stereocontrol.</p><!><p>The experimentals for compounds 5–10 and 12–17 have been previously published.4c</p><!><p>To a cooled (−78 °C) solution of dihydropyridone 20 (58.2 mg, 211 µmol) in THF (5.0 mL) was added n-BuLi (84 µL, 230 µmol, 1.1 equiv) dropwise. The solution immediately turned bright yellow and became heterogeneous. After 10 min the anion was rapidly quenched with freshly distilled benzyl chloroformate (60 µL, 420 µmol, 2.0 equiv). The solution was allowed to stir for an additional 2.5 h and then a 50% saturated aqueous solution of NaHCO3 (2.0 mL) was added. The aqueous phase was extracted with EtOAc (4 × 3.0 mL). The combined organic phases were washed with brine (5 mL), dried (MgSO4), filtered (Celite) and concentrated in vacuo. The crude oil was purified by radial PLC (silica gel, 75% EtOAc/hexanes, 2% TEA) to give 4 (82.7 mg, 96%) as a colorless oil. [α]D24 + 6.0 (c 0.81, MeOH); IR (film, NaCl) 2922, 1725 (C=O), 1672 (O-C=O), 1603 (C=C), 1327, 1192 cm−1; 1H NMR (300 MHz, CDCl3) δ 7.79 (d, J = 6.3 Hz, 1H) 7.40-7.37 (m, 5H), 5.35-5.31 (m, 2H), 5.21 (d, J = 11.7 Hz, 1H), 4.55 (br s, 1H), 2.81-2.71 (m, 2H), 2.45 (d, J = 16.5 Hz, 1H), 2.21 (s, 3H), 1.98-1.89 (m, 2H), 1.79 (m, 1H), 1.63-128 (m, 9H), 1.13-0.655 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 192.9, 152.5, 141.9, 135.0, 129.1, 129.0, 128.8, 107.2, 69.3, 62.7, 57.7, 52.2, 44.3, 43.0, 39.9, 38.3, 37.3, 34.5, 28.9, 26.6, 26.0, 25.6, 22.9; HRMS calcd for C25H34N2O3(M+H)+ 411.2648, found 411.2653.</p><!><p>To a rapidly stirred solution of dihydropyridone 7 (1.25 g, 4.18 mmol) in glacial acetic acid (38 mL) was added zinc powder (5.5 g, 84 mmol, 20 equiv) in small portions, to prevent the zinc from clumping. The mixture was allowed to stir for 17 h, filtered (Celite) and then concentrated in vacuo. The heterogeneous oil was purified by radial PLC (silica gel, 10–20% EtOAc/ hexanes) to provide piperidone 8 (1.17 g, 93%) as a colorless oil. Spectral data is identical to that previously reported.</p><!><p>The chiral N-acylpyridinium salt was prepared in situ by adding (−)-TCC chloroformate (253 mg, 901 µmol, 2.1 equiv) to a cooled (−45 °C) solution of 3-(triisopropylsilyl)-4-methoxypyridine (241mg, 901 µmol, 2.1 equiv) in toluene (27 mL). The solution was allowed to stir at −45 °C for 45 min followed by cooling to −78 °C before addition of the organometallic. The organometallic was prepared by dropwise addition of t-BuLi (559 µL, 944 µmol, 1.7 M in pentane, 2.2 equiv) to a cooled (−78 °C) solution of (4aR,5S,7R,8aR)-5-iodomethyl-1,7-dimethyl-1,2,3,4a,5,6,7,8,8a- decahydroquinoline4c (17) (132 mg, 429 µmol) in ether (9.0 mL). The solution was stirred for 45 min at −78 °C and then warmed to −42 °C (dry ice/acetonitrile) for 45 min. To the solution was added methylmagnesium iodide (154 µL, 429 µmol, 2.85M in ether, 1.0 equiv), and the solution was allowed to stir for an additional 10 min. This solution was added dropwise via cannula over 3 min to the cooled (−78 °C) N-acylpyridinium salt solution. This mixture was allowed to stir for 5 h at −78 °C followed by addition of 10% HCl(aq) (4 mL). The mixture was allowed to warm to rt and stirred for an additional 30 min. The aqueous phase was made basic with solid K2 CO3 and then extracted with EtOAc (5 × 3 mL). The combined organic phases were washed with brine (5 mL), dried (K2CO3), filtered (Celite), and concentrated in vacuo. The crude oil was purified by radial PLC (SiO2, 50% EtOAc/hexanes, 1% TEA) providing 19 (198 mg, 69%) as a mixture of isomers at the newly generated stereocenter; diasteriomeric excess was found to be 84% by HPLC. 1H NMR (400 MHz, CDCl3) δ 7.72 (s, 1H), 7.32-7.28 (m, 4H), 7.12 (t, J = 6.8 Hz, 1H), 4.89 (dt, J = 10.8, 4.4 Hz, 1H), 2.76 (d, J = 11.6 Hz, 1H), 2.64 (m,1H), 2.31 (dd, J = 15.6, 6.0 Hz, 1H), 2.19 (s, 3H), 2.05-1.99 (m, 4H), 1.88-1.74 (m, 3H), 1.61-1.19 (m, 23H), 1.06-1.01 (m, 24H), 071 (q, J = 12.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 196.8, 152.8, 152.2, 147.6, 128.3, 125.2, 125.1, 110.5, 77.8, 62.8, 57.7, 51.4, 50.7, 44.1, 43.0, 39.8, 39.5, 39.1, 37.5, 34.3, 33.8, 31.0, 28.9, 26.9, 26.8, 26.2, 26.0, 24.8, 23.0, 21.6, 19.0, 18.9, 11.3;</p><!><p>Sodium methoxide (734 µL, 3.2 mmol, 4.36 M in MeOH, 10 equiv) was added to a suspension of 19 (217 mg, 320 µmol) in methanol (20 mL). The mixture was brought to reflux affording a homogeneous solution. The solution was refluxed for 17 h, allowed to cool to rt, and concentrated in vacuo. The residual oil was taken up in THF (20 mL) and then 10% HCl(aq) (2.0 mL), was added. The mixture was allowed to stir for 3 h. The acidic mixture was carefully quenched with K2CO3(s) (~500 mg). Anhydrous K2CO3(s) (~5–7 g) was then added until the solution appeared to be dry. The clumps of K2CO3 were broken up with a glass rod, and the mixture was filtered through Celite. The filter pad was washed with hot EtOAc (5 × 20 mL), and the filtrate was concentrated in vacuo. The crude oil was purified by radial PLC (silica gel, 50–100% EtOAc/hexanes, 1% TEA) to give the dihydropyridone 20 (86.9 mg, 98%) as an oil that solidified upon standing, mp 141–142 °C (50% EtOAc/hexanes); 1H NMR (400 MHz, CDCl3) δ 7.16 (t, J = 7.6 Hz, 1H), 5.03 (d, J = 7.6 Hz, 1H), 4.72 (br s, 1H), 3.65 (m, 1H), 2.83 (br d, J = 11.2 Hz, 1H), 2.49 (dd, J = 16.0, 4.4 Hz, 1H), 2.54 (d, J = 16 Hz, 1H), 2.25 (s, 3H), 2.06 (m, 1H), 1.99 (m, 1H), 1.74-1.46 (m, 9H), 1.38 (m, 1H), 1.23 (m, 1H), 1.12 (dt, J = 13.0, 4.0 Hz, 1H), 0.91 (d, J = 6.4 Hz, 3H), 0.76 (q, J = 12.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 192.9, 151.6, 98.7, 62.8, 57.6, 52.4, 44.2, 42.9, 41.5, 39.8, 38.5, 35.0, 32.1, 29.1, 26.0, 25.9, 22.9.</p><!><p>Zinc powder was added slowly to a rapidly stirred solution of 4 (24.6 mg, 60 µmol) in glacial acetic acid (1.0 mL). The reaction mixture was allowed to stir for 11 h, and then filtered through Celite, and the filter pad was washed with methanol (5 mL). The filtrate was concentrated in vacuo. The resulting amorphous solid was suspended in dichloromethane (5 mL) and passed through a plug of basic alumina (activity 2). The plug was washed with a 10% solution of methanol in dichloromethane (5 × 5 mL). The solution was concentrated in vacuo to provide a colorless oil. The crude material was purified by flash chromatography on silica gel (50% EtOAc/hexanes, 1% TEA) to provide 21 (24.6 mg, 99%) as a colorless oil. [α]D23 −49 (c 0.69 , CHCl3); IR (film, NaCl) 2925, 2775 (N-Me), 1721 (C=O), 1698 (O-C(=O)N), 1422, 1233, 1104, 1005 cm−1; 1H NMR at 50 °C (300 MHz, CDCl3) δ 7.37-7.31 (m, 5H), 5.24 (d, J = 9.0 Hz, 1H), 5.13 (d, J = 9.0 Hz, 1H), 4.59 (br s, 1H), 4.38 (dd, J = 9.6, 6.0 Hz, 1H), 3.33 (ddd J = 11.2, 8.5, 2.9 Hz, 1H), 2.81 (br d, J = 8.4 Hz, 1H), 2.60 (dd, J = 10.8, 4.8 Hz, 1H), 2.50 (ddd, J = 11.5, 8.5, 5.2 Hz, 1H), 2.34 (br d, J = 10.8 Hz, 1H), 2.22 (s, 3H), 2.04-1.94 (m, 2H), 1.64-1.42 (m, 9H), 1.30 (m, 1H), 1.20-1.12 (m, 1H), 1.03 (m, 1H), 0.83 (d, J = 4.2 Hz, 3H), 0.70 (q, J = 9.0 Hz, 1H); 13C NMR at 50 °C (100 MHz, CDCl3) δ 207.5, 155.5, 136.7, 128.8, 128.5, 128.4, 68.0, 63.1, 57.9, 52.3, 44.6 (2C, determined by 13C NMR at 70 °C in benzene-d6 δ 45.5, 44.3), 43.0, 40.8, 40.1, 39.4, 39.1, 35.7, 31.2, 29.3, 26.2 (2C, determined by 13C NMR at 70 °C (in benzene-d6 δ 26.9, 26.4), 22.9; HRMS calcd for C25H36N2O3 (M+H)+ 413.2804, found 413.2797.</p><!><p>To a cooled (−78 °C) solution of piperidone 21 (10.8 mg, 26.3 µmol) and 2-(5-chloropyridyl)triflimide (33 mg, 84 µmol, 3.2 equiv) in THF (2.0 mL) was added potassium bis(trimethylsilyl)amide (3 × 56 µL, 84 µmol, 0.5M in toluene, 3 equiv) dropwise in three portions. The reaction mixture was allowed to stir at −78 °C for 30 min, saturated aqueous NaHCO3 (1 mL) was rapidly added, and the mixture was allowed to warm to rt. The aqueous phase was diluted with water until it became homogeneous (1 mL). The aqueous phase was extracted with EtOAc (5 × 1 mL). The combined organic phases were washed with brine (1 mL), dried (Na2SO4), filtered (Celite), and concentrated in vacuo. The crude oil was purified by flash column chromatography with basic alumina (activity 2, elution with 5% MeOH/CH2Cl2), and then a second column with silica gel (elution with 50% EtOAc/hexanes) to afford the product (13.8 mg, 96%) as an inseparable mixture of enol triflate isomers 22 (ratio of 3,4- to 4,5-enol triflate found to be 1.0 to 3.1 by 1H NMR). 1H NMR at 50 °C (400 MHz, CDCl3) δ 7.33 (br s, 5H, mixture of isomers), 5.81 (s, 1H, minor isomer), 5.75 (s, 1H, major isomer), 5.25-5.07 (m, 2H, mixture of isomers), 4.57-4.38 ( m, 2 H, mixture of isomers), 3.08 (m, 1H, minor isomer), 2.80 (d, J = 10 Hz, major isomer), 2.74-2.61 (m, 1H, mixture of isomers), 2.22-2.22 (m, 7H, mixture of isomers), 2.01-1.94 (m, 4H, mixture of isomers), 1.65-1.02 (m, 22H, mixture of isomers), 0.88-0.67 (m, 7H, mixture of isomers); HRMS calcd for C26H35F3N2O5S(M+H)+ 545.2297, found 545.2310.</p><!><p>A solution of the mixed triflates 22 (13.8 mg, 25 µmol), 5% platinum on carbon (14 mg) and lithium carbonate (18 mg, 240 µmol) in EtOAc was stirred under an atmosphere of hydrogen gas for 5.75 h. The solution was filtered through a plug of Celite and then concentrated in vacuo. The crude oil was purified by flash column chromatography (silica gel, 50% EtOAc/hexanes, 1% TEA) providing piperidine 23 (8.3 mg, 84%) as a colorless oil. [α]D24 –20.6 (c 0.65, MeOH ); IR (film, NaCl) 2930, 1694 (C=O), 1422, 1259 cm−1; 1H NMR (300 MHz, CDCl3) δ 7.35-7.25 (m, 5H), 5.18 (d, J = 12.3 Hz, 1H), 5.07 (d, J = 12.3 Hz, 1H), 4.23 (br s, 1H), 4.06 (br d, J = 13.2 Hz, 1H), 2.23-2.80 (m, 2H), 2.23 (s, 3H), 2.03-1.95 (m, 2H), 1.75-1.22 (m, 17H), 0.97 (dt, J = 13.4, 3.9 Hz, 1H), 0.82 (d, J = 6.0 Hz, 3H), 2.75 (q, J = 11.9 Hz, 1H); 13C NMR at 50 °C (75 MHz, CDCl3) δ 155.7, 137.2, 128.7, 128.2, 128.1, 67.2, 63.0, 57.8, 49.9, 44.7, 43.1, 40.1, 39.6, 38.5, 35.6, 29.2, 27.2, 27.0, 26.3, 26.0, 25.7, 23.1, 18.8; TLC Rf = 0.26 (30% EtOAc/hexanes, basic alumina); HRMS calcd for C25H38N2O2 (M+H)+ 399.3012, found 399.3010.</p><!><p>To a cooled (0 °C) solution of 23 (8.3 mg, 21 µmol) and lithium carbonate (2.1 mg, 21 µmol, 1 equiv) in chloroform (1.0 mL) was added cyanogen bromide (10 µL, 31 µmol, 3M solution in CH2Cl2, 1.5 equiv). The solution was allowed to stir at 0 °C for 30 min, warmed to rt, and concentrated in vacuo. The crude oil was purified by flash column chromatography (silica gel, 20% EtOAc/hexanes) to give 26 (8.2 mg, 96%) as a colorless oil. [α]D23 –30 (c 0.79, CHCl3); IR (film, NaCl) 2929, 2205 (C≡N), 1693 (CO), 1454, 1422, 1262 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.38-7.26 (m, 5H), 5.19 (d, J = 12.0 Hz, 1H), 3.05 (d, J = 12.0 Hz, 1H), 4.22 (br s, 1H), 4.07 (br d, J = 13.2 Hz, 1H), 3.41 (br d, J = 12.8 Hz, 1H), 2.95 (td, J = 11.4, 3.6 Hz, 1H), 2.85 (t, J = 12.8 Hz, 1H), 2.69 (br s, 1H), 2.01 (br d, J = 9.2 Hz, 1H), 1.67-1.25 (m, 16H), 1.07-0.85 (m, 5H); 13C NMR (75 MHz, CDCl3) δ 155.6, 137.0, 128.7, 128.3 (br s, 2C), 117.1, 67.3, 57.1, 51.3, 49.7, 43.9, 39.6, 39.2, 38.4, 35.2, 27.9, 26.9, 26.6, 26.0, 25.6, 25.2, 22.5, 18.8; TLC Rf = 0.21 (30% EtOAc/hexane); HRMS calcd for C25H35N3O2 (M+H)+ 410.2808, found 410.2814.</p><!><p>A mixture 26 and 6M HCl(aq) (1.0 mL) was heated at reflux for 3 h. The solution was cooled to rt and then extracted with ether (3 × 0.5 mL). The organic phase was discarded and the aqueous phase was made basic by careful addition of solid K2CO3. More potassium carbonate was added until the solution became saturated. The aqueous phase was then extracted with EtOAc (14 × 1.0 mL) until the extracts no longer contained product by tlc. The crude solid was purified by flash chromatography (basic alumina (activity 2), MeOH) providing 1a as a solid. The solid was then taken up in hexanes (1.0 mL) and passed through a plug of cotton providing phlegmarine (1a) (4.8 mg, 96%) as a solid. [α]D24 -29 (c 0.39, CHCl3); IR (film, NaCl) 3230 (N-H), 2923, 2852, 1120, 743 cm−1; 1H NMR (400 MHz, CDCl3) δ 3.08-2.99 (m, 2H), 2.63 (dt, J = 11.8, 2.7 Hz, 1H), 2.56 (dt, J = 11.9, 2.5 Hz, 1H), 2.45-2.35 (m, 2H), 1.86-1.46 (m, 11H), 1.45-1.23 (m, 5H), 1.07 (tt, J = 11.2, 3.8 Hz, 1H), 1.05 (dt, J = 12.4, 4.0 Hz, 1H), 0.99-0.90 (m, 2H), 0.88 (d, J = 6.0 Hz, 3H), 0.80 (q, J = 13.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 56.1, 55.5, 47.5, 47.0, 46.1, 43.2, 39.7, 35.5, 35.2, 32.5, 28.9, 27.6, 27.0, 26.1, 25.0, 22.8; LRMS: 250 (M+, 7), 235 (5) 167 (12), 150 (14), 124 (6), 110 (9), 97 (24), 84 (100), 70 (90) 56 (10), 44 (80) m/z; HRMS calcd for C16H30N2 (M+H)+ 251.2487, found 251.2485.</p><!><p>A solution of the mixed enol triflates 22 (17.7 mg, 32.5 µmol) and 20% palladium hydroxide on carbon (17.7 mg) in ethanol (1.0 mL) was stirred under an atmosphere of hydrogen gas for 6 h. The solution was filtered (Celite) and solid K2CO3 (100 mg) was added to the filtrate. The suspension was allowed to stir for 1 h, again filtered (Celite), and concentrated in vacuo. The residual oil was purified by flash chromatography (basic alumina (activity 2), 2% MeOH/CH2Cl2) to provide Nβ-methylphlegmarine (1b) (7.7 mg, 90 %) as a colorless oil. [α]D22 -65 (c 0.39 , CHCl3); IR (film, NaCl) 3276 (N-H), 2925, 2774 (N-Me), 1455, 1331, 1118, 1006 cm−1; 1H NMR (400 MHz, CDCl3) δ 3.06 (br d, J = 11.6 Hz, 1H) 2.82 (br d, J = 11.2 Hz, 1H), 2.63 (dt, J = 11.6, 2.8Hz, 1H), 2.41 (m, 1H), 2.24 (s, 3H), 2.06-2.00 (m, 2H), 1.80-1.58 (m, 9H), 1.45 (m, 8H), 1.04 (dt, J = 12.7, 4.6 Hz, 1H), 0.96 (m, 1H), 0.89 (d, J = 6.8 Hz, 3H), 0.72 (q, J = 11.9 Hz); 13C NMR (100 MHz, CDCl3) δ 63.0, 57.7, 56.1, 47.4, 44.6, 42.9, 39.9, 39.2, 35.5, 35.4, 32.3, 29.1, 26.8 (two carbons), 26.8, 24.9, 23.4; TLC Rf = 0.13 (5% MeOH/CH2Cl2, basic alumina); LRMS: 264 (M+, 20), 249 (22), 207 (16), 180 (12), 166 (55), 164 (44), 150 (10), 124 (40), 111 (38), 97 (28), 84 (100), 44 (68) m/z; HRMS calcd for C17H32N2 (M+H)+ 265.2644, found 265.2654.</p><!><p>Lithium aluminum hydride (400 µL, 400 µmol, 1.0 M in THF, 12 equiv) was added dropwise slowly into a solution of 26 (13.7 mg, 33.5 µmol) in THF (6.0 mL). The solution was then heated at reflux for 3 h, cooled to 0 °C, and water (10 µL) was added carefully followed by a 25% solution of NaOH (20 µL). The solution was warmed to rt and Celite (0.5 g) was added. After stirring for 1.5 h, the mixture was filtered through Celite, and the filter cake was washed with hot EtOAc (3 × 10 mL). Concentration of the filtrate gave a crude heterogeneous oil which was purified by flash column chromatography (basic alumina (activity 2), stepwise gradient from 75–100% EtOAc/hexanes then 5% MeOH/CH2Cl2) to afford Nα-methylphlegmarine (1c) (8.4 mg, 94%) as an oil. [α]D22 –77 (c 0.42, CHCl3); IR (film, NaCl) 3364 (N-H), 2926, 2777 (N-Me), 2852, 1455, 1371, 1026 cm−1; 1H NMR (300 MHz, CDCl3) δ 3.01 (br d, J = 12.2 Hz, 1H), 2.83 (br d, J = 11.4 Hz, 1H), 2.57 (dt, J = 12.2, 2.5 Hz, 1H), 2.42 (dt, J = 10.6, 3.6 Hz, 1H), 2.27 (s, 3H), 2.11 (dt, J = 11.4, 5.2 Hz, 1H), 1.84-1.4 (m, 15H), 1.39-1.05 (m, 4H), 1.00 (br t, J = 10 Hz, 1H), 0.89 (d, J = 6.4 Hz, 3H), 0.81 (q, J = 11.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 62.2, 57.3, 55.5, 47.1, 46.4, 43.4, 43.3, 38.4, 35.2, 30.7, 29.3, 28.9, 27.8, 26.2, 25.9, 24.3, 23.0; TLC Rf = 0.18 (5% MeOH/CH2Cl2, basic alumna); LRMS: 264 (M+, 0.07), 249 (0.5), 150 (5), 134 (1), 122(1), 110 (3), 98 (100), 82 (2), 70 (4), 54 (2), 44 (23) m/z; HRMS calcd for C17H32N2 (M+H)+ 265.2644, found 265.2643.</p><!><p>To a cooled solution (0 °C) of Nβ-methylphlegmarine (1b) (6.9 mg, 26 µmol) and pyridine (6.3 µL, 78 µmol) in dichloromethane (1.0 mL) was added acetyl chloride (5.6 µL, 78 µmol) dropwise. The solution was stirred at 0 °C for 30 min, warmed to rt for an additional 30 min, and then saturated NaHCO3 (0.5 mL) was added. The aqueous phase was extracted with EtOAc (5 × 0.5 mL), and then the combined organic phases were concentrated in vacuo to give a colorless oil. The crude material was purified by flash chromatography over basic alumina (activity 2, 2% MeOH/CH2Cl2) providing Nα-acetyl-Nβ-methylphlegmarine (1d) (7.6 mg, 95%) as a colorless oil. [α]D23 -75 (c 0.37, CHCl3); lit.7 [α]D - 11 (c 0.7, CHCl3) ; IR (film, NaCl) 2929, 2776 (NCH3), 1643 (C=O), 1424, 1263, 1005 cm−1; 1H NMR (300 MHz, CDCl3) δ 4.75 and 3.80 (2 br s due to rotamers, total 1H), 4.54 and 3.59 (2 br d due to rotamers, J = 13.0 Hz, total 1H), 3.15 and 2.63 (2 tt due to rotamers, J = 3.2, 13.2 Hz, total 1H) 2.83 (br d, J = 8.4 Hz, 1H), 2.25 and 2.23 (2 s due to rotamers, total 3H), 2.08 and 2.07 (2 s due to rotamers, total 3H), 2.02-1.90 (m, 3H), 1.68-1.24 (m, 18H), 1.09 and 1.02 (2 dt due to rotamers, J = 13.0, 3.8 Hz, total 1H), 0.92 (apparent t due to rotamers, J = 5.8 Hz, 3H), 0.75 and 0.71 (2 q due to rotamers, J = 11.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 169.0, 63.1 and 63.0 (doubled due to rotamers), 57.9 and 57.7 (doubled due to rotamers), 52.8 and 47.1 (doubled due to rotamers), 44.7 and 44.6 (doubled due to rotamers), 43.1, 42.3, 40.1 and 38.9 (doubled due to rotamers), 38.4 and 36.9 (doubled due to rotamers), 36.0 and 35.8 (doubled due to rotamers), 29.2, 27.3 and 27.2 (doubled due to rotamers), 26.8, 26.4 and 26.3 (doubled due to rotamers), 26.3 and 26.0 (doubled due to rotamers), 26.2 and 25.5 (doubled due to rotamers), 23.2 and 23.0 (doubled due to rotamers), 22.4 and 21.8 (doubled due to rotamers), 19.0 and 18.9 (doubled due to rotamers); TLC Rf = 0.23 (2.5% MeOH/CH2Cl2, basic alumina); LRMS: 306 (M+, 10), 291(7), 264 (1), 263 (4), 250 (2), 249 (11), 206 (3), 181 (1), 180 (6), 167 (13), 166 (100), 164 (7), 127 (3), 126 (32), 124 (5), 123 (4), 98 (2), 97 (10), 96 (8); HRMS calcd for C19H34N2O (M+H)+ 307.2749, found 307.2750.</p><!><p>To a cooled (0 °C) solution Nα-methylphlegmarine (1c) (8.4 mg, 32 µmol) and pyridine (8.6 µL, 110 µmol, 3.5 equiv) in dichloromethane (1.0 mL) was added acetyl chloride (7.5 µL, 110 µmol) dropwise. The solution was allowed to stir for 30 min at 0 °C and then warmed to rt for an additional 30 min. Saturated NaHCO3 (1.0 mL) was added and the phases were separated. The aqueous phase was extracted with dichloromethane (5 × 1 mL), and the combined organic phases were washed with brine (1.0 mL), dried (Na2SO4), filtered (Celite) and concentrated in vacuo. The crude oil was purified by flash column chromatography (basic alumina (activity 2), stepwise elution with 50–100% EtOAc/hexanes then 10% MeOH/CH2Cl2) providing Nα-methyl-Nβ-acetylphlegmarine (1e) (8.2 mg, 84%) as a colorless oil. [α]D23 –192 (c 0.40, CHCl3); IR (film, NaCl) 2928, 2777 (N-Me), 1640 (C=O), 1439, 1247, 1027 cm−1; 1H NMR at 50 °C (400 MHz, CDCl3) δ 3.71 (br m, 2H), 3.11 (br m, 1H), 2.82 (dt, J = 11.6, 3.4 Hz, 1H), 2.27 (s, 3H), 2.13 (dt, J = 11.6, 6.8 Hz, 1H), 2.05 (s, 3H), 2.03 (m, 1H), 1.92-1.55 (m, 13H), 1.42 (m, 1H), 1.35-1.02 (m, 6H), 0.94-0.85 (m, 1H), 0.91 (d, J = 6.4 Hz, 3H); 13C NMR at 50 °C (100 MHz, CDCl3) δ 169.9, 62.2, 56.8, 54.8, 43.2, 41.5, 39.6, 38.6, 37.9, 35.4, 30.7, 28.5, 26.8, 25.9, 24.1, 23.1, 23.0, 22.5, 22.1; TLC Rf = 0.29 (2.5% MeOH/CH2Cl2, basic alumina); LRMS: 306 (M+, 0.96), 291 (0.41), 263 (1.6), 207 (1.9), 192 (1.3), 150 (5.2), 110 (2.8), 98 (100), 70 (5.1), 55 (3.0), 44 (49) m/z; HRMS calcd for C19H34N2O (M+H)+ 307.2749, found 307.2747.</p><!><p>Structures of the four known phlegmarine alkaloids and derivative 1e.</p><p>Calculated lowest energy conformation of 10 (MMFF).</p>

PubMed Author Manuscript

Effects of pH on proteins: Predictions for ensemble and single molecule pulling experiments

Protein conformations change among distinct thermodynamic states as solution conditions (temperature, denaturants, pH) are altered or when they are subject to mechanical forces. A quantitative description of the changes in the relative stabilities of the various thermodynamic states is needed to interpret and predict experimental outcomes. We provide a framework based on the Molecular Transfer Model (MTM) to account for pH effects on the properties of globular proteins. The MTM utilizes the partition function of a protein calculated from molecular simulations at one set of solution conditions to predict protein properties at another set of solution conditions. To take pH effects into account we utilized experimentally measured pKa values in the native and unfolded states to calculate the free energy of transferring a protein from a reference pH to the pH of interest. We validate our approach by demonstrating that the native state stability as a function of pH are accurately predicted for CI2 and protein G. We use the MTM to predict the response of CI2 and protein G subject to a constant force (f) and varying pH. The phase diagrams of CI2 and protein G as a function of f and pH are dramatically different, and reflect the underlying pH-dependent stability changes in the absence of force. The calculated equilibrium free energy profiles as function of the end-to-end distance of the two proteins show that, at various pH values, CI2 unfolds via an intermediate when subject to f. The locations of the two transition states move towards the more unstable state as f is changed, which is in accord with the Hammond-Leffler postulate. In sharp contrast, force-induced unfolding of protein G occurs in a single step. Remarkably, the location of the transition state with respect to the folded state is independent of f, which suggests that protein G is mechanically brittle. The MTM provides a natural framework for predicting the outcomes of ensemble and single molecule experiments for a wide range of solution conditions.

effects_of_ph_on_proteins:_predictions_for_ensemble_and_single_molecule_pulling_experiments

Introduction<!>Molecular Transfer Model for pH effects on proteins under tension<!>Coarse-Grained models for proteins<!>Simulation details<!>Analysis<!>The Molecular Transfer Model for pH effects on proteins<!>The Molecular Transfer Model accurately models pH denaturation<!>Force-pH and force-temperature phase diagrams<!>Force midpoints<!>pH-dependent free energy profiles<!>pH and temperature dependent movements in transition state location suggest Hammond-Leffler behavior<!>Discussion

<p>Recent experimental advances, especially in single molecule techniques1,2, have made it possible to obtain detailed mechanistic insights into the folding of proteins over a wide range of external conditions. For example, singe molecule FRET (smFRET) experiments have been used to probe the characteristics of the ensemble of unfolded states under folding conditions, providing a glimpse of the nature of the collapse transition in proteins3. Developments in single molecule force spectroscopy, which probe the response of proteins subject to mechanical force (f), have been used to map the entire folding landscape of proteins described by the end-to-end distance of the molecule4–6. These experiments have been remarkably successful in measuring the roughness of the energy landscape7, estimating barriers to folding8, and characterizing minimum energy compact structures9,10 that are sampled during the folding process. In the majority of the smFRET experiments, folding or unfolding is initiated using chemical denaturants11,12, whereas in pulling experiments external force is applied to select points on the protein to control folding. More recently, the global response of proteins to f in the presence of osmolytes and denaturants, and pH changes have also been reported10,13. The wealth of data emerging from these studies demand computational models for which exhaustive simulations at conditions that mimic those used in experiments can be performed14.</p><p>Besides denaturants15–17, protein folding or unfolding can also be initiated by altering pH18. Although a number of experimental studies have reported the pH-dependence of protein stability19–22 there are very few theoretical approaches which have addressed the thermodynamic aspects of pH-dependent folding. In principle, all atom molecular dynamics simulations can be used to model pH-dependent effects on protein folding. Such an approach has found success in predicting and interpreting pKa values of titratable groups within the native and unfolded ensembles23–25. However, calculating other pH-dependent protein properties using all-atom models is difficult because of inaccuracies in force-fields26 and the inability to adequately sample both the folded and denatured conformational space.</p><p>One of the most widely used thermodynamic models for pH effects on proteins was created by Tanford and coworkers, who showed that from knowledge of the pKa values of titratable groups it is possible to predict the change in protein native state stability as a function of pH17. This thermodynamic model cannot be used to calculate the distribution of pH-dependent properties of proteins because it neglects the ensemble nature of folding. It is limited to making estimates of changes in the stability and the difference in the number of bound protons between the native and unfolded states.</p><p>The limitations of the thermodynamic model can be overcome using the Molecular Transfer Model (MTM), which we originally introduced to account for osmolyte effects on proteins27. Although the MTM can be used in conjunction with all-atom models for proteins, we used a coarse-grained representation of proteins27,28 so that the partition function of the system could be precisely computed at a given solution condition. Knowledge of the partition function can be used to compute any thermodynamic property at another solution condition by appropriate reweighting. The MTM utilizes the Tanford Model17,29 to estimate the free energy cost of transferring each microstate (i.e., protein conformation) from one solution condition to another. Thus, the MTM is a post-simulation processing technique, which allows for a rapid prediction of the thermodynamic properties of proteins under a wide range of external conditions by performing simulations at one solution condition27,30,31.</p><p>Here, we further develop the MTM to model pH effects on protein properties. We validate our approach by demonstrating that the methodology accurately predicts experimentally measured changes in the native state stability as function of pH. We further establish the efficacy of the MTM by calculating the pH-dependent response of proteins subject to an external mechanical force, f. Simulations at constant force and varying pH for chymotrypsin inhibitor 2 (CI2)32,33 and protein G34,35 show dramatically different behavior. Both the diagram of states as a function of f and pH and the free energy profiles depend on the protein. Our results are consistent with experimental data from a constant pulling speed force experiment36 and offer a number of testable predictions.</p><!><p>The MTM27 utilizes protein conformations from Cα side chain model (Cα – SCM) coarse grained simulations (see below), experimentally measured or theoretically computed amino acid transfer free energies and the Weighted Histogram Equations37 (WHAM) to predict how changes in external conditions alter the thermodynamic properties of a protein. The MTM equation for predicting the average of a quantity A at a given pH, temperature, and f value is</p><p> (1)〈A(pH2,T,f)〉=Z(pH2,T,f)−1∑k=1R∑t=1nkAk,te−βEP(k,t,pH2,f)∑m=1RnmeFm−βmEm(k,t)−fmx(k,t), where Z(pH2, T, f) is the partition function. In Eq. 1, R is the number of independent simulated trajectories, nk is the number of protein conformations in the kth simulation, Ak,t is the value of property A for the tth conformation, β = 1/kBT, where kB is Boltzmann constant and T is the temperature. The potential energy EP of the tth conformation in the kth simulation at a pH value denoted pH2, and under external force f is EP (k, t, pH2, f) = EP (k, t, pH1) + Gtr(k, t, pH2) − fx(k, t), where EP (k, t, pH1) is the potential energy of the system at pH = pH1, ie. the pH conditions at which the simulations are carried out in this study (see below). The free energy, ΔGtr(k, t, pH2), of transferring the tth conformation in the kth simulation from a solution at pH1 to a solution at pH2. f is the external pulling force and x is the end-to-end distance vector of the protein projected onto the direction of the applied force. In the denominator of Eq. 1, nm and Fm are, respectively, the number of conformations and the free energy of the mth simulation. The values of Fm are obtained self consistently at the simulated solution conditions as described in reference37.</p><p>To estimate ΔGtr(k, t, pH2) we use a model developed by Tanford and coworkers17 in which the free energy of transferring a titratable group p in conformation l of the protein from pH1 to pH2 is</p><p> (2)δgp,l=−kBTln[10pH2+ΘN(l)10pKN,p+ΘD(l)10pKD,p10pH1+ΘN(l)10pKN,p+ΘD(l)10pKD,p], where ΘN (l) and ΘD(l) are Heaviside step functions that identify a conformation l as being either native or denatured. ΘN (l) (ΘD(l)) equals 1 if conformation l is native (denatured) and 0 otherwise. pKN,p and pKD,p are the pKa values for group p in the native and denatured states respectively. We use pKN,p values that have been determined experimentally34,38 (see Table S1). Details on classifying conformations as native and denatured are given below. Finally ΔGtr(k,t,pH2)=∑p=1Npδgp,l, which is the sum of the δgp,l values that are calculated using Eq. 2.</p><!><p>We model the 65 residue long protein CI2, and 56 residue protein G using the Cα-SCM27,28 in which each amino acid is represented as two interaction sites, one of which is located at the α carbon position of the backbone. For all amino acids except glycine, the other interaction site is located at the center-of-mass of the SC. We use a Go model version39 of the Cα-SCM. Thus, side chains that are in contact or backbone groups that form hydrogen bonds in the crystal structure have attractive non-bonded Lennard-Jones interactions while all other non-bonded interactions are repulsive. Sequence dependent effects are modeled using non-bonded interaction parameters based on the Miyazawa-Jernigan statistical potential40. The excluded volume of an amino acid side chain is proportional to its experimentally measured partial molar volume in solution.</p><p>The potential energy EP of a Cα-SCM conformation is EP=EA+EHB+ENBN+ENBNN, which is the sum of potential energy terms corresponding to angles (EA), hydrogen bonds (EHB), and native ( ENBN) and non-native ( ENBNN) non-bonded interactions. We use the Shake algorithm41 to hold the bond lengths fixed in the simulations, hence there is no energy term corresponding to this constraint. The functional forms of the terms in the Cα-SCM force field are</p><p>On the right hand side (RHS) of Eq. 3, and from left to right, the summation corresponds, respectively, to bond angle, dihedral angle and improper dihedral angle energy terms. The improper dihedral term is used to model chirality about the Cα interaction site. On the RHS of Eq. 4 we model hydrogen bonds found in the crystal structure as a Lennard-Jones potential (first term) with a well-depth set to εHB and the positions of the minima rHB,i0 are set by the interaction site distance in the crystal structure. The second summation term in Eq. 4 accounts for native interactions between sites and is treated using the Lennard-Jones potential whose well-depth is set using the Miyazawa-Jernigan statistical potential and minima location rmin,i corresponds to the distance in the crystal structure. Non-native interactions (Eq. 5) between sites are treated as short-ranged and repulsive, with the rmin,i being proportional to the experimentally measured partial molar volume of the amino-acid type. The force-field parameters used for CI2 and protein G are in Table S2, and additional details on the model can be found in our previous study27. We use the crystal structures with PDB codes 2CI242 and 1GB143 for CI2 and protein G, respectively.</p><!><p>We use Hamiltonian Replica Exchange (HREX)44,45 in the canonical (NVT) ensemble to obtain equilibrium simulations of CI2 and protein G at constant force (f) applied in the positive x-direction to the C-terminal Cα interaction site of the protein. In the simulations, the N-terminal Cα interaction site is fixed at the origin. In the HREX simulation independent trajectories (replicas) are simulated at different temperatures and at different f values using Langevin dynamics46 with a damping coefficient of 0.8 ps−1 and an integration time-step of 6 fs. The non-bonded interactions were truncated at 20 Å with a switch function applied starting at 18 Å. We use CHARMM (version c33b2) to simulate the time evolution of the replicas47. Every 5,000 to 7,000 integration time-steps the coordinates of the proteins were saved for each replica and then exchanged, either between neighboring temperatures or between neighboring external tensions (i.e., Hamiltonians) according to exchange criteria that preserve detailed balance44. It total, 90,000 exchanges, alternating between temperature and force values, were attempted. The first 10,000 exchanges were discarded to allow for equilibration.</p><p>For CI2, five temperature windows (300, 317, 330, 345, 380) K and eight f values (f = 0.00, 0.35, 3.47, 8.68, 9.03, 9.38, 9.73, 10.42, 13.89) pN were used for a total of forty replicas. For protein G four temperature windows (310, 320, 330, 370 K) and ten f values (f = 0.00, 0.35, 1.60, 2.85, 4.10, 5.35, 6.60, 7.85, 9.10, 10.42, 13.89 pN) were used for a total of forty replicas. Swap acceptance ratios of between 10 and 40% were achieved in the HREX runs. The equilibrium properties of the proteins at temperatures and constant pulling forces other than those values explicitly simulated were calculated using Eqs. 1 and 6.</p><!><p>A conformation is native if the root-mean-squared-distance (RMSD) of the Cα interaction sites are within 5 Å, for protein G, or 11 Å, for CI2, of the corresponding Cα atoms in the crystal structure, otherwise it is classified as denatured. These RMSD thresholds were determined as the upper limit on the integral of the RMSD probability densities at the melting temperature (i.e., the maximum in the heat capacity trace) that yielded a value of 0.5. This method is illustrated in Fig. S1. This means we assumed that at the melting temperature CI2 is a two state system. Structurally, CI2's larger threshold arises from the disordered random coil regions in the native state ensemble (see Fig. 1A).</p><p>A key step in applying the MTM is in the choice of the reference pH ('pH1' in the equations above) in the post-simulation analysis. To choose the reference pH we first calculated the native stability of these two proteins at 300 K. For CI2 we identified a pH value for which the calculated and experimentally measured stabilities are similar38. Using this criterion we obtained a value of 3.5 for the reference pH. We then determined the temperature at which the calculated stability exactly equaled the experimental value of 6.0 kcal/mol at pH 3.5. This occurs at a simulation temperature of 302 K. In this way we set the overall free energy of this system to match the experiment at a single pH value. This procedure provided a reference solution condition (T=302 K, pH 3.5) from which predictions at all other pH values are made by reweighting of the partition function using the MTM procedure in Eqs. 1 and 6. Thus, despite the fact that there are no hydrogens in the coarse-grained simulation model, the thermodynamic effects of differential proton binding to N and D can still be accounted for within the MTM theory, as demonstrated by the successful comparisons between simulations and experiments. Experimental data of pKa values and stability versus pH for wild-type protein G does not exist. Therefore, we chose a simulation temperature (318 K) that resulted in a native stability typical for such small proteins (−3.0 kcal/mol) and set pH1 to 2.3. The trends and conclusions presented below are insensitive to the choice of reference pH, especially when such stability matching is carried out.</p><p>Two-dimensional native state stability phase diagrams (e.g., ΔGND(f, pH), etc.) are computed by rewriting Eq. 1 in terms of the probability of being folded as a function of f and pH using48</p><p> (6)PN(f,pH)=Z(pH2,T,f)−1∑k=1R∑t=1nkΘN(k,t)e−βEP(k,t,pH2,f)∑m=1RnmeFm−βmEm(k,t)−fmx(k,t), where ΔGND(f, pH) = −kBTln(PN (f, pH)/(1 − PN (f, pH))). All terms in Eq. 6 are the same as in Eq. 1 except we use the Heaviside step function ΘN (k, t), which is one if conformation (k, t) is native and zero otherwise. We calculate fm using PN (fm, pH) = 0.5.</p><!><p>The theory of the MTM hinges on the observation that if the partition function Z(A)(= Σj e−βE(j,A)) is known at some solution condition A, and if the free energy cost ΔGtr(A → B) of transferring each protein conformation from A to an arbitrary solution condition B is known, then the partition function in B is Z(B) = Σje−βE(j,A)−βΔGtr(j,A→B). In other words, the potential energy of the jth conformation in B is the sum of potential energy in A (E(j, A)) and the reversible work of transferring conformation j from A to B. In the current study, A and B differ in pH. In practice, the precision of the MTM, which is a mean field-like approximation to the exact partition function, is limited only by the accuracy of the protein model Hamiltonian (i.e., the force field), the errors in the ΔGtr model, and the extent of sampling in A.</p><p>We use the Aune-Tanford pH model49, which is among one of the most widely used theories to account for pH effects on protein stability17, to compute the free energy, ΔGtr(pH1 → pH2), of transferring a protein conformation from pH1 to pH2. The change in the experimentally measured native (N) state stability with respect to the denatured (D) state (ΔGND), due to a change in pH is fit using,</p><p> (7)ΔGtr(l,pH1→pH2)=−kBT∑k=1Ntln[10pH2+10pKk,l10pH1+10pKk,l], where the summation is over the Nt titratable groups and pKk,l is the pKa value of the kth titratable group in the lth protein conformation. It can be shown that Eq. 7 is a mean field result obtained by integrating over all possible protonation states of a protein with Nt independent titratable groups in the native and denatured states50. The success of Eq. 7 in modeling experimental ΔGND versus pH not only offers insight into the mechanism of pH denaturation, but also provides a means to estimate the free energy cost of transferring individual protein conformations from one solution pH to another.</p><p>To implement the MTM for pH effects, we use Hamiltonian Replica Exchange simulations44 of the Cα side-chain coarse grained model28 of protein G and CI2 to calculate the partition function Z(A). We use experimental pKa values (Table S1), to estimate the free energy cost of transferring the conformations (Eq. 7) sampled in the simulations that we classify as belonging to either the native or denatured ensembles based on suitable order parameters. To optimize the accuracy of the calculated partition function from the simulations we use the Weighted Histogram Equations (see Methods for details)37.</p><!><p>We first calculated the thermodynamic properties of CI2 and protein G as a function of pH at f = 0. The MTM prediction of the dependence of ΔGND(pH) on pH for CI2 is in excellent agreement with experiment (Fig 1A)38. Just as in the experiment, we find that the stability of CI2 decreases monotonically in a sigmoidal fashion as pH decreases. Although there are small differences between the experimental and simulation ΔGND(pH) data at pH less than 2, the overall agreement with experiment shows that the MTM accurately models pH effects on the thermodynamics of folding and unfolding.</p><p>We also calculated ΔGND as a function of pH (Fig 1B) for wild-type protein G for which experimental data are not available. However, pH-dependent ΔGND(pH) for a triple mutant (T2Q, N8D, N37D) of protein G35 has been measured. Although mutations can alter the native state stability, it is likely that the response of ΔGND to pH for the wild-type and the mutant will be qualitatively similar. With this caveat, we note that the overall shape of the calculated ΔGND as a function of pH for wild-type protein G is similar to the experimental data from the triple mutant (Fig. 1B). They both exhibit non-monotonic trends with minima located in the pH range of 3 to 4. In addition, the difference in stability at two different pH values, ΔGND(pH = 7) − ΔGND(pH = 3.4) is similar for the wild type and the triple mutant, with values of 1.3 and 1.7 kcal/mol, respectively. This suggests that the three mutations to titratable groups in the wild type protein do not drastically alter the characteristics of the thermodynamic response of protein G to pH changes. The non-monotonic dependence of ΔGND on pH observed for protein G contrasts with the monotonic-dependence observed for CI2 (Fig. 1A).</p><!><p>The versatility of the MTM is illustrated by probing the response of protein G and CI2 when, as is done in constant force single molecule pulling experiments, a tensile force f is applied to their N and C termini at various pH values. Such constant force pulling simulations are at equilibrium. We calculated the phase diagram for both CI2 and protein G as a function of pH and f (Fig. 2). Based on the destabilization of the native state of CI2 at acidic pH at f = 0 (Fig. 1A) we expect that the midpoint force required to unfold CI2 should decrease as pH decreases. This expectation is borne out in the pH range from 2 to 9 (Fig. 2A). For CI2, decreasing pH facilitates force unfolding by stabilizing the denatured state. As a result, the force required to unfold CI2 decreases as pH decreases (Fig. 2A).</p><p>The [f,pH] phase diagram for protein G (Fig. 2B) differs qualitatively from the one for CI2. In contrast to CI2, for protein G increasing the pH above 3.4 destabilizes the native state ensemble, which implies that smaller forces are needed to unfold protein G (Fig. 2B). These results show that the mechanical response of proteins are strongly pH-dependent and can have opposite trends, which reflects the underlying stability of the proteins.</p><p>We also calculated for CI2 the [f,T] phase diagram at two pH values (Fig. 3). The locus of points separating the folded, partially folded (see below), and unfolded structures is reminiscent of previously calculated [f,T] phase diagrams for simpler lattice and off-lattice models51,52. Not surprisingly, the region of stability increases as temperature decreases (compare the extent of blue regions in Figs. 3A and 3B). The force required to destabilize CI2's native state increases as the temperature is lowered. Furthermore, at T = 280 K and pH 3.5 only at f > 12 pN (Fig. 3B) is the native state unstable, whereas at pH = 1.0 this occurs for f > 8 pN.</p><!><p>From the phase diagrams the pH-dependent midpoint unfolding force, fm, can be determined using ΔGND(fm,pH) = 0. Similarly, T-dependent fm can be computed using ΔGND(fm, T) = 0. At high (pH > 5) and low pH (pH < 2) values we find fm is largely unchanged for both proteins (Fig. 4). For CI2, the interplay between native state stabilization with increasing pH and the counteracting f-induced destabilization results in a population of partially structured conformations at f < fm and pH > 4 (see blue regions in Fig. 2A). At intermediate pH values (2 < pH < 5) fm for CI2 is an increasing function of pH (Fig. 4), which is a reflection of the enhanced stability of the native state at f = 0 (Fig. 1A). In contrast, at intermediate values of pH fm for protein G exhibits non-monotonic behavior with a maximum at pH = 3.4, which coincides with the pH at which the native state stability is largest when f = 0 (Fig. 1B).</p><p>Although the dependence of fm on pH differs greatly for the two proteins the T-dependent fm results exhibit qualitatively similar behavior (blue curves in Fig. 4). Increasing temperature affects all interactions whereas changing pH only affects titratable groups. Temperature effects are global and pH effects are more localized. As a consequence, ΔG(f,pH) and ΔG(f, T) are different, which leads to the predictions in Fig. 4.</p><!><p>Single molecule force experiments are routinely used to obtain the f-dependent free energy profiles G(x) where x is the end-to-end distance of the protein projected onto the pulling direction53,54. The free energy profiles in principle can yield both the barrier height to unfolding and the location of the transition state assuming that x is an appropriate reaction coordinate. It is also possible to extract the intrinsic ruggedness of the folding landscape at f = 0 using the f-dependent kinetics of unfolding at different temperatures55,56. This would require doing explicit kinetic simulations or experimentally measuring the unfolding rates. We first calculated G(x) at several pH values for CI2 at f=8.4 pN and protein G at f=4.2 pN (Figs. 5A and 5B).</p><p>For CI2, we observe three basins of attraction in G(x) over a range of pH values (Fig. 5A), which suggests that CI2 undergoes a two-stage force induced unfolding transition in which a partially folded state is populated between the fully folded and fully unfolded basins. To obtain structural insights into the nature of the intermediate we calculated the fraction of native contacts for various structural elements within the native topology of CI2 (see structure in Fig. 1A) as a function of x. The analysis indicates (Fig. S2) that the transition from the native to the intermediate basin (located between 2 and 6 nm in G(x)) corresponds to the unfolding of β-strand 3 (residues 75 and 76 in PDB 2CI2) resulting in the loss of tertiary interactions with β-strand 2 (residues 65 to 71) and the α-helix (residues 32 to 42). The transition to the unfolded basin (located at x > 7 nm) corresponds to the unfolding of the rest of the structural elements in the protein (i.e., β-strands 1–2, and interaction of these strands with the α-helix). Sample structures of the native, intermediate and unfolded conformations from the simulations of CI2 under these conditions are consistent with this analysis (Fig. 5E).</p><p>The pH-dependent free energy profiles for protein G under tension have only two basins of attraction (Fig. 5B), which implies that force induced unfolding occurs in a single step. The free energy barrier to unfolding increases from 2.1 kcal/mol at pH 6.0, to 3.4 kcal/mol at pH 3.5. Because the curvature near the native basin and barrier top are roughly independent of pH (Fig. 5B) it follows from Kramers's theory that transition rates between the folded and unfolded states are determined entirely by the barrier height. Thus, the calculated G(x) profiles in conjunction with Kramers's theory predict that the unfolding rate kU (f) increases by a factor of 10 as pH increases from 6.0 to 3.5. The predictions for free energy profiles and the inferred changes in unfolding rates are amenable to experimental tests.</p><p>In order to ascertain the generality of our conclusions we show in Fig. 6 the free energy profiles over a wide range of forces. Just as in Fig. 5 we find that for CI2 there is force-induced folding intermediate, suggesting that the two transition states persist at all relevant f values. Remarkably, the invariance of the location of the TS is preserved at all forces. Both these figures show in a rather dramatic manner that the mechanical responses of CI2 and protein G are very different, which reflects the underlying variations in the native topology52 (see below for additional discussions).</p><!><p>According to the Hammond-Leffler postulate57 the transition state (TS) should resemble the least stable species in the reaction. Although originally proposed for reactions of small organic molecules, Hyeon and Thirumalai58 showed that the Hammond postulate is also applicable to force unfolding of biomolecules regardless of the nature of the reaction coordinate. For proteins under tension this implies that the location, xTS, of the TS, should either be independent of f or move towards the native state when f increases.</p><p>The G(x) profiles for CI2 at f=8.4 pN (Fig. 5A) show that there are two transition states, one between the native and intermediate, whose distance is ΔxN–TS with respect to the location of the native state, and the other between the intermediate and fully unfolded ensemble, whose distance is ΔxI–TS. Fig. 5C shows that ΔxN–TS and ΔxI–TS are independent of pH when pH exceeds 3.5. As pH increases, resulting in enhanced stability of both N with respect to I and I with respect to U (Fig. 5A), ΔxN–TS and ΔxI–TS increase with a dramatic jump at a pH=3.0. These results imply that the locations of the two transition states move closer to the less stable species, which is in accord with the Hammond-Leffler postulate. As a corollary, we expect and find (Fig. 5C) that upon an increase in temperature ΔxN–TS and ΔxI–TS should decrease as both the folded and intermediate states are destabilized relative to the unfolded state. Although these observations do not establish the adequacy of the one-dimensional reaction coordinate to describe f-induced unfolding of CI2 they support the generality of the Hammond-Leffler postulate for interpreting force spectroscopy results55.</p><p>The abrupt change in ΔxN–TS and ΔxI–TS at pH 3 range from about 1.7 to 2.0 nm (Fig. 5C). Similarly, the change in ΔxN–TS is 2.3 nm as the temperature is increased from 300 to 340 K. Such large changes in TS locations are not typically observed in constant loading rate AFM experiments. For example, the maximum value of xN–TS observed in filamin ≈ 0.7 nm59. The observed changes for CI2 are similar to the values obtained in the transition from intermediate to the unfolded states in RNase H using laser optical tweezer experiments4. The large variations in ΔxN–TS and ΔxI–TS for CI2 shows that besides experimental conditions the native state topology must also play a critical role in response to f.</p><p>In sharp contrast to CI2, the TS changes in the unfolding of protein G are dramatically different. The TS location ΔxN–TS is independent of pH (Figs. 5B and 5D), which implies that protein G behaves as a brittle material when subjected to f at all pH values. As the temperature increases ΔxN–TS decreases (Fig. 5D) in two steps, one at 305 K and the other at 320 K. In comparison, to CI2 the values of ΔxN–TS are roughly in the range observed for several proteins using AFM experiments. The value of ΔxN–TS changes by 0.4 nm as the temperature increases from 280 K to 340 K. The decrease in ΔxN–TS, reflecting the movement of the TS closer to the native state, as temperature increases is consistent with the Hammond-Leffler postulate.</p><!><p>We have introduced a way to account for pH effects on proteins within the framework of the Molecular Transfer Model. Our formulation overcomes the key limitations of the Tanford Thermodynamic model49, which is restricted to predicting only changes in protein stability due to changes in pH. Besides accomplishing this goal, the MTM also offers a molecular interpretation of folding and unfolding over a broad range of external conditions, including the response to f and pH. In principle, the MTM can be combined with all-atom simulations to calculate pH effects on proteins. As a matter of practice, however, currently such simulations under sample the partition function of proteins and therefore do not yield statistically significant results for the self-assembly of proteins.</p><p>Applications of the f-dependent response to pH of proteins using the MTM have revealed a number of surprising predictions. In particular, we found that fm had a non-linear dependence on pH; fm increases at acidic pH for protein G where as it decreases for CI2. These results correlate with the pH-dependence of ΔGND at f = 0, a conclusion also reached from single molecule force experiments36. We have also shown that the movement of the transition state location follows Hammond-Leffler behavior at all forces and solution conditions examined here. Large or discontinuous changes in transition state location inferred from the free energy profiles provide structural evidence for plasticity or brittleness of forced-unfolding of CI2 and protein G. We note that the sequence of events during an unfolding event cannot be directly calculated from the Hamiltonian replica exchange simulations, which are restricted to obtaining the measurable equilibrium free energy profiles. Brownian dynamics or all all-atom molecular dynamics simulations have to be performed to obtain the force-induced unfolding kinetics. Nevertheless, using our previous study52, which established a link between the structure of the native state and potential unfolding pathways, we can suggest the plausible structural origin of the brittleness of protein G. Our earlier work52 showed that upon application of force unfolding occurs by a shearing-type motion of β-sheets that are arranged in an anti-parallel manner. Using this result we surmise that protein G unfolds by shearing (or sliding) of the strands in the β-sheets (most likely the C-terminal strands) with respect to each other. Hence, the transition is abrupt involving a f-independent transition state (Figs. 5 and 6). In contrast unfolding is gradual in the plastic protein CI2 in which the TS move in response to f (Figs. 5 and 6). Explicit kinetic simulations are needed to enumerate the force-induced unfolding pathways, and to further confirm the drastically different responses to f predicted for these proteins.</p><p>Our results can be compared at a qualitative level to single molecule constant pulling speed experiments on ubiquitin36 in which pH effects were studied. Those experiments found36 that the unfolding force of ubiquitin is a constant over a range of pH values (6 to 10) and decreased at acidic pH. These findings are qualitatively consistent with our results for CI2 (Figure 4A). A major prediction from our results is that such pH-dependent trends depend critically on the specific protein under study. For example, fm for protein G shows the opposite trend observed in CI2; fm increases slightly at more acidic pH values (Figure 4B).</p><p>In single molecule pulling experiments, with x as the only experimentally accessible coordinate, identification of the TS location with the ensemble of TS structures in the multidimensional landscape is a challenging problem. It is possible that at f > fm the pulling coordinate is a good reaction coordinate because at large forces the molecule is likely to be aligned along the f direction, thus forcing it to unfold along the coordinate conjugate to f. Recently, we showed that the suitability of x as a reaction coordinate is determined by the interplay between compaction (determined by protein stability) and tension (dependent on xTS and the barrier to unfolding)60. A test of the adequacy of x as a reaction coordinate is captured by the experimentally measurable molecular tensegrity parameter, s=fcfm, where the unfolding critical force, fc, equals ΔG‡xTS, and ΔG‡ is the height of the free energy barrier. For CI2, with its two transition states, the values of s1 (N → TS) and s2 (I → TS2) are 0.019 and 0.005 at pH = 3.0. The theory of Morrison et. al.60 predicts that at this pH x is a good reaction coordinate for both the transitions because it is likely that the ensemble of conformations starting from ΔxN–TS (ΔxI–TS) would reach I and N (I and U) with equal probability (pfold ≈ 0.5). For protein G at pH = 6.0, s = 0.058, which also lies in the range for which x is likely to be a good reaction coordinate as assessed by the theory outlined in60.</p><p>A number of assumptions underlie our application of the MTM, including the temperature independence of pH transfer free energies (Δ Gtr(k, t, pH2)), and the use of the independent site model of titration. In addition, there are other assumptions17,50 that are inherent to the Aune-Tanford model that should be kept in mind in specific applications of our theory. However, the excellent agreement between experiments and simulations demonstrated here (Fig. 1) and in previous applications of MTM27,31 suggests that this assumption is reasonable for the proteins and solution conditions studied here.</p><p>To predict pH effects on proteins we utilized experimentally measured pKa values of titratable side-chains of protein G and CI234,38. In the absence of such experimental data pKa values calculated using quantum chemical methods can be utilized. Alternatively, the MTM could potentially be utilized to solve the inverse problem of predicting pKa values from either simulation structures alone, or from known changes of protein properties as a function of pH61. The MTM could also be used to test different functional forms that go beyond the two-state mean-field assumption of Eq. 750.</p><p>The Molecular Transfer Model is a significant advance in our ability to model in a natural way the effects of osmolytes and pH on the folding of proteins. By combining thermodynamic models and physio-chemical data the MTM incorporates the effects of osmolytes and pH into simulations in a physically transparent and theoretically rigorous manner. Consequently, reliable simulations can be performed to predict measurable quantities, which enables a direct comparison to experiments27,30,31,62.</p>

PubMed Author Manuscript

From molecular adsorption to decomposition of methanol on various ZnO facets: A Periodic DFT study

Methanol is an interesting and important molecule to study because of its potential to replace existing fuels. It is also a prominent hydrogen source which can be used to generate hydrogen in-situ. ZnO is widely used as catalyst in synthesis of methanol from CO 2 at industrial scale. In this work, we demonstrate that the same catalyst could be used for MeOH decomposition. We have carried out a systematic study of interaction of methanol with various flat and stepped facets of ZnO by employing Density Functional Theory (DFT). Two flat [(1010) and (1120)] and two stepped [(1013) and (1122)] facets are investigated in detail for methanol adsorption. Chemisorption of MeOH with varying strength is common to all four facets. Most importantly spontaneous dissociation of O-H bond of methanol is observed on all facets except (1120). Our DFT calculations reveal that molecular adsorption is favored on flat facets, while dissociation is favored on step facets. Also, (1010) facet undergoes substantial reconstruction upon MeOH adsorption. Activation of C-H bond along with strengthening of C-O bond on ZnO facets suggest partial oxidation of methanol. With our DFT investigations, we dig deeper into the underlying electronic structure of various facets of ZnO and provide rationale for the observed facet dependent interaction of ZnO with MeOH.

from_molecular_adsorption_to_decomposition_of_methanol_on_various_zno_facets:_a_periodic_dft_study

Introduction<!>Computational Details<!>Results and Discussion<!>Positions<!>SI4-(b)<!>Conflicts of interest

<p>Methanol is one of the most important chemical for industrial reactions and a primary feed-stock for energy production. Due to easier transportation and compatibility with the existing infrastructure, methanol attracts considerable attention. Methanol is also a promising hydrogen source because of its high hydrogen content. [1,2] However, use of methanol as a source of hydrogen requires breaking of its O-H and C-H bonds with substantial bond dissociation energies, viz. 96.1 kcal/mol and 104.6 kcal/mol respectively. [3] Over the past two decades, extensive studies of activation and decomposition of methanol on various metal surfaces, [4,5,6,7,8,9,10,11,12,13,14] metal alloys, [15,16,17,18,19,20] metal clusters, [21,22,23,24] metal oxides, [25,26,27,28,29,30,31] mixed metal oxides, [32] and zeolites [33,34,35] have been carried out. In general metal oxides turn out to be better catalyst for activation of methanol due to the presence of oxygen on the surface, which acts as an active site.</p><p>ZnO is considered as a very active catalyst for many reactions because of its mixed covalent and ionic bonding. [ Zn terminated ZnO(0001) surfaces using high-resolution electron energy loss spectroscopy (HREELS) in conjunction with temperature programmed desorption (TPD). They found that for all three ZnO surfaces, methanol adsorb dissociatively at room temperature which leads to the formation of hydroxyl and methoxy species. Upon heating to higher temperatures (370K and 440K), the dissociated and intact methanol species on ZnO(1010) predominantly undergo molecular desorption releasing CH 3 OH. While on both polar surfaces, thermal decomposition of CH 3 OH occurs to produce CH 2 O, H 2 , CO, CO 2 , and H 2 O at temperatures higher than 500K. [41] Although ZnO is used extensively as a catalyst in many reactions, its potential is not truly realized. To the best of our knowledge only non-polar (1010) and polar (0001) facets of ZnO have been studied for methanol activation. XRD pattern shows that (1120), (1013), and (1122) are also prominent facets. These facets are hardly studied for methanol activation. In the present work, we have systematically studied the interaction of methanol with various flat (1010), (1120) and stepped surfaces (1013), (1122) by employing periodic DFT. We report not only molecular adsorption and activation of O-H bond of methanol on these facets but also spontaneous dissociation of its O-H bond leading to formation of methoxy species. The quenched C-O bond-length in methanol along with partial double bond type character indicates onset of oxidation of methanol. Finally, we demonstrate various possibilities regarding interaction of MeOH with ZnO and bring out the rationale behind the reactivity in terms of electronic structure of these facets.</p><!><p>All the calculations are carried out within the Kohn-Sham formalism of Density Functional Theory. Projector Augmented Wave potential [42,43] is used, with Perdew Burke Ernzerhof (PBE) [44] approximation for the exchangecorrelation and generalized gradient approximation, [45] as implemented in planewave, pseudopotential based code, Vienna Ab initio Simulation Package (VASP ). [46,47,48] The bulk unit cell is taken from the materials project. [49] The bulk lattice parameters upon optimization are a = 3.28 Å and c = 5.30 Å demonstrate excellent agreement with the experimentally measured (a = 3.24 Å, c = 5.20 Å) lattice parameters. [50,51] Two flat facets, (1010) and (1120) of ZnO are modeled as slabs by cleaving a surface with 3x3 periodicity in x and y direction with 4 layers using Quantumwise-VNL-2017.1. [52] Two stepped facets, (1013) and (1122) are also cleaved by taking 3x1 and 2x2 periodicity respectively in the x and y direction with 6 layers. In every model, bottom layer is fixed and rest all layers and adsorbate are fully relaxed. Van der Waals corrections are applied to account for dynamic correlations between fluctuating charge distribution by employing Grimme method (DFT-D2). [53] It is observed that 20 Å of vacuum is sufficient to avoid interaction between adjacent images of planes along the z-direction. Geometry optimization is carried out with a force cutoff of 0.01 eV/ Å on the unfixed atoms and the total energies are converged below 10 −4 eV for each SCF cycle. A Monkhorst-Pack grid of 3x2x1 for (1010) and 3x3x1 for (1120) slabs is used. For both stepped surfaces, Monkhorst-Pack grid of 2x2x1 is used. The difference in energies is less than 4meV/atom for every system upon refining the K mesh further. Entire surface is scanned by placing MeOH molecule at all available unique sites. To compare the interaction of methanol at these sites, interaction energy is calculated using the formula: E int = E system -(E surf ace + E molecule ) where E system is energy of the system when MeOH is placed on the surface, E surf ace is energy of the bare surface and E molecule is energy of the MeOH molecule. To understand the electronic structure of these facets, total Density of States (tDOS ) are calculated with denser k-mesh using LOBSTER. [54,55,56,57] Mulliken charges are computed for all the atoms on the surface.</p><!><p>Bulk ZnO crystallizes in the hexagonal wurtzite structure consisting of hexagonal Zn and O planes stacked alternately. Both oxygen and zinc atoms are coordinated by four zinc and oxygen atoms respectively. Polar ((0001) and ( 0001)) and non-polar ((1010), ( 1011), (1120)), (1013), and (1122)) facets have prominent peaks in XRD. [58,59] In this work, we have studied the interaction of methanol with two flat ((1010), (1120)) and two stepped ((1013), ( 1122 Another flat facet that we studied is (1120). This is a highly symmetric facet with less number of inequivalent sites on the surface as shown in Fig. SI4-(a).</p><!><p>Interestingly, when MeOH is placed on any sites except 4 th , upon optimization it gets chemisorbed at one specific site as schematically represented in Fig.</p><!><p>. The orientation of methanol on this site is shown in Fig. SI4-(c).</p><p>E ads for methanol at this site is -1.</p><!><p>"There are no conflicts to declare".</p>

ChemRxiv

FRET Pumping of Rhodamine-Based Probe in Light-Harvesting Nanoparticles for Highly Sensitive Detection of Cu 2+

In this work we presented novel strategy for increasing the performance of popular fluorescent probes on the basis of rhodamine-lactam platform. This strategy is based on the incorporation of probe molecules into the light-harvesting nanoparticles to pump modulated optical signal by Förster resonant energy transfer. Using the commercially available Cu 2+ probe as a reference chemical, we have developed an efficient approach to significantly improve its sensing performance. Within obtained nanoparticles coumarin-30 nanoantenna absorbs excitation light and pumps incorporated sensing molecules providing bright fluorescence to a small number of emitters, while changing the probe-analyte equilibrium from liquid-liquid to solidliquid significantly increased the apparent association constant, which together provided a ~100-fold decrease in the detection limit. The developed nanoprobe allows highly sensitive detection of Cu 2+ ions in aqueous media without organic cosolvents usually required for dissolution of the probe, and demonstrate compatibility with inexpensive fluorometers and the ability to detect low concentrations with the naked eye.

fret_pumping_of_rhodamine-based_probe_in_light-harvesting_nanoparticles_for_highly_sensitive_detecti

Introduction<!>Chemicals and instruments<!>Synthesis and characterization of d98<!>Conclusions

<p>In recent years, colorimetric and fluorescent probes have become an effective analytical tool due to unique capability for sensitive monitoring of metal ions [1,2], anions [3,4], reactive oxygen species [5,6] and biomolecules [7,8]. Among the various chromophore scaffolds, lactam derivatives of rhodamines represent one of the most developed platform for the design of selective colorimetric and fluorescent probes due to their high extinction coefficients, high quantum yields, and excellent chemical and photostability [9,10]. Typically, such probes are rhodamine dyes modified with a selective receptor capable of interacting with the analyte. The sensing mechanism is based on the structural change of a molecule from a spirocyclic lactam to an open-ring amide [9,10]. Without analyte the probe exists in a colorless non-emissive spirocyclic form, while interaction of ligand group with specific molecule or ion results in ring opening and appearance of fluorescence and pink coloration.</p><p>Obviously, the sensitivity of analyte determination depends on the brightness of the probe (absorptivity x fluorescence quantum yield), as well as the equilibrium constant of probe-analyte interaction. The molar absorptivity of rhodamine 6G is approximately 10 ^5, and in a standard cuvette with an optical path of 10 mm, a change in [probe-analyte] complex concentration of 10 nM will correspond to change in optical density of ~0.001, which is equal to the measurement error of a typical spectrophotometer. The binding constants of real probes are in the range 10 3 -10 5 , and the typical working range of analyte detection is ~1μm-0.1mM. Of course, fluorescence measurements are more accurate than colorimetric, but they do not provide a significant increase in sensitivity, since they have exactly the same fundamental limitations. Modern approaches for ultrasensitive chemical sensing on the basis of surface-enhanced Raman scattering [11][12][13][14] or surface-enhanced fluorescence [15][16][17][18], allow determination of significantly lower analyte concentrations, but require powerful and expensive experimental equipment, both for measurements and for fabrication of nanostructured enhancing substrates.</p><p>It is clear that to improve the performance of fluorescent probes, it is necessary to significantly enhance the response signal by pumping the probe with a much brighter luminescent nanoparticle via Förster resonance energy transfer (FRET), however, nanoparticles are generally not efficient FRET donors because their sizes are beyond the FRET radius (1-10 nm), and in case of semiconductor nanocrystal quantum dots, 10-50 acceptor molecules are required to ensure efficient FRET [7]. New possibilities appeared with recently introduced light-harvesting FRET nanoparticles on the basis of cationic dyes separated by bulky hydrophobic counterions that prevent dye self-quenching [19][20][21][22]. In these NPs a short inter-fluorophore distance controlled by the counterion enable ultrafast dye-dye excitation energy migration on a femtosecond time scale through the whole particle within the fluorescence lifetime until it reaches a donor close to the acceptor leading to FRET. Therefore, the energy can be transferred beyond Förster radius from multiple donors to a single acceptor, providing a basis for signal amplification [7].</p><p>Inspired by recent works on the topic demonstrating the capabilities of this method, we aimed to enhance the response signal of popular rhodamine-based probes. Using the commercially available Cu 2+ probe as a reference chemical, we have developed an efficient approach to significantly improve its sensing performance, which requires chemical modification of the probe and incorporation it into lightharvesting FRET nanoparticles to amplify its fluorescence signal. Within these particles coumarin 30 nanoantenna absorbs excitation light and pumps incorporated sensing molecules providing bright fluorescence to a small number of emitters, while changing the probe-analyte equilibrium from liquid-liquid to solidliquid significantly increased the apparent association constant, which together provided a ~100-fold decrease in the detection limit. The developed nanoprobe allows highly sensitive detection of Cu 2+ ions in aqueous media without organic cosolvents usually required for dissolution of the probe, and demonstrate compatibility with inexpensive fluorometers and the ability to detect low concentrations with the naked eye.</p><!><p>Rodamine 6G (99%), Coumarin 30 (99%), 4-(dimethylamino)benzaldehyde (99%), hexane (95%), chloroform (99%), ethyl acetate (99.8%), N 2 H 4 *H 2 O (98%), sodium tetrakis [3,5-bis(trifluoromethyl)phenyl]borate, sodium tetrakis[3,5-bis(1,1,1,3,3,3hexafluoro-2-methoxy-2-propyl)phenyl]borate trihydrate, silica gel (100/200 μm) were purchased from Sigma-Aldrich and used as received. All other reagents were of analytical grade and used without purification. All aqueous solutions were prepared using deionized water.</p><p>The Fourier transform infrared radiation (FT-IR) spectra of the compounds in the range 400-4000 cm -1 were recorded using a Perkin Elmer Spectrum 100BX II spectrometer in KBr pellets. 1 H, 13 C NMR spectra were performed on a Bruker Avance 400 with the frequency of proton resonance 400 MHz using CDCl 3 as the solvent and tetramethylsiliane as the internal reference. UV-VIS and fluorescence measurements were performed on Shimadzu UV-2600 spectrophotometer and Shimadzu RF-6000 spectrofluorophotometer using 1 cm path length cuvettes at room temperature. The size and electrokinetic potential of fluorescent particles were determined using ZetaSizer Nano ZS analyzer (Malvern Instruments Ltd.) The pH measurements were carried out using a Sartorius Professional Meter PP-50.</p><!><p>Rhodamine 6G hydrazide was prepared as described in [23]. Yield 80%. 1 H NMR (400 MHz, CDCl 3 , ppm, δ): 7.96 (m, 1H), 7.45 (m, 2H), 7.06 (m, 1H), 6.39 (s, 2H), 6.26 (s, 2H), 3.58 (s, 2H), 3.54 (br.s, 2H), 3.22 (q, 4H), 1.92 (s, 6H), 1.32 (t, 6H); Elemental Analysis data: Calc. C, 72.87; H, 6.59; N, 13.07; Expt. C, 72.97; H, 6.66; N, 12.89. d98. A 300 mg (7E-4 mol) of rhodamine 6G hydrazide and 220 mg (1.4E-3 mol) of 4-(dimethylamino)benzaldehyde were dissolved in 15 ml of ethanol (95%). The mixture was refluxed for 6 h. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography on a silica gel with hexane/ethylacetate (v/v = 2/1) as the eluent to afford the product as a crystal powder (290 mg, 74%). 1 pH range (2-10), however fluorescence of acidic solutions was higher than that of basic ones. Since the work was focused on improving the performance of the rhodamine-lactam platform, all further experiments were performed at pH = 5, the maximally acidic environment in which the response of such probes is not crossinfluenced by hydrogen ions [26].</p><p>In the search for the optimal C30/counterion ratio, which ensures the stability of resulting colloidal solution and a high FLQY, we varied the excess of the counterion at a fixed dye concentration. Fig. 2(a, c, d) shows that with a sufficient excess of corresponding f-TPB, the absorbance of the resulting solution is significantly higher than that of coumarin aqueous solution of the same concentration, while the absorption band is red-shifted (Fig. 2a, solid curves). This spectral shift clearly indicates protonation of coumarin and redistribution of electronic density, which was also confirmed by acidification of the coumarin with hydrochloric acid, where a similar redshift was observed (Fig. 2a, dashed curves). In the case of an equimolar ratio or a slight excess of the counterion (less than 5), the resulting solutions are much less stable and partially precipitate within a few hours, while at higher counterion concentration the solutions remained stable for at least several weeks.</p><p>DLS measurements (Fig. 2(c, d), blue curves) revealed that the size of the formed particles also changes with an increase in counterion excess. In the case of F12, the size sequentially decreases from 140 to 30 nm at a 20-fold excess and remains unchanged (Fig. 2c, blue curve), while in the case of F6 it gradually increases from 60 to 100 nm (Fig 2d, blue curve). This difference can be explained by difference in solubility of corresponding anion precursors. Thus, NaF12 is soluble in water, the resulting NPs mainly consist of the insoluble salt C30/F12, while the excess of anions is adsorbed on solid surface, forming the first negatively charged layer of the electric double layer (more anions stabilize smaller particles). The solubility of NaF6 in water is much lower and it coprecipitate with C30/F6 forming large mixed particles. It is noteworthy that in both cases a higher excess of counterions provides the dye a higher FLQY (Fig. 2(c, d)): at pH=5, the maximum quantum yields were ~ 40% and 20% for F12 and F6, respectively, whereas in an aqueous solution without counterions, FLQY of coumarin was 0.08 at pH=7 and 0.04 at pH=5 (for comparison, in EtOH, FLQY=0.8). A similar impressive fluorescence light up has been noted for rhodamine B, a cationic dye, and was attributed to unique hydrophobic environment within the nanoparticles [21], however, for a neutral dye that is quenched upon protonation even in good solvent (Fig. S2), this is the firsttime report. We then obtained FRET nanoparticles with the fixed C30/counterion ratios (C30/F6=1/50, C30/F12=1/20) and a variable amount of the model acceptor (R6G) in order to estimate the donor/acceptor ratio, ensuring a noticeable energy transfer within nanoparticles. As shown in Fig. 3(a, b, d, e FRET efficiency in F12-and F6-based NPs, respectively. It should also be noted that with an increase in acceptor loading, FLQY of obtained NPs slightly increases (Fig. 3f) and reaches maximum at a ratio of R6G/C30 = 1/10. Thus, taking into account d98 has 2 binding sites -hydrazone and aninoaromatic fragments (marked with red circles in Fig. 4). The hydrazone fragment has affinity for both Cu 2+ and H + , while the aminoaromatic one only for H + . In an acidic medium, both sites are occupied and d98 is colored and fluorescent, while in the presence of Cu 2+ only the hydrazone fragment is occupied, and d98 is colored and non-fluorescent. In a moderately acidic medium, the copper ion displaces both protons from the molecule, and thus its concentration can be determined from the change in the fluorescence intensity of the solution at constant absorption (i.e., by decrease in FLQY).</p><p>To test whether a similar effect will take place inside the nanoparticles, lightharvesting nanoprobe were synthesized with an optimized ratio of precursors the same. To confirm that observed quenching is a result of d98-Cu 2+ interaction, we analyzed the stoichiometry of binding event according to Job`s method. Fig. 5c clearly demonstrate 1:1 binding stoichiometry between d98 and Cu 2+ both in the case of d98 solution and in the case of NPs containing d98. All these data reliably confirm the successful implementation of the key idea of the work: to increase the performance of the probe by pumping it with multiple donor molecules; however, the achieved increase in performance turned out to be much higher than the optical pumping factor. We believe that such behavior is associated with a change in the type of chemical equilibrium. Fluorescent probes are usually poorly soluble in water and require organic co-solvent, which provides the probe solubility and reversible probe-complex equilibrium in homogeneous solution. In our case, solid nanoparticles act as a cation exchanger; thus, the observed response is the result of a heterogeneous sorption process with much higher equilibrium constant. F6-and F12-based NPs provide 10-and 100-fold better sensitivity than d98 in solution.</p><!><p>To conclude, we have presented novel strategy for increasing the performance of very popular fluorescent rhodamine probes, which is based on the incorporation of probe molecules into the light-harvesting nanoparticles to pump optical signal by Förster resonant energy transfer.</p><p>According to published works, for the formation of bright FRET particles (without aggregation caused quenching), cationic dyes should be associated with bulky hydrophobic anions, however, organic cationic dyes emit in the yellow-NIR spectral range, which means that the blue-green absorbing dyes can be used only as donors, but not as acceptors. We have shown that neutral dyes containing amino groups act as cationic dyes and can also be used in the ion association method for preparation of fluorescent nanoparticles to pump blue-green absorbing cationic dyes.</p><p>Finally, on the basis of commercially available Cu 2+ probe we have developed FRET-nanobrobe with sub-nM detection limit which demonstrate compatibility with inexpensive fluorometers and the ability to detect low concentrations of Cu 2+ with the naked eye.</p>

ChemRxiv

Proline provides site-specific flexibility for in vivo collagen

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13 C-13 C correlation NMR spectroscopy on 13 C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril "expansion joints" to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.The dominant protein components of the extracellular matrix are ordered fibrillar collagens. These collagens must provide well-defined binding sites for many matrix proteins and cell-adhesion receptors, exemplified here by integrins. These same collagen fibrils also constitute the main mechanical component of the extracellular matrix, constantly subjected to local forces from adherent cells, which induce local collagen molecular movements likely to disrupt collagen-ligand bindings. How the collagen molecular and fibrillar structures are able to fulfil at first sight contradictory ligand binding and mechanical roles is an important question for both biology and materials scientists developing biomimetic implant materials.Fibrillar collagens are triple-helical proteins and, with the exception of their short N-and C-terminal telopeptides, consist entirely of G-X-Y (or Gly-Xaa-Yaa) triplet repeats where X is most commonly the cyclic imino acid, proline, and Y, hydroxyproline (O/Hyp), a post-translational modification of proline [1][2][3][4] . The hydroxylation of a Yaa position P, occurring immediately after synthesis of the collagen precursors, causes the resulting O ring to strongly favour the exo conformation (ring Cγ pointing away from the residue C=O group), which in turn pre-organises the peptide chain structure at the O residues towards the polyproline II helix that is required in each strand of the collagen triple helix; thus, GPO triplets in collagens are widely viewed as being essential for triple helix folding and stabilizing the triple helix structure 5,6 .The X-position P rings (P X ) in GPO triplets of short model collagen peptides occupy metastable structures, and endo and exo ring conformations (Fig. 1) are almost equally favoured at biologically-relevant temperatures. Endo and exo conformations have significantly different backbone geometry, and, moreover, flipping between them occurs on a nanosecond timescale 7 . Proline rings in purified collagen preparations are also dynamic on a nanosecond timescale [8][9][10][11][12][13][14] ; in native hard and soft tissues, both backbone dynamics 15,16 and proline flipping dynamics 17 are still retained. Taken together, these data suggest that native collagen GPO triplets are actually flexible, rather than rigid, structural regions. The high GPO triplet abundance in native collagen sequences led us to speculate that GPO P X flexibility could be key to allowing collagen to simultaneously provide essential mechanical properties and structurally well-defined protein binding sites for their biological roles.

proline_provides_site-specific_flexibility_for_in_vivo_collagen

Results and Discussion<!>GPO Pro(X) flexibility may be necessary for the biomechanical function of collagen.<!>Role of GPO triplets in binding of collagen fibrils to other ECM components. Different types of<!>Asymmetrical distribution of GPO triplets may facilitate functional bending of the triple helix.<!>GPO clusters can tolerate pathological mutations in collagen type I.<!>Methods<!>Isolation of fetal sheep osteoblasts.

<p>Proline(X) rings are flexible in biologically-derived samples. A significant source of P X flexibility arises from ring flipping between endo and exo conformations. Solid-state NMR spectroscopy provides an accurate method for assessing the distribution of collagen proline ring endo-exo conformations in intact tissues. In previous work, we utilised the 13 C chemical shift of proline Cγ to determine the distribution of proline P X ring endo-exo conformations in model collagen peptides 7 . There, we deduced that the collagen GPO P X 13 C shift is about 24 ppm for the endo conformation, and 25.7 ppm for the exo conformation. Proline rings in crystalline proline 18 and in model collagen peptides 7 rapidly flip between endo and exo conformations at room temperature and above, while by measuring dipolar coupling order parameters in native collagen, proline rings have been shown to undergo greater angular fluctuations 17 compared to glycine and hydroxyproline 13 . Rapid P X ring flipping means that the observed 13 Cγ isotropic shift is a population-weighted average of the endo and exo conformation 13 Cγ chemical shifts, allowing the endo:exo population ratios for P X rings to be determined from the population-weighted average shift 7 . In our previous work, we have demonstrated the presence of rapid endo-exo flips for proline rings in model collagen-like peptides containing the GPO/POG motif at room and physiological temperatures, and our simulations indicate that these puckering motions are coupled to the backbone motion of the triple helix. In the present work, we look for evidence of similarity in proline conformation dynamics between model peptides and more biologically-relevant samples to extend our findings to native collagen fibrils in the extracellular matrix.</p><p>Resolving the GPO proline 13 Cγ signals for model collagen peptides where specific 13 C labelling is readily achieved is straightforward. However, resolving this signal in intact tissue samples is not as easy; at natural 13 C abundance, the 13 C NMR spectrum of an intact collagenous tissue contains signals from all amino acid residues, including collagen GPY triplets, where Y is any residue not hydroxyproline (O), as well as the GPO proline signals of interest. Here we use 13 C, 15 N-enriched mouse bone and in vitro sheep osteoblast matrix, both generated as described previously 19,20 , as models for fibrillar collagen type I in intact tissues. 1D NMR spectra of all samples reported in this manuscript can be found in Supplementary Fig. S2.</p><p>Isotopic enrichment is necessary to use two-dimensional 13 C NMR spectroscopy for resolving the proline 13 Cγ chemical shift distributions in these intact tissues through 13 C- 13 C double-quantum-single quantum (DQ-SQ) correlation spectra. In this type of spectrum, pairs of strongly dipolar-coupled (i.e. spatially close) 13 C nuclei give signals in the double-quantum spectrum at the sum of the chemical shifts for the two 13 C nuclei. These DQ signals are correlated in the other spectral dimension with the normal (single) quantum 13 C NMR spectrum for the respective pair of 13 C nuclei. Horizontal slices through the DQ-SQ correlation spectra thus yield one-dimensional 13 C spectra of pairs of spatially close carbon nuclei. 2D 13 C DQ-SQ spectra from three 13 C, 15 N-labelled collagen/ collagen-like samples are shown in Fig. 2 for comparison: mouse bone, cultured osteoblast matrix and a model triple-helical collagen peptide, ((GPO) 5 (G*P*O)(GPO) 5 ) 3 , where * indicates a U- 13 C, 15 N-labelled residue (in each and every chain of the triple helix). As Fig. 2 shows, the connectivity of bonded carbons in the proline ring can be traced out in the DQ-SQ spectrum. By comparison with the signal connectivities in the model peptide spectrum, we can make straightforward assignment of collagen proline P X signals in the 13 C, 15 N-labelled mouse bone and in vitro matrix spectra.</p><p>We then need to separate 13 C signals from P X in general GPY triplets, where Y ≠ O (233 per collagen triple helix) from those in GPO triplets (103 per triple helix) in the mouse bone collagen DQ-SQ spectrum. We utilise the fact the 13 C signals from P X in GPO triplets are subject to the proline effect 21,22 , which manifests as a 1-2 ppm reduction in 13 C chemical shift for Cα, Cβ and C′ for residues that precede an imino acid, as in the case of P X in GPO triplets, where P X precedes Hyp/O, but not for P X in GPY triplets. The GPO P X Cα-Cβ correlation in the bone DQ-SQ spectrum can be distinguished from the corresponding GPY P X correlation using this criterion, as indicated in Fig. 2.</p><p>From the GPO P X Cα-Cβ correlation, the GPO P X Cβ-Cγ correlations are found, as shown in Fig. 2 and Fig. 3, from which the mean 13 Cγ chemical shift for the bone GPO P X rings is determined as 24.8 ppm, with partial overlap with the main population of GPY centred at 25.3 ppm. We note that the strong similarity of the proline signal chemical shift distribution between the mouse bone and in vitro matrix samples for all proline signals is a clear indication that the collagen structures in the two samples are similar, unlike the peptide proline distribution, which clearly only overlaps with a subset of that found in bone. As discussed above, this 13 Cγ chemical shift represents a population-weighted average between the 13 Cγ chemical shifts expected for the endo (23.8 ppm) and exo (25.7 ppm) proline ring conformations 7 . The mean 13 Cγ chemical shift of 24.8 ppm for bone collagen GPO P X , while only a small change compared to the 13 Cγ of 25.3 ppm for the (GPO) 11 model collagen peptide 7 , indicates the mean population distribution for collagen GPO P X in the intact tissues is slightly more skewed towards endo compared to the collagen model peptide. Nonetheless, this observed 13 Cγ chemical shift (24.8 ppm) is still distinct from a "pure" endo chemical shift (23.8 ppm), indicating that the proline rings in GPO triplets in bone do not strongly favour the thermodynamically more stable endo conformation [23][24][25] , and instead show a distribution between endo and exo conformations, with fast exchange over the NMR time scale, i.e. they are flexible.</p><p>To confirm our assignment, we note that the intensity of the signals arising from GPO triplets compared to the GPY (where Y ≠ O) should match the expected ratio in the sequence for mouse collagen type I. Figure 4 shows 1D slices taken from the SQ-DQ experiment in Fig. 2, at the C′-Cα, Cα-Cβ, and Cβ-Cγ DQ (sum) frequencies. The GPY:GPO triplet ratio in the sequence is 233:103, which means we expect to observe a GPY signal that is approximately 2.3 times more intense than that of GPO (assuming similar linewidths, if not then the integral of the signals should match this ratio). From Fig. 4, we can see that this is indeed true. The effect is clearer in the case of the osteoblast matrix cultured in vitro than the bone spectrum, as the bone spectrum is labelled in other amino acids apart from proline and glycine, giving rise to overlapping signals that can skew the observed ratio.</p><p>We expected that GPO P X would occupy a much larger range of conformations for fibrils in intact tissues compared to model collagen-like triple helical peptides. However, this is not the case, as evidenced by the NMR lineshapes for different samples shown in Fig. 4. The relative line widths are most easily assessed for the model peptide and in vitro osteoblast matrix, because the Pro 13 C signals are not obscured by signals from other labelled amino acids as they are for the bone sample.</p><p>There is some overlap between GPO and GPY proline 13 C signals for the osteoblast ECM sample, particularly for C′, which needs to be taken into account when comparing the Pro 13 C lineshapes (GPY C′ sites give a shoulder on the high frequency side of the GPO C′ signal). Nevertheless, it is clear that the GPO proline Cα and Cβ lineshapes are highly similar between model peptide and in vitro matrix collagen. The Cα lineshapes are directly comparable. In the Cβ spectrum, there is some additional small signal to low frequency for the in vitro matrix sample, but the majority signal has a similar linewidth to that for the model peptide.</p><p>The lineshapes allow us to directly compare the range of conformations for GPO P X between collagen in an intact tissue and the model collagen peptide. The single-quantum NMR lineshapes are in effect the sum of 13 C DQ-SQ correlation spectra of mouse calvarial bone (green), in vitro osteoblast extracellular matrix (blue) and model collagen peptide (orange). All spectra were obtained at 10 kHz MAS and 297 K, and only the proline carbon regions are shown in this figure (full spectra can be found in Supplementary Fig. S3). The peptide and osteoblast ECM samples are isotopically enriched specifically in glycine and proline residues. In the bone sample, ~20% of essential amino acids and glycine are U-13 C, 15 N labelled. The GPO proline signals are labelled in the figure and the 13 C- 13 C correlations between them are traced (purple line) for the model peptide spectrum, starting with the amide carbon at around 172 ppm. The same trace is overlaid on the bone and osteoblast matrix spectra to show how we arrived at the assignment of the collagen GPO proline signals in those spectra, with a slight difference in that the Cγ signal in the spectra obtained from biologically-derived samples is at 24.8 ppm rather than 25.3 ppm in the peptide spectrum (pink line). The GPY (where Y ≠ O) connectivity is shown to be separate from that of GPO (grey line). The arrows on the bone spectrum indicate the two sets of signals corresponding to Cβ-Cγ, where the difference between the GPO (purple) and GPY (grey) signals can be most clearly observed. The black dotted line in each spectrum indicates the DQ-SQ diagonal.</p><p>individual lineshapes from each GPO P X residue in the sample, each having an isotropic shift determined by the local molecular geometry for each individual P X . These individual lineshapes are not resolved, but the width of their sum signal, i.e. the observed signal in the SQ dimension of the DQ-SQ spectra, retains information on the range of GPO P X isotropic chemical shifts and thus on the distribution of time-averaged P X ring conformations.</p><p>Apaft from the distribution of proline ring geometries, the NMR linewidths can be affected by by other factors: T 2 (transverse) relaxation, homonuclear 13 C- 13 C dipolar coupling and molecular motion all contribute to the homogeneous linewidth. Hydration and sample temperature also play a role. While we have carried out our experiments with a fairly low level of hydration in order to capture a maximum range of conformations (lyophilization generally traps conformations and increases heterogeneous broadening), and maintained them at similar experimental temperatures, we cannot resolve the different contributions to the 13 C lineshapes. However, correspondence of the overall 13 C lineshapes implies some similarity in the distribution of proline residue conformations, molecular motion and T 2 relaxation time (which itself depends on molecular motion). 3 is shown with a green arrow on the bone GPO Cβ-Cγ slice. Vertical scaling was normalised on the larger signal within a GPO/GPY pair, maintaining the intensity difference between GPO and GPY to scale, but the vertical scale between pairs of GPO/GPY and between different samples are not shown to a unified scale. All spectra are presented on the same horizontal scale, enabling comparison of the chemical shift and also the linewidth at half height.</p><p>We further confirmed similarity in the distribution of GPO geometries between the in vitro matrix collagen and the collagen model peptide by following the build-up of G-P 13 C correlation signal intensity in a series of 2D 13 C-13 C proton-driven spin diffusion (PDSD) correlation NMR experiments in which the mixing time for 13 C to 13 C magnetization transfer is varied (see Supplementary Fig. S4).</p><p>In summary, we see that P X in GPO exhibits a mix of endo and exo conformations, in a distribution that is similar to what we see in the model peptide. In our previous work 7 , we have shown that collagen-like model peptides exhibit a dynamic equilibrium of endo and exo conformations, with the equilibrium positioned at a ratio of near 50:50 at biologically-relevant temperatures. With the experimental data summarised thus far, we propose that the same distribution of endo and exo conformations (with very little bias towards endo) is observed in biological samples.</p><p>The Pro(X) rings in GPO triplets have greater local conformational flexibility than in GPY sequences. From the NMR data presented above, we determined that P X in GPO in native collagen samples has a distribution over endo and exo conformations. However, it is also clear that P X in GPY (where Y ≠ O) exhibits a 13 Cγ chemical shift that is also greater than that in a pure endo case. In previous work 7 , we used the potential energy landscape approach 26 to demonstrate the inherent flexibility or frustration of helix parameters, which is coupled to P X endo-exo flips in GPO sequences. We use the same approach here to analyse whether the flexibility of P X rings in GPO triplets differs in any way from GPY triplets, where Y is a conformationally less constrained amino acid. Specifically, we predict the structural behaviour of P X rings in a GPA triplet compared with those in a GPO triplet. A GPA triplet was selected since it is the most common collagen type I GPY triplet in which Y is an amino rather than an imino acid.</p><p>All the calculations involve geometry optimisation to characterise local minima and the transition states and pathways that connect them on the potential energy landscape. By applying appropriate structural perturbations to a ground state conformation, this procedure allows for sets of energy minima to be computed that span particular regions of conformational space. For the present purposes we are interested in the subspace of minimum energy conformations that can be adopted by perturbing the backbone dihedral angles of a single GPY triplet while it is embedded within in a larger collagen triple helix. Flexibility at a residue comes from the presence of a range of energetically-accessible conformations for that residue. The relative number of distinct minima found in each subspace and the corresponding range of dihedral angles are taken as a measure of the likely flexibility. If many alternative conformations exist, separated by thermally accessible barriers on the experimental time scale, then we predict greater flexibility.</p><p>The structural perturbations to the ground state structure were carefully designed to uniformly sample the space of backbone dihedral angles and proline endo and exo conformations for the sixth triplet in the trailing chain in both (POG) 12 and (PAG) 12 superstructures. A single perturbation consisted of choosing a pair of atoms separated by a linear set of covalent bonds, and rotating as a rigid body all the intervening atoms by an angle randomly selected within a maximum amplitude. When such rotations occur between two backbone atoms the local ring geometry and endo/exo structures are conserved, but the backbone dihedrals, in which the atom pair are involved, change. Rotations applied to the atoms between the Cβ and Cδ atoms in the proline rings can force a subsequent relaxation into either endo or exo conformations, depending on the angle of rotation, which also modifies the backbone dihedrals of the proline and neighbouring residues. Further details of the particular groups considered can be found in the Methods.</p><p>By repeating our structural perturbations for each triplet, an ensemble of feasible conformations, corresponding to local potential energy minima, were generated. This analysis yielded only four minima in the case of (PAG) 12 , and 64 minima in the case of (POG) 12 . Rather than presenting the whole database of structures, which would be unwieldy, we illustrate the backbone dihedral angles of two neighbouring P X and Y residues in the centre of the sequence for each structure in a Ramachandran plot (Fig. 5). For ease of viewing, each residue (proline and alanine, or proline and hydroxyproline) is presented on different panels of the figure. In the case of P X in (POG) 12 , we have further divided the plot into two, based on whether the hydroxyproline in the structure has an endo or an exo pucker. Thus, each point in each Ramachandran plot represents a minimum in the potential energy landscape, i.e. an alternative accessible conformation.</p><p>For P X rings in the GPA triplet, all the perturbed minima in the case of P X endo pucker have nearly identical dihedral angles, resulting effectively in just two possible backbone conformations, one associated with endo pucker at P X , and the other with exo pucker. There are therefore, two well-defined, accessible backbone conformations at GPA triplets. To deform the GPA triplet, or in other words, to access GPA P X backbone structures with significantly different φ/ψ angles to these endo or exo minima, would require large energy perturbations, corresponding to thermally inaccessible barriers at biologically relevant temperatures.</p><p>In striking contrast, the P X ring in a GPO triplet can move between local energy minima that exhibit a range of φ/ψ angles for both endo and exo P X conformations. The diversity of conformations accessed via local structural perturbations for GPO P X rings depends on whether the neighbouring Hyp is in the endo or exo conformation. The combination of endo and exo conformations for proline and hydroxyproline in GPO gives rise to four clusters, as illustrated in Fig. 5(d,e), where P X (and Hyp) endo-exo ring conformations are accompanied by systematic shifts in backbone conformation corresponding to extension-compression of the helical chain.</p><p>Combined with the NMR results presented in the first section, we conclude that the GPO proline rings in native collagen proteins exhibit flexibility in the form of endo-exo pucker, just as previously observed in model peptides. Our computational results further demonstrate that these endo-exo pucker motions are not limited locally at the proline ring, but are coupled to the backbone, and can lead to overall extension and compression of the triple helix at GPO triplets. In the context of heterotrimeric collagens and fibrils formed from collagen triple helices, it is likely that these extension and compression motions will lead to bending of the triple helix and the fibril.</p><!><p>We next asked what is the role of such flexibility at GPO triplets, and specifically whether it could impact on fibril as well as molecular flexibility. To determine the location of the evolutionarily conserved GPO triplets in the fibril, we carried out sequence alignment across diverse species for collagen type I and generated a consensus sequence for each chain. Using the database of collagen type I sequences, we also calculated the conservation of each GPO triplet. Full results of the calculations for collagen type I are presented in Supplementary Table S1 and Fig. S5.</p><p>From our sequence analysis, we find that there are 33 highly conserved GPO sites across collagen α1(I) chains out of 43 in the consensus sequence, and 20 highly conserved GPO triplets across collagen α2(I) chains out of 33 in the consensus sequence. If we consider only mammalian sequences, these values increase to 41 out of 43 GPO triplets in the α1(I) chain, and 24 out of 33 in the α2(I) chain being defined as highly conserved at the same conservation threshold (75%).</p><p>The distribution of GPO sites in the experimentally-determined model 27 of collagen type I fibrils and also the consensus sequence is shown in Fig. 6. For ease of comparison, the consensus sequence for the collagen type I α1 Figure 5. The energy landscape simulation results presented as Ramachandran plots. For each different, accessible conformational structure, the dihedral angles of two consecutive residues, proline and the residue subsequent to proline, are plotted. These backbone conformations arise from perturbing a single Pro residue in (PAG) 12 and (POG) 12 whilst maintaining the overall ring conformation as either endo or exo. For (PAG) 12 , only four accessible structures were found, three of which were very similar in backbone dihedral angles in the current scaling, and therefore overlap nearly perfectly. (a) shows the backbone dihedral angles for the alanine residue of these four structures and (b) shows the backbone dihedral angles for the proline residues, showing a clear separation in backbone dihedral angles for endo and exo ring conformations. For (POG) 12 , 64 accessible structures were found. (d) shows the backbone dihedral angles for the hydroxyproline for all 64 structures, split into two populations according to the conformation of the hydroxyproline ring. The preceding proline ring conformations were plotted for the case where hydroxyproline is exo (e) and endo (f). Panel (c) illustrates the dihedral angle change in terms of overall backbone conformation using structures generated for a GGG tripeptide (glycine was used for clarity) of constant dihedral angle using Avogadro 1.1.1 54 , ranging from a fully extended backbone conformation (180°, 180°) to a much more compressed coiled conformation (−20°, 120°). It is clear that as φ and ψ increase, the peptide backbone is increasingly extended, while as the dihedral angles tend towards lower values, the backbone is increasingly compressed. Although the dihedral angle changes presented in the simulation represent a level of change that is smaller than that shown in the middle two peptides in (c) (a length difference of under 5%), the absolute extent of expansion/compression will scale with the length of the peptide. As previously reported 7 , the idealized 7/2 (tighter) and 10/3 (looser) triple helices both have dihedral angles that more closely match endo P X in (b,e and f). and α2 chains is arranged by D-period. The consensus sequence for type I collagen is provided as a larger PDF image in the Supplementary data.</p><p>From Fig. 6, it is clear that there is a spatial correlation of GPO sequences; far from being randomly scattered over the fibril structure, these sequences occur primarily as banded clusters across the fibril structure that persist across different D-periods, exemplified in the XRD-derived collagen type I structural model 28 in Fig. 6a and the sequence representation in Fig. 6b.</p><p>The distribution of GPO triplets varies within the fibril structure. Collagen fibrils have two zones: the overlap zone, where all five D periods overlap, and the hole zone, where the short D5 period leads to a gap or hole in the fibrillar structure. There are clearly more GPO triplets present in the overlap zone, and fewer in the hole/gap zone. The banding of GPO triplets is also more obvious in the overlap zone.</p><p>Bearing in mind the conformational flexibility of the GPO triplet structure, these clusters of GPO triplets in the fibril represent regions where the fibril structure itself can rapidly extend, compress or bend, and so serve as flexible joints for both the molecular and fibrillar structure, without significantly increasing the energy of the system. Hence, we propose that these GPO triplets act as "expansion joints", especially when viewed in the context of their distribution over the fibril. While Y-position hydroxyproline is known to increase the thermal stability of a collagen triple helix 29 , and to promote PPII formation in the backbone 5,30 , the secondary effect of placing the preceding proline into a conformationally frustrated state (i.e. with many pucker conformations of similar energy) 7 provides local flexibility at these conserved locations of the triple helix and therefore also of the fibril. Such local flexibility can be important for the overall integrity of the fibril, thus explaining the observation that thermal stability is associated with total imino acid content (proline and hydroxyproline) and not hydroxyproline alone 31 .</p><p>We consider three important implications arising from our proposal of GPO sites as expansion joints in collagen fibrils: firstly, the role of of GPO triplets in collagen binding by considering the distribution of GPO relative to known binding sites on collagen proteins; secondly, the possibility that asymmetrical distribution GPO triplets may aid larger angle bending and functional displacement of the C terminus in the D5 period; and thirdly, the consequences of known mutations of conserved GPO sites.</p><!><p>binding are likely to exert different mechanical forces on the collagen fibril. The integrin α2β1 domain-peptide co-crystal (PDB 1DZI) 32 describes a bend in the model collagen peptide upon ligation, suggesting that molecular distortion may be necessary for optimal interaction of native collagen with the integrin. Such molecular distortion Figure 6. The distribution of GPO sites within a type I collagen fibril, from diffraction data (a) and in the consensus sequence (b). In (a), the collagen fibrillar structure (from PDB 3HR2 27 ) is modelled from X-ray diffraction data on rat tail tendon 28 with the Cα atoms in GPO triplets represented as red spheres. In (b) the consensus sequence based on the most frequent amino acid at each position is shown. The three chains of collagen are staggered by one residue with respect to each other, based on a previous study on the VWF A3 domain binding 55 . The D period arrangement shows a good match to the experimentally-derived fibril structure model above. Highly conserved (75%+) GPO triplets are highlighted in red; others in pink. High affinity integrin binding sites are in blue and the DDR/ VWF binding site in green.</p><p>upon binding to the integrin would affect the integrity of the collagen fibril, and if replicated many times by the multiple collagen-integrin interactions presented by a cell, significant disorder of the collagen fibril would result.</p><p>We note that the high affinity integrin binding sites, shown in Fig. 6, all occur in close proximity and on the N-terminal side of conserved GPO triplets. On the other hand, the main DDR and VWF binding site, also occurring close to multiple strongly conserved GPO triplets, is found on their C-terminal side. We hypothesise that the clusters of GPO triplets serve to protect the structural integrity of the adjacent sites when the fibrillar structure is subjected to external forces. The multiple highly conserved GPO triplets close to the DDR/VWF binding site in the fibril (as seen in the D-period arrangement of the consensus sequence in Fig. 6b) may provide additional controlled flexibility for this binding site.</p><p>We speculate that local structural distortion is necessary to maintain the organization of collagen molecules within the fibrillar structure when ligands bind, and that clusters of GPO triplets across the fibril in close proximity to the collagen-ligand binding site provide controlled, reversible local distortion of the structure.</p><!><p>The distribution of GPO triplets between the three chains of the collagen triple helix can affect its accessible overall motion range. Where a GPO sequence occurs at the same locus of all three chains, proline ring endo-exo flips will allow extension-compression in all three chains, permitting concerted movement at this position. The stagger between the three chains dictates that extension-compression is never restricted to the axis of the triple helix, but will always include a bending component.</p><p>For collagen type I, a heterotrimer containing two α1 chains and one α2 chain, there are many cases where GPO triplets do not occur at the same locus in both the α2 and α1 chains. Chain extension-compression can then only occur asymmetrically, bending the triple helix in a specific direction, through an angular range defined by the change in chain backbone structure upon proline endo-exo ring flip, typically a change of around 15° in proline dihedral angle.</p><p>Interestingly, the majority of the GPO triplets in the fibril hole zone only occur in either the α1 or α2 chains. The X-ray fibre diffraction-derived model of the collagen fibril structure 27 shows the collagen triple helices twisting around one another through the hole zone. If this molecular twisting were to occur via homogeneously flexible regions in the collagen triple helices, molecular ordering would be compromised, as there would be no control over which direction (or by how much) a collagen triple helix can bend. However, an appropriate distribution of GPO triplets within the hole zone could readily allow well-defined bending in a specific direction, which would allow the collagen triple helices to twist around one another without the possibility of molecular disordering. Such considerations, exemplified here by collagen I, will not apply to the homotrimeric fibrillar collagens, II, III, XXIV and XXVII, but will be relevant to the heterotrimeric collagens V and XI.</p><p>The (GPO) 5 and (GPO) 4 sequences at the C termini of the α1 chains and α2 chains, respectively, are well-conserved in the fibrillar collagens. The triple helix extension-compression ability that these sequences confer would allow the C-terminus some flexibility in its spatial location, thus our hypothesis that the GPO sequences play the role of "expansion joints". All other GPO triplets in D5 that precede the (GPO) 5 sequence occur only in the α1 chains, so that D5 has controlled freedom to bend at these three points. It has previously been hypothesized that the C-terminal telopeptide protects underlying ligand binding sites in the collagen fibril molecular structural arrangement 33 ; the (GPO) 5 sequences and D5 period that precede it in primary sequence may assist in such a function, by controlling the possible displacements of the C-terminal, but at the same time allowing it to be displaced, thus allowing ligand binding.</p><!><p>While the GPO positions are evolutionarily conserved, we assessed the known mutations at these sites that lead to human disease. Using the NCBI ClinVar database 34 , we assessed missense mutations in collagen, and the overlap of these mutations with locations of conserved GPO triplets, to understand the functional and likely pathological consequences. We are aware that this type of analysis is prone to survivor bias. With this caveat, we will offer a few interpretations of our results.</p><p>As far as the collagen type I triple helical region is concerned, there are 95 unique single base mutations for the α1(I) chain, and 110 for the α2(I) chain. For α1(I), we found 33 highly conserved GPO sites in the consensus sequence, of which 10 correspond to the reported mutation sites (human variants). Conserved GPOs account for 9.8% of the total helix sequence, and 10.5% of their locations overlap with possible mutation sites. For α2(I) we found that there are 9 GPO locations out of 20 highly conserved that overlap with reported mutation sites. 5.9% of the chain consists of conserved GPO triplets, and 8.2% of the 110 mutations overlap with conserved GPOs.</p><p>By inspecting the sites where mutations overlap with bands or clusters of conserved GPOs, we note that 16 mutations (in both chains α1(I) and α2(I)) occur within a GPO band/cluster, while six mutations occur in relatively isolated GPOs. However, given the fact that many more GPO triplets occur in clusters within the triple helix or across the fibril, and relatively few are isolated, it is difficult to draw firm conclusions from this observation.</p><p>The diseases associated with the reported mutations appear to vary by chain. Most mutations in α1(I) are associated with osteogenesis imperfecta, and in α2(I) with Ehlers Danlos syndrome.</p><p>These results show that although mutations occurring in GPO triplets in collagen type I can be tolerated, in the sense that it is possible to survive past embryonic developmental stages, they lead to pathology in most cases. A more detailed analysis including more collagen types will be required to separate the effects of GPO (or lack thereof) on biomechanical properties of the fibril and its effects on biosynthesis and embryonic development.</p><!><p>Peptide synthesis and purification. ((GPO) 5 (G*P*O)(GPO) 5 ) 3 was synthesized as previously reported 7 .</p><p>Briefly, the peptide was synthesized (0.1 mmol scale) as C-terminal amides on a TentaGel R RAM resin (loading of 0.19 mmol/g, Rapp Polymere) following the standard Fmoc-based solid-phase peptide synthesis strategy on a microwave-assisted automated peptide synthesizer (Liberty ™ , CEM), and purified by reverse phase HPLC. Pure peptides were characterised by matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF) mass spectrometry (Supplementary Fig. S1).</p><p>Labelling of mouse tissue. Our feeding and euthanasia methods were unregulated procedures under the UK Animals (Scientific Procedures) Act 1986 and therefore were not subjected to formal ethics review. Our methods complied with the review processes of the University Biomedical Service of the University of Cambridge, which is overseen by the Animal Welfare Ethical Review Body of the University of Cambridge. All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986.</p><p>The procedure is based on our previous work 19 . Briefly, in vivo labelling was achieved with 2000 g of a gel diet (modified Classic A03 Geldiet, SAFE, Augy, France) to minimize in-cage spillage, comprising 50 g 13 C, 15 N-labelled Celtone powder (Cambridge Isotope Laboratories, Andover, MA, U. S. A.), 67 g fish hydrolysate, 410 g protein free diet (mainly corn starch), 72.9% water, 2.1% preservatives and texture additives, which was packaged in 100 g packs and irradiated at 10 kGy. Three young adult female C57Bl/6 mice, housed together, were fed ad libitum for ca. 3 weeks until the labelled diet was consumed, humanely euthanized using a Schedule 1 method and tissues harvested. Bone tissues from all animals were examined by solid-state NMR to ensure we were not assigning small biological variations between animals, and did not show major variation.</p><!><p>Fetal sheep osteoblasts were isolated from a fetus removed from an 18 weeks pregnant sheep sacrificed for an unrelated study. Femurs were removed from the fetus. After washing several times with 1% trigene (Medichem International), the femur was stripped of muscle and non-osseous tissue to expose the bone which was sectioned into small longitudinal pieces and washed with 70% ethanol followed by repeated washings with Minimum Essential Medium (MEM; Invitrogen) to remove all traces of ethanol. Bone strips were then transferred to Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) containing bacterial collagenase A (0.5 mg/mL) and dispase II (3 mg/mL) both from Roche Diagnostics. A total of 100 mL of enzyme-media mixture was used for bone sections taken from 3 limbs. Bone strips were incubated at 37 °C in a shaking water bath for 3 hours to release osteoblasts into the medium. After incubation the cell suspension was transferred to a fresh tube and the bone sections were rinsed in DMEM with 20% fetal calf serum (FCS; Invitrogen) to stop the enzymatic digestion. Rinse medium and cell suspension were pooled and passed through a 40 μm mesh filter (Appleton Woods). The cell suspension was then centrifuged at 1000 g for 5 min at room temperature to pellet the cells. The pellet was resuspended in DMEM complete medium and transferred to two T-175 cm 3 culture flasks (Nunc) and placed in a 37 °C CO 2 incubator. When the cultures were almost confluent, cells were detached with 10 ml of 0.25% trypsin containing 1 mM EDTA (SigmaAldrich) and incubated for 5 min at room temperature. The flasks were tapped at the end of incubation period to completely dislodge the cells from the flask. Trypsin was neutralized by adding 15 mL of DMEM complete media to the culture flask. The cell suspension was centrifuged in a 50 mL tube (Greiner) at 1200 rpm for 5 min and resuspended in 10 mL of DMEM. The cells were transferred into T-175 cm 3 culture flasks and were expanded to passage 3 for subsequent experiments.</p><p>Basal Medium Eagle (BME) complete medium was prepared by adding 10% FCS, 30 μg/mL L-ascorbic acid 2-phosphate (Sigma), 10 mL/L L-glutamine-penicillin-streptomycin (200 mM L-glutamine, 10,000 units/ml penicillin, and 10 mg/ml streptomycin in 0.9% sodium chloride; Sigma). DMEM complete medium was prepared by adding 10% FCS, 30 μg/mL L-ascorbic acid 2-phosphate, and 10 mL/L L-glutamine-penicillin-streptomycin. All supplements were filter sterilized (0.22 μm filter, Appleton Woods) before addition.</p><p>Culturing osteoblasts with labelled compounds. The procedure is based on our previous work 19 .</p><p>Briefly, osteoblasts were cultured to confluence in T-175 flasks containing 25 mL BME complete medium. Labelled (U-13 C 5 , 15 N) proline (Cambridge Isotope Laboratories) and (U-13 C 2 , 15 N) glycine (Cambridge Isotope Laboratories) were added to a final concentration of 46 mg/L and 30 mg/L respectively after filter sterilization (0.22 μm filter). The cultures were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 . The culture medium with isotope labelled supplements was renewed every two days until the cells and matrix began to detach from the culture flask, by which time enough ECM had formed for solid-state NMR. Refinement of the cell culture method to produce ECM that was a similar as possible to the native mouse bone tissue as judged by their respective 2D solid-state NMR spectra included adjusting the cell culture medium, the frequency with which the medium was changed, the concentration of ascorbic acid and the manner in which the cultures were handled (so as to produce minimal shear forces on the cells during changes of medium produced the optimal ECM). Samples from more than 20 batches using the final optimized protocol were prepared using isotope-enriched amino acids and all characterized by solid-state NMR to ensure reproducibility of results.</p><p>Harvesting ECM from cell culture. The matrix was harvested after nine days of culture, when the cells produce a dense matrix which started to peel off the surface of the tissue culture flask. The medium was removed and the cells were washed with 20 mL 1 x phosphate buffered saline. The flask was placed in a freezer at −80 °C for 24 hours and the cells were lysed by thawing the flasks at room temperature for 30 minutes. The debris produced by cell lysis was removed by repeated washes with PBS. The decellularized ECM was dislodged by gently swirling the flask in the presence of 20 mL PBS. The matrix collected in PBS was transferred to a fresh 50 mL tube and centrifuged at 1200 rpm for 5 min at room temperature. The supernatant was poured off and the ECM dehydrated in an oven at 37 °C overnight. The samples were stored at −20 °C until NMR analysis. ECM of mouse tissue was used directly in solid-state NMR experiments without extraction, purification, or excessive processing.</p><p>Rotor packing for solid-state NMR experiments. All samples were placed into Kel-F inserts for Bruker 4 mm rotors prior to being placed into a full-length (17 mm), normal wall thickness (1 mm) Bruker 4 mm rotor. The inserts provide the advantage of restricting the sample length to the region of the coil in the NMR probe that has optimal RF homogeneity.</p><p>The peptide ((GPO) 5 (G*P*O)(GPO) 5 ) 3 and the ECM obtained from cell culture were lyophilised prior to packing into the Kel-F insert. The dry mass of the samples used for solid-state NMR experiments were 20.0 mg of pure peptide and 14.2 mg of ECM.</p><p>The mouse calvaria bone was not subject to any drying or dehydration procedures, and was simply broken into clips without cryomilling prior to packing into the Kel-F insert. We expect that this will lead to the bone sample being slightly more hydrated than the peptide or the cell culture ECM samples, though no discernible amounts of water was observed visually after the experiment, nor was a significant reduction in sample mass observed. 12.6 mg of mouse bone was used for solid-state NMR experiments.</p><p>Solid-state NMR Spectroscopy. All solid-state NMR spectra were recorded on a Bruker Avance I NMR spectrometer with a 9.4 T superconducting magnet, operating at 400 MHz ¹H, 100 MHz ¹³C and 40 MHz 15 N frequencies. For all experiments, magic angle spinning rate was set at 10 kHz, and the sample temperature is 297 K, unless otherwise specified. 13 C cross-polarisation ( 13 C CP) experiments: the standard cross polarisation sequence in the Bruker pulse programme library was used: ¹H 90° pulse length 2.5 μs, contact time 2.5 ms, with a ramped pulse on ¹H. During acquisition, SPINAL-64 35 decoupling at 100 kHz was applied on ¹H.</p><p>2D 13 C-13 C double quantum (DQ)-single quantum (SQ) correlation NMR experiment: initial cross polarisation parameters were the same as in 13 C CP experiments. At 10 kHz MAS, 70 kHz POST-C7 pulse sequence 36 was applied on 13 C to excite double quantum coherence in 0.4 ms. Magnetisation was returned to zero quantum by another 0.4 ms of POST-C7 sequence. During DQ evolution and reconversion, 100 kHz Lee-Goldburg decoupling was applied on ¹H. During acquisition 100 kHz SPINAL-64 decoupling was applied on ¹H. The pulse sequence used was an adapted version of the Avance I large sweep width POST-C7 experiment in the Bruker library. For in vitro ECM 128-256 scans and for the heavy mouse bone samples 432 scans per t 1 -slice were recorded.</p><p>2D 13 C-13 C proton-driven spin diffusion (PDSD) correlation NMR experiment: initial cross polarisation parameters were also the same as in 13 C CP experiments. At 10 kHz MAS, the magnetisation was allowed to evolve at single-quantum coherence during the incremental delay, and returned to zero quantum coherence by a 13 C 90° pulse with a length of 3.8 μs. ¹H decoupling was switched off during the mixing period to allow transfer of 13 C magnetisation via dipolar coupling and spin diffusion 37 , with a 13 C 90° readout pulse at the end of the mixing period. During both the incremental delay and acquisition periods, SPINAL-64 decoupling was applied at 100 kHz. The pulse sequence used was an adapted version of the Avance I CP spin diffusion experiment in the Bruker library. Mixing periods of between 5 and 200 ms were recorded. For in vitro ECM 64-400 scans (depending on the amount of sample) and for the heavy mouse bone samples 256 scans per t 1 -delay were recorded.</p><p>Analysis of energy landscapes. The calculations employed a standard atomistic force field, namely Amber9 with implicit solvent (igb = 2), and the FF99SB parameter set 38 . In the computational potential energy landscape approach we employ geometry optimisation techniques to calculate local minima and the transition states and pathways that connect them, to construct a kinetic transition network 26 . The low energy region of the landscape was first sampled using basin-hopping global optimisation 39,40 . These minima were then connected using double-ended transition state searches, which identify new minima and pathways, progressively augmenting the database. Observable properties were extracted using standard methods of statistical mechanics and unimolecular rate theory, and the underlying database was refined using additional connection attempts until the quantities of interest appeared to have converged. Details of these procedures can be found in recent reviews 41,42 The simulations conducted here, which seek to explain the results of the ssNMR data relating to flexibility of P X prolines adjacent to hydroxyproline residues, required parameters for the Amber9 force field obtained from experimentally derived properties of hydroxyproline with post-translational modifications 43 . The initial starting points for (POG) 12 were derived from PDB 1V7H 44,45 ; those for (PAG) 12 were generated from (POG) 12 by replacing the hydroxyprolines with alanine.</p><p>For each of the two peptide sequences, (POG) 12 and (PAG) 12 , 10000 geometry perturbations were applied to the equivalent PYG group (the 6th group of the trailing chain) in the central region of each collagen triple helix. The perturbed structures were then relaxed to local minima. The 10000 resulting minima were filtered to identify the unique structures, which correspond to the space of accessible conformations for the backbone in the given PGY triplets. The unique minima were then connected in further transition state searches, which helps to ensure that all the relevant structures in the accessible configuration space were located.</p><p>We ensured uniform exploration of the backbone dihedral space by randomly rotating rigid groups of atoms between carefully chosen atom pairs. For the (PAG) 12 system the pairs were: the Cβ to Cδ axis of the proline tip, the Cα-Cα axis of the peptide bond between the proline and alanine, the N-C axis of the alanine residue, and the Cα to Cα axis of the peptide bond that trailed the alanine residue. This combination of rotation groups ensures that psi and phi dihedrals of the proline, the psi and phi dihedrals of the alanine, the phi angle of the trailing glycine, and the psi angle of the preceding glycine residue are sampled. In addition, the full range of endo and exo conformations of the P X ring are encountered. For the (POG) 12 system, the corresponding groups were chosen, except that the C-N rotation of the alanine was replaced by a Cβ-Cδ rotation of the hydroxyproline tip, again inducing exploration of the endo/exo states of the O Y residue and corresponding backbone dihedrals.</p><p>The uniform sampling of the full set of backbone dihedral angles can generate local minima that include peptide bond rotations and stereochemical rotations, both of which do not correspond to a biologically-feasible collagen triple helix structure. For our analysis, we are only interested in dihedral angle distributions that can be found in a collagen triple helix 46,47 . Therefore, only those structures where all the perturbed residues relax into the range of phi = −50° to −90° and psi = 130° to 170° were accepted.</p><p>Sequence alignment and analysis. For our analysis of the conservation of the GPO positions, we built multiple sequence alignments across diverse species for collagen α1(I) and α2(I) sequences. For each alpha chain, a set of orthologous sequences was acquired using the National Centre for Biotechnology Information (NCBI) Protein Database resources (https://www.ncbi.nlm.nih.gov/protein) 48 . The NCBI Reference Sequence Database (RefSeq) 49 was used as the main resource for protein sequence data. A small proportion of the data was obtained from GenBank and the EMBL databases 50,51 . The initial sets of sequences identified from these databases were filtered to remove duplicates (including isoforms), leaving a single representative sequence for each species. Each set of sequences was aligned using the software program MUSCLE (http://www.drive5.com/muscle/) 52,53 . The final sequence sets used can be found in Supplementary Table S2-S4.</p><p>Conservation of each GPO triplet was determined by calculating the probability of the occurrence of the GPO triplet across all aligned sequences. The amino acids with the highest frequency at each alignment position was used to generate a consensus sequence.</p><p>To analyze the effect of missense mutations on GPO sites, we used the consensus sequences generated in the same procedure as described above. We mapped mutation sites reported for human variants into the consensus sequences and assessed how they align with conserved GPO positions on the collagen type I primary sequence. The National Centre for Biotechnology Information (NCBI) ClinVar database (https://www.ncbi.nlm.nih.gov/ clinvar/) was used as a mutation data resource 34 . The search for mutation sites across procollagen alpha chains was performed using the relevant gene names and the following criteria: molecular consequence: missense mutation; variation type: single nucleotide; review status: at least one star. The GPO triplet is counted as being present at a mutation site if one position of any of its residues (G, P or O) overlaps with a reported mutation site. The analysis of correlation between mutation sites and conserved GPO locations was carried out for the major fibrillar alpha chains (type I, II and III) and for collagen type V alpha chains.</p>

Scientific Reports - Nature

Ovalbumin Epitope SIINFEKL Self-Assembles into a Supramolecular Hydrogel

Here we show that the well-known ovalbumin epitope sIINFeKL that is routinely used to stimulate ovalbumin-specific T cells and to test new vaccine adjuvants can form a stable hydrogel. We investigate properties of this hydrogel by a range of spectroscopic and imaging techniques demonstrating that the hydrogel is stabilized by self-assembly of the peptide into nanofibres via stacking of β-sheets. As peptide hydrogels are known to stimulate an immune response as adjuvants, the immunoactive properties of the sIINFeKL peptide may also originate from its propensity to self-assemble into a hydrogel. This finding requires a re-evaluation of this epitope in adjuvant testing.Fibrillar nanostructures can be assembled by a number of natural and designed peptides, which usually possess distinct biophysical properties, such as propensities for β-sheet folds. Nanofibrils can also form as a result of protein or peptide misfolding, which is associated, for example, with pathogeneses of neurodegenerative disorders such as Parkinson's and Alzheimer's diseases 1 . In biological settings, peptide nanofibrils can have important functional roles, the extracellular amyloid fibrils of E. coli, for example, are used for cell propulsion 2 . More recently, it has been possible to tune and employ the fibril-forming properties of peptides in the design and manufacture of functional nanomaterials that can be used in disease treatment and prevention 3,4 . Self-assembled peptides in this case possess several advantages, which include multi-valency, defined synthetic composition, tuned specificity, and ease of further functionalisations 5,6 . These advantages have allowed peptide nanofibrils, as well as the associated hydrogels, to be successfully used as scaffolds in regenerative medicine, cell culture matrices and vehicles for drug delivery 7,8 . Certain peptides that constitute fragments of full-length proteins have been described to form fibrils and hydrogels. These include mouse laminin a-1 9 , human transthyretin 10 and human troponin C 11 . Peptide fragments of each of these proteins assemble into nanofibrils networks in vitro, with further assembly leading to formation of stiff hydrogels.A new and exciting application of fibrillar peptide assemblies is in adjuvanting of subunit vaccines 12 . Although subunit vaccines show remarkable promise in treatment and prevention of deadly diseases, they suffer from poor immunogenicity, which substantially limits their efficacies 13 . Adjuvanting or enhancing the immunogenicities of subunit vaccines is therefore a significant challenge in biomedical research. A range of materials is currently under development as vaccine adjuvants and among them are hydrogels consisting of peptide nanofibrils. Peptides in this case can be conjugated to specific antigens or epitopes, often themselves peptides, and subsequently used in targeted immunisations [14][15][16][17] . The disease antigens and individual peptide epitopes are identified through an analysis of a given immune response against an immunogen, which lead to the individual peptide fragments that directly interact with the cells of the immune system 18 . Some of these peptide epitopes are capable of eliciting an immune response in the absence of the parent pathogen but such activity is heavily dependent on the addition of proper adjuvants 19 .During the development of adjuvants, well-known and well-characterised peptide antigens are used to evaluate the adjuvant efficacies. Ovalbumin (OVA) has been historically a popular source of such antigens, since OVA can induce both humoral and cellular immune responses based on well-characterised peptide epitopes 20,21 . The OVA 257-264 octapeptide was one of the first OVA epitopes to be characterised, it has an amino acid sequence

ovalbumin_epitope_siinfekl_self-assembles_into_a_supramolecular_hydrogel

<!>Results and Discussion<!>Conclusion<!>Methods<!>Rheology.<!>Microscopy.

<p>SIINFEKL, which is recognised by cytotoxic T lymphocytes 18 . Immunisation with the adjuvanted SIINFEKL peptide induces long-lasting CD8+ T cell immunity in mice 22 .</p><p>Here we report on the properties of the OVA epitope SIINFEKL to self-assemble into fibrillar nanostructures that lead to formation of a peptide hydrogel. The properties of this peptide assembly have been analysed by use of rheology, electron microscopy, small angle X-ray scattering (SAXS), circular dichroism (CD) and infrared (IR) spectroscopies. The molecular analysis of the peptide fold has been carried out with peptide nuclear magnetic resonance (NMR) measurements. It is demonstrated that SIINFEKL forms fibrillar assemblies similar to other peptide hydrogels. The immunoactive properties of this peptide can therefore be related to its self-assembling nature.</p><!><p>The OVA 257-264 octapeptide SIINFEKL was prepared via standard Fmoc-SPPS and purified via HPLC (Fig. SI-1). Gel formation was observable first during precipitation of this peptide in diethyl ether following cleavage with trifluoroacetic acid (TFA). The gel forming property was confirmed by preparing a 1% (w/v) solution of the purified peptide in Millipore water, where the gel formed immediately after dissolving the peptide (Fig. 1a). A hydrogel also forms with 0.5% peptide in water following overnight incubation. A typical peptide concentration used to prepare peptide-based vaccines is 8 mM [14][15][16] and the case of SIINFEKL the concentration of 8 mM would amount to 7.7 mg/ml or 0.77% (w/v) solution, which is well within the gelation conditions.</p><p>The mechanical properties of the SIINFEKL hydrogel at these two concentrations were evaluated by use of rheology after incubation at room temperature for 24 h. Constant sheer strain (γ = 1%) and angular frequency (ο = 6 rad/s) were applied in time sweep experiments. Both gels exhibited properties typical of peptide hydrogels, where elasticities (storage modulus G') are higher compared to the corresponding viscosities (loss modulus G") (Fig. 1a,b) 23 . In the case of the 1% gel, G' was more than 6 times stronger than G", which is a characteristic feature of peptide hydrogels 6 . The value of G' also indicates moderate mechanical stability.</p><p>The micro-and nanoscale properties of the hydrogel material were studied by electron microscopy and small angle X-ray scattering (SAXS). The presence of fibrillar networks with fibrils on the micrometer range and widths of less than 25 nm is apparent in micrographs of the 1% hydrogel (Fig. 2). SAXS allows for measurement of nanoscale changes in density of a given sample and it is therefore a common technique in evaluation of nanoscale properties of different materials 24 . SAXS measurements of the SIINFEKL hydrogel showed an intensity increase towards small q-values (scattering vector) and a constant intensity towards higher q-values (Fig. SI-2). The data was normalised to time and beam intensity. A decrease of the total X-ray intensity was visible for each subsequent measurement of the sample. This could be caused by increased fibrillisation of the sample, which would decrease the number of scattering bodies. The radius of the fibrils was evaluated from Guinier theory, modified for long rod-like objects with circular cross-section 24,25 . Calculating a weighted mean of the three measurements resulted in a mean radius of the cylindrical www.nature.com/scientificreports www.nature.com/scientificreports/ objects of 12.3 ± 1.2 nm. The calculated radius correlates well with the diameter of the narrowest long peptide fibrils observed by TEM (22 nm, Fig. 2).</p><p>The peptide conformation within the hydrogel was evaluated by CD and IR spectroscopies. Due to their specific optical activity, secondary structure of different biomolecules can be analysed by use of CD 26 . The corresponding spectra of the peptide hydrogel diluted in water show a combined profile with properties of both a random coil (slope at 200-210 nm, Fig. 3a) and a β-sheet (slope at 210-230 nm). The CD curve was fitted using a β-structure selection algorithm, which predicted 55% of the sample to be in a random coil conformation, 20% anti-parallel β-sheets, and 19% β-turns (fitted curve, Fig. 3a, RMSD = 0.0738) 26 . In conjunction with CD, FTIR is a technique that is often used for evaluation of biomolecular secondary structure. The FTIR spectrum of the SIINFEKL hydrogel was measured following chloride ion exchange in D 2 O. Although broad, a signal spectrum at 1632 cm-1, can be observed (Fig. 3b), which can also be attributed to the amide I band of anti-parallel β-sheets 27 .</p><p>The precise secondary arrangement of the individual amino acids can be obtained by use of NMR 28 . NMR spectra of the SIINFEKL hydrogel were recorded from the 5% hydrogel in 1:5 D 2 O/H 2 O. Although hydrogel formation was visible, the NMR spectra did not display any line broadening (Fig. SI-6). By use of TOCSY, it was possible to assign the proton shifts corresponding to the amide, αand most of the β-hydrogens of the individual amino acids as well as the αand β-carbons. Most of the identified backbone NMR shifts fell within the range of the random coil conformation with the exception of αand β-carbons (Fig. 3c,d, respectively) of the isoleucine residues at positions 2 and 3. These shift differences from the random coil structure indicate the β-sheet arrangement for the two isoleucing residues 28 .</p><p>In order to evaluate the role of the N-terminal isoleucine residues on the gelation properties of the peptide, an analogue with alanine substitutions was prepared (peptide sequence: SAANFEKL). When subjected to conditions, at which SIINFEKL peptide undewent gelation, the solution of the new analogue remained liquid even after a 72 hour incubation at room temperature (Fig. SI-3). Another analogue was prepared, this time with replacement www.nature.com/scientificreports www.nature.com/scientificreports/ of the C-terminal hydrophilic residues by alanines (peptide sequence: SIINFAAL). This analogue proved challenging to dissolve in aqueous buffers due to its increased hydrophobicity, which made it impossible to evaluate the hydrogel forming properties of this peptide.</p><p>The propensity of SIINFEKL to form hydrogels has been previously briefly described by Lowenheim and co-workers 29 , who showed that the peptide conjugate can serve a scaffold for neurite outgrowth. However, no analysis of the propertied of the SIINFEKL hydrogel beyond scaffolding was carried out. In this work we show that the morphology of SIINFEKL fibrils is similar to that of other peptide hydrogels, as it consists of a dense network of apparently flexible fibrils but with a rather large diameter of 20-25 nm as opposed to 2-5 nm usually observed for peptide fibrils, as for example for the troponin C peptide fragment VEQLTEEQKNEFKAAFDIFVLGA 11 . TEM micrographs show that the individual SIINFEKL nanofibres further assemble into micro-scale fibres with distinct branched structures. The size and shape of the nanofibres were confirmed by SAXS measurements. Assembly into fibrils is often facilitated by a distinct fold adapted by a portion of the monomer peptide, where β-sheets are by far the most common motifs 6 . Under our experimental conditions the SIINFEKL peptide also adopts a partial β-sheet fold, which is in contrast to the native fold of this peptide within the context of crystalline ovalbumin, where the termini of SIINFEKL are involved in two separate α-helices, the center of the peptide is in a random coil, and no portion of the peptide forms a β-sheet 30 . Our NMR data indicates that only the two N-terminal isoleucine residues of the peptide are not in a random coil conformation. Due to their side-chain hydrophobicity, isoleucine residues are known to facilitate self-assembly of short peptides, as is the case for the peptide IIIK that forms stable nanofibers in aqueous solution via β-sheet formation combined with molecular amphiphilicity 31 . The continued assembly of IIIK leads to formation of a soft peptide hydrogel. The fibrillar structure of SIINFEKL assemblies is also likely to be facilitated via such amphilicity, as the N-terminal half of the peptide contains two hydrophobic residues (IleIle), whereas the C-terminal half contains two adjacent hydrophilic amino acids (GluLys).</p><!><p>As the peptide SIINFEKL is a very well-characterised epitope, it is often used in proof-of-principle studies aimed at demonstrating the efficacies of new adjuvant systems. It has been employed in evaluation of, among others, monophosphoryl lipid A 32 , bacterial membrane vesicles 33 and HMGB1 peptide 34 . SIINFEKL has also been used to study the immune response to the Bacillus Calmette-Guérin vaccine, which is commonly used against tuberculosis 35 . It is now, however, well-known that peptide hydrogels consisting of nanofibres can themselves act as adjuvants 14,36 , and therefore, our results should caution the use of the SIINFEKL peptide during evaluation of adjuvant efficacies because its ability to form fibrils and a hydrogel.</p><!><p>peptide synthesis and gel formation. Solid phase peptide synthesis (SPPS) was done using fluorenylmethoxycarbonyl (Fmoc) chemistry on 100 µmol scale. Peptides were deprotected and cleaved in a mixture of trifluoroacetic acid, triisopropylsilane and water (38: 1: 1) for 2 hours at room temperature. The peptide was precipitated with diethyl ether and centrifuged. After washing twice with ether, the precipitated peptide was dissolved in water and lyophilised. The peptide was purified by RP-HPLC using a semi-preparative Kromasil C18 column. Analysis of the purified peptide SIINFEKL (38 mg, 39% yield), was carried out using an analytical Kromasil C4 column with a gradient of 5% buffer B (acetonitrile + 0.08% TFA) in buffer A (water + 0.1% TFA) to 65% B in A over 20 min at 1 mL/min and UV measurement at 214 nm (Fig. SI-1). Peptides SAANFEKL (Fig. SI-4) and SIINFAKL (Fig. SI-5) were synthesised and analysed in a similar manner.</p><p>Hydrogel formation was monitored visually as a function of pH, peptide and buffer concentration (Table SI-2). In the standard gelation procedure, water was added to the peptide and the resulting mixture mixed until the sample assumed a homogeneous consistency. Gelation was apparent minutes after dissolving, with final pH 4.2. The pH was then adjusted to 7 by adding aliquots of an appropriate NaOH aqueous solution and the volume was adjusted to give the target final concentration. The solutions were allowed to stand at room temperature overnight. Similar experiments were carried out at a fixed peptide concentration of 1 wt% and samples were incubated at different temperatures.</p><!><p>The rheological measurements were performed on a stress-controlled rheometer (TA Instruments HR2) fitted with a 50 mm diameter plate geometry, with a gap of 0.2 mm. The sample was allowed to anneal at 25 °C for 1 hr prior to time-sweep experiments in linear regime for both strain and frequency as discussed in the manuscript.</p><!><p>For scanning electron microscopy (SEM), peptide hydrogel was applied to a Thermanox ™ coverslip and air dried. The coverslips were sputter coated with gold in high vacuum (Bal-Tec SCD 005). SEM images were recorded with Zeiss SEM Supra 55 VP operating at 20 kV.</p><p>For transmission electron microscopy (TEM), peptide hydrogel was applied onto carbon coated copper grid and subsequently viewed with Philips CM200 at 200 kV. TEM images were acquired with OriusTM SC600 Gatan CCD camera.</p><p>Circular Dichroism. CD spectra were recorded on peptide solutions at 0.5% by weight in water using a 1 mm quartz cuvette. Solutions had been incubated at room temperature for a minimum of 24 h. Scans were performed in 1 nm increments with 3 s scans at 20 °C and averaged over 5 scans.</p><p>FtIR. FTIR-ATR was recorded on peptide solutions at 1% by weight in D2O using Agilent Cary 630 with a single bounce diamond ATR-cell and potassium-bromide optics. The spectral resolution was set to 2 cm -1 , with zero filling factor 2, which resulted in a formal resolution of 0.46892 cm -1 . (2019) 9:2696 | https://doi.org/10.1038/s41598-019-39148-8 www.nature.com/scientificreports www.nature.com/scientificreports/ sAXs. SAXS measurements were performed using X-rays from a Nanostar (Bruker AXS) system, operating at λ = 0.1542 nm (CuKα-radiation) and equipped with a two-dimensional detector (Våntec 2000). X-ray patterns were radially integrated to obtain the scattering intensity of the peptide in dependence on the scattering vector q = (4π/λ) sinθ, with 2θ being the scattering vector. The samples were filled into capillaries and the solvent sample was subtracted as background from the peptide sample dissolved in the solvent. Sample measurement time was 3, 6 and 12 hours. NMR. NMR spectra were acquired with a 700 MHz Bruker Avance III HD NMR spectrometer on peptide sample dissolved in 20% D 2 O in H 2 O at 5 mg/ml. For H-H Total Correlation Spectroscopy (TOCSY) 16 transients were collected using 16 dummy scans with spectral width of 10 ppm in both dimensions.</p>

Scientific Reports - Nature

Improved photodecarboxylation properties in zinc photocages constructed using m-nitrophenylacetic acid variants

The methoxy-and fluoro-derivatives of meta-nitrophenylacetic acid (mNPA) chromophores undergo photodecarboxylation with comparable quantum yields () to unsubstituted mNPA, but uncage at red-shifted excitation wavelengths. This observation prompted us to investigate DPAdeCageOMe (2-[bis(pyridin-2ylmethyl)amino]-2-(4-methoxy-3-nitrophenyl)acetic acid) and DPAdeCageF (2-[bis(pyridin-2-ylmethyl)amino]-2-(4-fluoro-3nitrophenyl)acetic acid) as Zn 2+ photocages. DPAdeCageOMe has a high  and exhibits other photophysical properties comparable to XDPAdeCage ({bis[(2-pyridyl)methyl]amino}(9-oxo-2-xanthenyl) acetic acid), the best perforiming Zn 2+ photocage reported to date.Since the synthesis of DPAdeCageOMe is more straightforward than XDPACage, the new photocage will be a highly competitive tool for biological applications.

improved_photodecarboxylation_properties_in_zinc_photocages_constructed_using_m-nitrophenylacetic_ac

<!>Conflicts of interest

<p>We are developing decarboxylation reactions of metanitrophenylacetatic acid (mNPA) and xanthone (XAN) groups to design photocages that release Zn 2+ upon irradiation with light. 1,2 Such photocaged complexes block the biological activity of metal ions until the Zn 2+ release is initiated by exposure to light of a specific wavelength. 3 We recently described DPAdeCage (1) and XDPAdeCage (2), two Zn 2+ photocages that incorporate a dipyridyl amine (DPA) chelating group to selectively bind Zn 2+ over other common loosely bound metal ions found in cells (Error! Reference source not found.). 2 Irradiation at or near the max initiates a photoreaction that results in the loss of the carboxylic acid functional group as CO2, which shifts the Zn 2+ binding equilibrium toward unbound metal ion (Scheme 1). The transformation from a 4-coordinate to a 3coordinate chelator leads to a 10 5 -fold reduction in binding affinity (Kd). For biological applications, a rapid synthesis to produce photocages in high yields would be ideal. The photophysics of XAN chromophores are superior to those of mNPA groups, [4][5][6] but the low-yielding synthesis of the XAN scaffolding limits the pace of photocage development. To overcome these limitations, we functionalized several mNPA chromophores to develop DPAdeCage derivatives with improved photophysical properties. In addition to those improvements, the added functional groups facilitate selective nitration at the 3-position of the aromatic ring simplifying the purification procedure for the mNPA chromophore that will facilitate the rapid generation of photocages. Designing effective photocages requires avoiding short wavelength excitation (max), which can induce cellular photodamage, while still delivering sufficient energy to break chemical bonds. Compared to our mNPA-based DPAdeCage, the max of XAN-derived XDPAdeCage is red-shifted by over 80 nm (Error! Reference source not found.). Furthermore, upon coordination to Zn 2+ the photoreactivity of DPAdeCage is significantly reduced. The XAN chromophore offers significant photophysical advantages that include maintaining a high  upon Zn 2+ binding. Accessing the XAN chromophore necessary to make XDPAdeCage requires a 2-step synthesis with a maximum 20% yield. By comparison, the nitration step to synthesize the mNPA chromophore has a much higher yield, increasing the total material obtained (~70% yield of mNPA). The difference in reaction time, 24 h and 1 h respectively, also impacts the rate of photocage synthesis. We hypothesize that the relative ease of making mNPA derivatives compared to the XAN chromophore, as well as the numerous readily available starting materials, will facilitate the rapid production of mNPAbased photocages for numerous applications. Furthermore, if functionalization of the mNPA group could shift the abs sufficiently, we can prepare and screen Zn 2+ photocages for biological applications more quickly as well as expand the toolbox of tunable chromophores. Measurements obtained in 40 mM HEPES buffer (pH 7.5, 100 mM KCl). Quantum yields were obtained in solutions containing 30% methanol to maintain photoproduct solubility. a. Hammet constants indicating the relative electron withdrawing or donating properties of each R group. b. 3e lacks a red-shifted absorbance band; = 3264 M -1 cm -1 at 300, 356 M -1 cm -1 at350.</p><p>In previous studies on ortho-nitrobenzyl photocages (oNB), introducing electron donating groups (EDGs) or electron withdrawing groups (EWG) produced photocages with different rates of photolysis, quantum yields and excitation wavelengths. [7][8][9][10][11] Tsien, Kaplan, and others reported functionalization of the oNB ring with methoxy groups shifted the max to longer wavelengths, 7,8 suggesting we might benefit from a similar approach. Modification of the nitrobenzyl ring, however, can also impact the electronics of the aromatic ring and therefore the nature of the photoreaction. 10,12 Unlike oNBs that undergo a Norrish type II photoreaction, 13,14 the mechanism for photodecarboxylation of mNPA chromophores involves an electron transfer from the nitro group to the methylene bridge of the phenylacetate to initiate the photodecarboxylation. [15][16][17] This suggests increasing the electron-density around the aromatic ring would impede electron-transfer to the methylene bridge, which could reduce photolysis quantum yields (). Thus, we predicted EDGs would exhibit reduced photoactivity compared to mNPAs functionalized with EWGs. By adjusting the electronic-structure of the mNPA chromophore we hoped to access a DPAdeCagederivative that will maintain a high  upon coordination to Zn 2+ .</p><p>A series of mNPA chromophores were prepared with various EDGs and EWGs at the 4-position of the aromatic ring (Error! Reference source not found.). We hypothesized that functional groups with heteroatoms would extend the conjugated system and red-shift the max. Functionalization resulted in the appearance of a new red-shifted absorbance feature that was not observed in the parent mNPA 3e (Figure 2). The red-shifted feature appears as a shoulder of the strong UV band centred at circa 270 nm in most of the compounds, but the band shifts sufficiently in the methoxy-(3f) and hydroxy-derivatives (3g) to appear as a distinct new absorbance feature. While all the derivatives possess a weaker extinction coefficient () of the large absorbance band below 300 nm compared to 3e, the compounds also absorb more strongly at wavelengths greater than 320 nm (Error! Reference source not found.). The photolysis quantum yields () were determined under simulated physiological conditions using LC/MS to monitor the loss of 3a-3i. Despite the red-shifted band, the photoactivity of the acetyl (3a), chloro (3b), and amide-functionalized (3d) compounds is diminished relative to 3e. The only EWGcontaining derivative that exhibits a  similar to 3e is the fluoroderivative 3c. Although 3b has a similar chloro group, the observed photoreactivity is diminished relative to 3c. Notably the absorptivity of 3c is stronger than 3b across the entire spectrum and likely contributes to the observed difference in (Error! Reference source not found.). While 3c exhibits photoactivity comparable to XDPAdeCage, the max is blueshifted by over 30 nm.</p><p>The mass peaks observed in the LC/MS spectrum of 3a-f after photolysis indicate tolyl and aldehyde derivatives analogous to those previously observed with mNPA-based photocages predominate the photoproducts (Scheme 1). 1,2 Compounds containing EDGs (3f-3i) exhibit a bathochromic shift in max compared to the parent compound 3e; however only 3f retained photodecarboxylation activity. The hydroxy compound 3g exhibits a higher (2989 M -1 , cm -1 ) and a more red-shifted absorbance (max = 427 nm) but lacks photoactivity compared to the corresponding methoxy derivative (3f). Excited state proton transfer does not appear to be responsible for the inactivity of 3g as the compound remains photochemically inert under strongly basic conditions (pH 14). Although 3f contains an electron-donating methoxy group, the photodecarboxylation and red-shifted max is the most comparable to XDPAdeCage.</p><p>While the methoxy functional group is electron-donating by resonance, inductive effects provide the most plausible explanation for the anomalous behaviour in the EDG series.</p><p>The model methoxy-and fluoro-compounds 3f and 3c exhibited the most comparable photophysical properties to XDPAdeCage, therefore we chose these chromophores to build new DPAdeCage derivatives (Error! Reference source not found.). Our initial attempts to prepare these photocages via our established route were unsuccessful due to oxidative decomposition during the final nitration reaction. Alternatively, starting with 3f and 3c provided 4 (DPAdeCageOMe) and 5 (DPAdeCageF) in 11.6% (5 steps) and 11.8% (4 steps) respectively. The only significant difference between the two synthetic pathways is the final ester hydrolysis. DPAdeCageF required acidic conditions at elevated temperature, while the basic conditions used to prepare DPAdeCage successfully provided DPAdeCageOMe. A combination of product decomposition in the harsher deprotection reaction as well as the more difficult isolation of DPAdeCageF from acidic solution, reduced the overall yield of the final product. A comparison of the synthetic pathways required to develop the new mNPA photocages and XDPAdeCage reveals both DPAdeCageOMe and DPAdeCageF can be more rapidly prepared and in greater amounts than XDPAdeCage. The photophysical properties of DPAdeCageOMe and DPAdeCageF are nearly identical to the parent compounds 3f and 3c under aqueous conditions (Error! Reference source not found.). Notably, the max of both DPAdeCageOMe and DPAdeCageF are red-shifted relative to DPAdeCage. The methoxy-functionalized DPAdeCageOMe (342 nm) has a max nearly identical to XDPAdeCage (347 nm). The addition of Zn 2+ to the photocages results in a slight hypsochromic shift of max, but relatively little change in absorptivity (Table 1). An analysis of the LC/MS spectrum of DPAdeCageOMe and DPAdeCageF following irradiation, indicate successful photodecarboxylation following irradiation. In the absence of Zn 2+ , both compounds appear to successfully release the carboxylate group based on the change in mass. In DPAdeCage, 2 a decrease in  is observed upon coordination to Zn 2+ and DPAdeCageF exhibits an even more dramatic decrease upon Zn 2+ coordination. Only extended irradiation times (>30 min) of [Zn(DPAdeCageF)] + resulted in evidence of photolysis, which severely limits the potential applications of the photocage. The addition of Zn 2+ does not appear to affect the resulting photoreaction of DPAdeCageOMe; however, DPAdeCageF forms a wider slate of photoproducts in the presence of Zn 2+ after long irradiation. Some higher mass, emissive photoproducts suggest that Zn 2+ facilitates the formation of coupled DPAdeCageF products TD-DFT calculations were performed to probe if the electronic structure contributed to the observed difference in photoreaction of [Zn(DPAdeCageOMe)] + and [Zn(DPAdeCageF)] + . The structure of DPAdeCageOMe, DPAdeCageF, [Zn(DPAdeCageOMe)] + and [Zn(DPAdeCageF)] + were optimized using DFT and the frontier molecular orbitals contributing to the electronic transitions found in the TD-DFT calculations were visualized. In all cases, the lowest energy excitation involves excitation from the HOMO to the LUMO. The LUMOs are localized around the nitro group and phenyl ring for all compounds, consistent with previous studies of mNPA chromophores (Error! Reference source not found.). 17 We believe that promoting an electron into a LUMO localized on the nitro group is necessary for photolysis to occur, suggesting [Zn(DPAdeCageF)] + should be capable of undergoing photodecarboxylation based on the calculated electronic structure. A closer examination of the observed oscillator strengths (f) representing the molar absorptivity of Zn(DPAdeCageOMe) + (f = 0.04) and Zn(DPAdeCageF) + (f = 0.01) reveal a nearly 4-fold decrease in the oscillator strength of the lowest energy transition for Zn(DPAdeCageF) + ,which may explain a decrease in observed photolysis, although other factors probably contribute to the difference given the large drop in  observed experimentally. Through a study of model mNPA chromophores, we were able to identify derivatives with comparable photophysical properties compared to the decarboxylation reaction observed with XAN groups. Specifically, fluoro-and methoxy-groups introduce a distint red-shifted absorption band; however, only the methoxy derivative retains sufficient photodecarboxylation activity when integrated into a Zn 2+ photocage. We were pleased to see DPAdeCageOMe maintains a high  when coordinated to Zn 2+ and has nearly identical photocaging properties to XDPAdeCage. A methoxy group in the 4-position also provides a potential site for modification of future photocages through an ether linkage that should not impact Zn 2+ binding significantly and will be the subject of future investigations.</p><!><p>There are no conflicts to declare.</p>

ChemRxiv

Multivalent and multifunctional polysaccharide-based particles for controlled receptor recognition

Polysaccharides represent a versatile class of building blocks that are used in macromolecular design. By choosing the appropriate saccharide block, various physico-chemical and biological properties can be introduced both at the level of the polymer chains and the resulting self-assembled nanostructures. Here, we synthetized amphiphilic diblock copolymers combining a hydrophobic and helical poly(γbenzyl-L-glutamate) PBLG and two polysaccharides, namely hyaluronic acid (HA) and laminarin (LAM). The copolymers could self-assemble to form particles in water by nanoprecipitation. In addition, hybrid particles containing both HA and LAM in different ratios were obtained by co-nanoprecipitation of the two copolymers. By controlling the self-assembly process, five particle samples with different morphologies and compositions were developed. The interaction between the particles and biologically relevant proteins for HA and LAM, namely CD44 and Dectin-1 respectively, was evaluated by surface plasmon resonance (SPR). We demonstrated that the particle-protein interaction could be modulated by the particle structure and composition. It is therefore suggested that this method based on nanoprecipitation is a practical and versatile way to obtain particles with controllable interactions with proteins, hence with the appropriate biological properties for biomedical applications such as drug delivery.Polysaccharides represent an important class of polymers for the design of functional biomaterials, especially towards biomedical 1-4 and cosmetic applications [5][6][7] . Compared to synthetic polymers, they provide better biocompatibility, biodegradability, and also diverse bioactivities depending on their structures [8][9][10][11][12] . Furthermore, as bio-sourced polymers, polysaccharides are fully renewable ingredients, which make them particularly relevant in the context of green chemistry [13][14][15] . In addition, their production can be completed in an eco-friendly way with minimal environmental impact, which perfectly matches the criteria of sustainable development for industry.Among all the available polysaccharides, hyaluronic acid (HA), a non-sulfated glycosaminoglycan (GAG), is composed of alternative units of D-glucuronic acid and N-acetyl glucosamine linked by β-1,3 and β-1,4 glycosidic bonds. HA is one of the main components of the extracellular matrix (ECM), and as such, is highly abundant in the ECM-rich tissues such as synovial fluid, vitreous body of eyes or dermis 16,17 . With a highly hydrophilic structure, HA plays important roles for water retention in skin and eyes 18,19 . In addition, the molecular weight of native HA being large (>1000KDa), the prepared solutions are generally very viscous with a shear-thinning character 20 . These properties make HA the unique space-filling material in tissues to maintain their morphology and homeostasis 21 . This is also the reason why HA is widely used in the dermal fillers for plastic surgery 5 .In addition to the functions related to its physicochemical properties, HA has numerous biological effects, especially via its specific interaction with HA binding proteins (hyaladherins) 22 , such as CD44, RHAMM, Stabilin-2, LYVE-1 and aggrecan. The interaction between HA and CD44 has been widely explored, mainly because CD44 is a glycoprotein expressed at the surface of most cell types including skin keratinocytes 23 and fibroblasts 24 , and is involved in a number of signal transduction pathways 25 . By interacting with CD44, HA exhibits activities in biological processes such as cell proliferation/migration 26 , wound healing 27 and tissue regeneration 28 . Compared to other hyaladherins, CD44 attracts researchers' high interest due to its involvement in cancer 29 . Overexpression of CD44 can be observed in cancer cells including breast, pancreas, gastric, prostate, ovarian and colon [30][31][32][33][34][35] , making CD44 + a biomarker of cancer cells 36 . Targeting overexpressed CD44 in cancer is becoming

multivalent_and_multifunctional_polysaccharide-based_particles_for_controlled_receptor_recognition

<!>Result and Discussion<!>Interaction of HA free chains and HA-based particles with CD44 observed by SPR.<!>Materials<!>Measurements<!>Methods

<p>one of the important strategies in cancer therapy 37 . As the main ligand of CD44, HA is now increasingly used in the nanomaterial design for drug delivery 38 . It has been demonstrated that the interaction of HA with targeted proteins and the relevant effects depends on its molecular weight. Indeed, native HA with high molecular weight in ECM contributes to skin integrity and prevents inflammation and angiogenesis, whereas fragmented HA is generated and involved in a range of immunological processes during the tissue injury and inflammation [39][40][41] . LAM is another important oligosaccharide that may present relevant biological activity. Indeed, as a β-D-glucan, LAM is a ligand of dectin-1 42 , a pattern-recognition receptor of immune system involved in immune response initiation during fungal infection 43,44 . By interacting with dectin-1, LAM can be used to modulate immune system and bring biological activities including immunostimulatory and antitumorous effects 45,46 .</p><p>In this study, particles were designed by self-assembly of polysaccharide-b-polypeptide block copolymers. Based on the synthesis strategy previously established in our group 47,48 , two copolymers, based on HA and LAM, were obtained. We further developed a nanoprecipitation process to obtain a range of particles with different morphologies and compositions, including particles containing only HA or LAM and hybrid particles containing both of them in different ratios. The interaction with CD44 was measured by using surface plasmon resonance (SPR). We investigated the interaction of the particles with CD44 in comparison with linear HA of different molecular weights and CD44. We demonstrated that our particles exhibit an enhanced interaction with CD44 compared to linear HA, and that the particle-CD44 interactions could be modulated by changing the ratio of HA in the hybrid particle. Furthermore, a practical method was developed based on SPR to observe the dual functionality of the hybrid particles and their ability to associate simultaneously with CD44 and Dectin-1. Altogether, our experiments demonstrated that our unique copolymer design and self-assembly approach allowed both for an increase of ligand efficacy and dual functionality of particles.</p><!><p>Polysaccharide-b-polypeptide block copolymer synthesis. A three-step synthesis based on copper-catalyzed Huisgen cycloaddition "click chemistry", previously reported by our group, was developed to obtain the polysaccharide-b-polypeptide di-block copolymer 47 . A poly(γ-benzyl-L-glutamate) with azide functional group (PBLG-N 3 ) was obtained by the ring-opening polymerization of the corresponding N-carboxyanhydride (NCA) initiated by 3-azido-1-propanamine primary amine (Fig. 1a). This controlled and living polymerization process led to the formation of PBLG-N 3 with a degree of polymerization DP = 30 (corresponding to 6.6 kDa) and low dispersity (Đ = 1.1). Propargyl functionality was introduced to the reducing end of both HA (5 kDa) and LAM (5 kDa) by reductive amination with propargylamine (Fig. 1b). Besides HA 49 and LAM 50 , other polysaccharides such as xylan, pullulan, dextran or galactan can also be functionalized in the same way [51][52][53][54][55] . The two building Chemical modifications of polysaccharides are often introduced on the lateral functional groups on their backbones such as -OH and -COOH. However, the modification ratio can only be controlled statistically and consequently brings additional variability to the resulting product. Furthermore, the biological activities of polysaccharides can be significantly altered by such side chain modifications 56,57 . We can notice that the structure of HA and LAM in our block copolymer is nearly intact compared to its natural structure. Each polysaccharide chain is modified once on the reducing end and the full conversion can be confirmed by 1 H-NMR. In this way, the chemical structure of the copolymer can be precisely controlled. The resulting nanostructures based on these copolymers can also maintain the properties of HA and LAM to associate with their target biological receptors, respectively CD44 and dectin-1, as shown later in the SPR study.</p><p>Amphiphilic copolymer self-assembly and particle formation. The two synthesized amphiphilic copolymers HA-b-PBLG and LAM-b-PBLG were self-assembled in water by using a nanoprecipitation approach, adapted from previous reports 47,58 . First dissolved in DMSO, a good solvent for both blocks, the copolymer solution was then diluted with water. The self-assembly was driven by the insolubility of the hydrophobic PBLG segments in water, that formed the particle core during solvent-displacement. Covalently attached to the PBLG in the copolymer structure, the hydrophilic polysaccharide moieties remained at the particle surface. In this way, we obtained stable self-assembled particles in water. The particle morphology could be modulated by process parameters such as mixing rate and water phase composition 59 . In our study, two process protocols, named fast nanoprecipitation and slow nanoprecipitation, have been optimized, as detailed in the Method section. Three monofunctional particle samples were thus obtained: HA-b-PBLG-30nm, HA-b-PBLG-150nm, LAM-b-PBLG-30nm as listed in Fig. 2.</p><p>The two copolymers have similar molecular structures, with the same ratio between the hydrophilic polysaccharide block and the hydrophobic polypeptide block. The resulting particles obtained by fast nanoprecipitation had consistently very similar sizes between 30 nm and 50 nm. In this study, we have identified the experimental conditions to co-nanoprecipitate HA-b-PBLG and LAM-b-PBLG copolymers together from their mix solution in DMSO during a fast nanoprecipitation process. Indeed, in these conditions, the particles were rapidly formed and instantly "frozen" during the solvent displacement. Such an out-of-equilibrium process favors the homogeneous mixture of the two copolymers in the particles, since the potential phase separation between the copolymers is minimized. The resulting hybrid particles were stable in water, containing both HA and LAM moieties. By changing the initial concentration of each copolymer from the DMSO solution, the composition of the particle can be continuously modulated. In the present study, after extensive dialysis against pure water in order to remove any trace of DMSO, particles with the same size range (around 40 nm), with relatively low polydispersity (PDI < 0.2) and negative zeta-potential (ξ ranging from −38 to −29 mV), but with composition of 90 wt% and 50 wt% of laminarin were obtained, and named Hybrid-nano 90%LAM and Hybrid-nano 50%LAM respectively (Fig. 2). All the relevant characteristics of the developed particles that will be further studied are summarized in Fig. 2.</p><!><p>Previously reported studies exhibited the capability of HA conjugates and particles functionalized with HA to recognize CD44 receptors [60][61][62][63][64] . In our group, polymer vesicles based on HA-b-PBLG have been proven efficient to target the overexpressed CD44 in cancer cells and deliver the loaded actives to limit cancer progression [65][66][67] . In the present study, the interaction between CD44 and HA-based compounds (HA of different molecular weights and the HA-containing particles) was investigated in detail by surface plasmon resonance (SPR). All the analyses were performed in non-saturated conditions, meaning that the CD44 receptors were never fully occupied by the sample. In the block copolymer, the molecular weight of the polysaccharide moiety was 5 kDa and that of polypeptide is 6.6 kDa. As a result, the copolymer and the particles formed by the copolymer, contained only 43 wt% polysaccharide, which is the active moiety to interact with the receptors. In a SPR analysis, the binding signal represents the entire mass of compounds attached to the bioreceptor-functionalized surface 68 . In case of particle-receptor interaction study by SPR, the polypeptide (PBLG) blocks, forming the particle cores, contributed to the SPR signal once the particle was attached to the surface, even they were not involved in the interaction with the receptors. In order to subtract the contribution of PBLG, the SPR signal of the particles was normalized by multiplying by 0.43, the polysaccharide weight ratio in the particle as shown in Fig. 3a. The resulting normalized signal revealed uniquely the quantity of bioactive polysaccharide involved in the interaction. However, the mass percentage of the particle samples (23ppm) in the tests was larger than that of HA samples (10ppm), so that all the samples contained the same quantity of active moiety for the interaction, which was the polysaccharide (Fig. 3b). It is important to notice that all the comparisons in this study were performed in these "double normalized conditions", meaning that the samples that we used contained the same concentration of HA, and we compare the quantity of normalized SPR signals revealing the quantity of HA associated with the surface by CD44.</p><p>As shown in Fig. 3b, the interaction between HA and CD44 increases with the molecular weight of HA, observed by enhanced SPR binding signals. The SPR signal of a high molecular weight HA (1000 kDa) was almost 5-fold stronger than that of a small molecular weight HA (5 kDa). These observations are consistent with previous studies from Dan Peer's group, also obtained by SPR 69 , and that of Ritchter and coll. through quartz crystal microbalance (QCM-D) analysis 70 . To synthesize the HA-b-PBLG copolymer, a low molecular weight HA (5 kDa) was used and consequently the resulting particles were covered with the same units of HA-5kDa. In SPR analysis (Fig. 3b), the particle sample showed a much stronger interaction with CD44 compared to a HA-5kDa sample with the same HA concentration. Even with slower kinetics, the binding signal of the particle sample was even higher than that of the highest molecular weight HA samples (100 kDa and 1000 kDa). With the same HA-b-PBLG copolymer, the particle interaction with CD44 could be modulated by its morphology, and therefore by the formulation process. Indeed, as observed in Fig. 3c, the SPR signal of larger HA-particles (150 nm) was significantly higher than that of those of smaller diameters (30 nm). These observations are consistent with recent in vitro experiments performed on different lung cancer cell lines expressing different level of CD44 71 . Richter's lab also recently observed the multivalent interaction between CD44 and high molecular weight HA by a multivalent interaction model 72 . They especially demonstrated by single molecule force spectroscopy that CD44/HA bonds have a high tensile strength despite their low affinity, and that multiple bonds along an HA chain rupture independently under load. Our experiments suggest that this interaction enhancement with the HA particle performed with the same principle. As schematically illustrated in Fig. 3d, a free HA-5kDa moiety in the solution can probably associate with only one or a very low number of CD44, whereas the HA particles were able to bind simultaneously a larger number of CD44. These multivalent interactions strengthen the interfacial force, and hence the binding signal in SPR analysis. This was also the reason why we were unable to measure the association and the dissociation constants. Indeed, to obtain these constants by curve fitting algorithms, it is essential to know the precise interaction model (1:1, 1:2 etc.) between the receptors and the analytes in the sample, which is very challenging with any particle systems.</p><p>Interaction modulation by controlling the particle composition. The interaction of the three HA-containing particles with CD44 was further compared with different HA content toward LAM (meaning HA-b-PBLG-30nm, Hybrid-nano 50%LAM and Hybrid-nano 90%LAM). All of them presented significant interaction signals with CD44, but the binding level was reduced with the decreasing ratio of HA in the particle as shown in Fig. 4. The three different particle samples were obtained by the same fast nanoprecipitation with similar sizes (Fig. 4), so that they were able to get in contact with a similar quantity of CD44 on the SPR surface. However, LAM was unable to associate with CD44. By introducing LAM on the particle surface, the density of HA on each particle was reduced, and so was the multivalency degree of the interaction with CD44 as well. As a consequence, the interaction strength with CD44 can be weakened by this dilution and modulated by the 'dilution' factor, which explains the SPR signal reduction in Fig. 4.</p><p>The dual functionality of the hybrid particle confirmed by SPR. The hybrid particles were obtained by co-nanoprecipitating the both HA-b-PBLG and LAM-b-PBLG copolymers from a common solution. By formulating polysaccharide moieties in a nanomaterial, it is possible to modulate the bioactivities of the resulting structure such as biological process regulation and inflammatory responses 73 . However, it was important to confirm that the two copolymers self-assemble together in our process and the resulting particle sample was not a mixture of the monofunctional particles. For this purpose, a two-step assay based on SPR was designed to check the coexistence of HA and LAM on the hybrid particle surface, hence its dual functionality. As illustrated in Fig. 5a, the CD44 surface in this assay can interact with the HA moieties and capture the hybrid particle in the first step. A short natural dissociation step was applied to confirm the stable interaction between the sample and the CD44 surface. Then, a second injection was directly performed with dectin-1, a specific biological receptor of LAM 44 . Dectin-1 can associate with LAM units on the hybrid particle but not with HA, and form the sandwich-like structure shown in Fig. 5a. The adsorption of dectin-1 at the surface of the particles can generate a positive binding signal during the injection. As shown in Fig. 5b,c, both hybrid particle samples gave positive binding signals as expected during the two injection steps. Meanwhile as a negative control, the same assay was applied to a mixture of monofunctional particles based on HA and LAM (Fig. 5d). The CD44 surface captured the HA particles and gave a binding signal during the first injection. However, no binding signal was observed during the second injection of Dectin-1, since no LAM was present on the surface of the HA particles. These observations really proved that (i) only particles with HA can bind to CD44 and that there is no unspecific adsorption of LAM based particles, and (ii) the LAM polysaccharide chains present at the surface of hybrid particles are still available for interaction with Dectin-1. By increasing the ratio of LAM on the particle surface, the interaction signal with CD44 decreased whereas that with dectin-1 increased. Through this assay, the dual functionality of the hybrid particles to interact simultaneously with CD44 and dectin-1 is confirmed and thus the concept of interaction modulation by changing the particle composition.</p><p>In summary, we have reported a versatile strategy to design particles with tunable interaction with targeting proteins by the combination of different amphiphilic copolymers in a nanoprecipitation process. Using two different polysaccharide-b-polypeptide copolymers, namely HA-b-PBLG and LAM-b-PBLG, a broad range of particles was obtained by changing the formulation process parameters. The control of the nanoprecipitation and co-nanoprecipitation processes allows the design of particles with controlled sizes and compositions in an accurate and reproducible manner. As demonstrated using SPR as a fast and efficient screening method, the interaction of the HA-particle with CD44 was far stronger than that of linear HA on its surface, which can be explained by a multivalent interaction between HA segments on the particle surface and CD44. The "multivalent-degree" can be controlled by changing the particle morphology and composition, hence leading to the modulation of the interaction strength. The combination of the two copolymers results in the formation of hybrid particles with the functionalities from both HA and LAM, as confirmed by an original method based on SPR analysis. All these observations and experimental results strongly suggest that such a simple co-nanoprecipitation process can be a versatile and practical way to design multifunctional particles with tunable biological activities.</p><!><p>Hyaluronic acid (HA) sodium salt of different molecular weights (research grade, HA-5kDa, HA-20kDa, HA-100kDa, HA-1000kDa) was purchased from LifeCore Biomedical (Cheska, MN, USA). Laminarin from Laminaria Digitata and all the chemicals used in the copolymers synthesis were purchased from Sigma Aldrich (St. Louis, MO, USA). Recombinant human CD44 and dectin-1 were purchased from R&D system (Minneapolis, MN, USA). CM5 chip and all the solvents and reagents for SPR analysis including the running buffer, N-hydroxysuccinimide (NHS), ethyl-3(3-dimethylamino)propylcarbodiimide (EDC), 1 M ethanolamine solution pH 8.5, the regeneration solution were purchased from GE Healthcare (Uppsala, Sweden) and used as suggested. Ultrafiltration discs were purchased from EMD Millipore (Billerica, MA, USA).</p><!><p>1 H-NMR spectra were obtained with a Bruker Avance 400 MHz spectrometer (Rheinstetten, Germany). Dynamic light scattering was measured Malvern Zetasizer NANO ZS (Worcestershire, UK). Surface plasmon resonance analysis was performed with Biacore T200 (Uppsala, Sweden). IR spectra were recorded on Perkin Elmer Spectrum One FT-IR (Shelton, CA, USA).</p><!><p>Synthesis of HA-b-PBLG and LAM-b-PBLG. HA-b-PBLG was prepared as reported elsewhere 47 , and LAM-b-PBLG was prepared using the same synthetic approach. The polysaccharide blocks have a 5 kDa molecular weight and the same PBLG of 6.6 kDa was used for the click reactions. The copolymers were analyzed by Particle preparation by nanoprecipitation-induced self-assembly. As mentioned in Fig. 2, the experimental conditions of two processes, fast and slow nanoprecipitation, have been optimized as reported below:</p><p>Fast nanoprecipitation: 9 ml PBS buffer (10 mM, pH = 7.4, 154 mM ionic strength) was heated to 50 °C and stirred at 500 rpm by a magnetic rotor. 1 ml copolymer solution in DMSO (1 wt%), previously heated to 50 °C, was added dropwise to the PBS buffer. The resulting solution was further stirred at 50 °C for 30 min then cooled down to room temperature. DMSO was removed by ultrafiltration with the PBS buffer against a MWCO = 100 kDa filter. The particle size was measured by DLS with a scattering angle at 173°. Slow nanoprecipitation: 1 ml copolymer solution in DMSO (1 wt%) was maintained at 60 °C and stirred at 500 rpm. 9 ml PBS buffer (10 mM, pH = 7.4, 154 mM ionic strength) was added dropwise for 400 seconds by a syringe pump. The resulting solution was further stirred at 60 °C for 30 min then cooled down to room temperature. DMSO was removed by ultrafiltration with the PBS buffer against a MWCO = 100 kDa filter. The particle size was measured by DLS with a scattering angle at 173°.</p><p>HA-b-PBLG-30nm was obtained by using a 1 wt% solution of HA-b-PBLG in DMSO with fast nanoprecipitation.</p><p>HA-b-PBLG-150nm was obtained by using a 1 wt% solution of HA-b-PBLG in DMSO with controlled nanoprecipitation.</p><p>LAM-b-PBLG-30nm was obtained by using a 1 wt% DMSO solution of LAM-b-PBLG in DMSO with fast nanoprecipitation.</p><p>Hybrid-nano 90%LAM was obtained by using a DMSO solution containing 0.9 wt% LAM-b-PBLG and 0.1 wt% HA-b-PBLG with fast nanoprecipitation.</p><p>Hybrid-nano 50%LAM was obtained by using a DMSO solution containing 0.5 wt% LAM-b-PBLG and 0.5 wt% HA-b-PBLG with fast nanoprecipitation. SPR analysis: surface functionalization and interaction analysis. CM5, a carboxymethylated dextran sensor chip from GE Healthcare, was used for SPR analysis. Recombinant human CD44 was immobilized on the CM5 chip by using a standard amine coupling protocol of Biacore T200. Briefly, the sensor chip flow cell was activated by an EDC/NHS mixture for 420 seconds. CD44 was dissolved in 10 mM acetate buffer pH 4 at 10 µg/ ml then injected to the activated surface for 300 seconds. About 4000RU of CD44 was immobilized on the chip during the injection. The remaining activated positions on the chip were then deactivated by 1 M ethanolamine pH 8.5. A blank flow cell was prepared as a reference by the same protocol without CD44 injection.</p><p>HBS-EP+ buffer, proposed by GE Healthcare, was chosen as the running buffer to perform all SPR analysis in this study. All the sample injection was performed at a rate of 30 µl/min. For the study in Fig. 4, the solutions of HA of different molecular weights (5 kDa, 20 kDa, 100 kDa, 1000 kDa) was prepared at 10 ppm in the running buffer, whereas those of HA-b-PBLG-30nm and HA-b-PBLG-150nm were prepared at 23ppm so that all the samples contain the same quantity of HA for comparison. For the same reason, HA-b-PBLG-30nm was prepared at 100ppm, Hybrid nano-50%LAM was prepared at 200ppm and Hybrid nano-90%LAM was prepared at 1000ppm for the study in Fig. 4. The particle samples were injected at 100ppm and dectin-1 solution was added at 10 µg/ml in the study shown in Fig. 5. The responses on the blank flow cell were systematically subtracted from the signal obtained on the CD44 coated flow cell to remove the contribution of unspecific interaction independent from CD44.</p><p>The surface was regenerated by adding 50 mM NaOH for 30 seconds after each analysis. The full removal of the analyte attached to CD44 was confirmed by the baseline level after the regeneration.</p>

Scientific Reports - Nature

Molecular recognition using receptor-free nanomechanical infrared spectroscopy based on a quantum cascade laser

Speciation of complex mixtures of trace explosives presents a formidable challenge for sensors that rely on chemoselective interfaces due to the unspecific nature of weak intermolecular interactions. Nanomechanical infrared (IR) spectroscopy provides higher selectivity in molecular detection without using chemoselective interfaces by measuring the photothermal effect of adsorbed molecules on a thermally sensitive microcantilever. In addition, unlike conventional IR spectroscopy, the detection sensitivity is drastically enhanced by increasing the IR laser power, since the photothermal signal comes from the absorption of IR photons and nonradiative decay processes. By using a broadly tunable quantum cascade laser for the resonant excitation of molecules, we increased the detection sensitivity by one order of magnitude compared to the use of a conventional IR monochromator. Here, we demonstrate the successful speciation and quantification of picogram levels of ternary mixtures of similar explosives (trinitrotoluene (TNT), cyclotrimethylene trinitramine (RDX), and pentaerythritol tetranitrate (PETN)) using nanomechanical IR spectroscopy.

molecular_recognition_using_receptor-free_nanomechanical_infrared_spectroscopy_based_on_a_quantum_ca

D<!>Results<!>Discussion<!>Methods

<p>etection, speciation, and quantification of extremely small concentrations of explosive vapors with high selectivity and sensitivity have immediate applications in many areas such as national security, forensics, and humanitarian demining 1 . Microfabricated chemical sensors are being actively investigated as potential sensor platforms capable of mass deployment. Despite having many advantages such as miniature size, high sensitivity, low-cost, and low power consumption, the microfabricated sensors suffer from poor chemical selectivity. Miniature sensors rely on adsorption-induced changes in physical variables such as adsorbed mass, surface stress, refractive index, resistance, capacitance, and temperature which are sensitive indicators of molecular binding. Since the changes in physical properties due to molecular adsorption are not chemically specific, sensors are usually modified with receptors (chemical interfaces) which can provide selectivity. Immobilized chemoselective interfaces, however, can only provide partial selectivity due to the unspecific nature of chemical binding; especially those based on weak intermolecular interactions such as hydrogen bonding. The interference from other chemical vapors which cause unacceptable levels of false positives is a major challenge for all sensors based on analyte interactions with immobilized chemical interfaces. Therefore, achieving chemical selectivity in a mixture of unknown chemical vapors is extremely difficult without resorting to molecular separation. Even approaches based on sensor arrays immobilized with unique chemical interfaces and subsequent analysis of array response using pattern recognition algorithms fail when it comes to ternary mixtures [2][3][4][5][6] .</p><p>Adding to the challenge is the low vapor pressure of explosives, which severely limits the number of molecules reaching the sensor surface in an acceptable detection time which requires extremely high sensitivity. In addition, explosive vapors can be present with a variety of vapors in the sensing environment and this can interfere with the selectivity of detection. Therefore, an urgent need exists for developing techniques which are selective, sensitive and quantitative for different mixtures of explosive vapors. In order to be effective, the sensor must have the ability to differentiate between the explosive molecules and other similar compounds. For surface adsorption-based sensors, interfering chemicals which have a high vapor pressure, such as volatile organic compounds, do not cause interference since they do not adsorb well on the surface at room temperature 7 . However other materials with very low vapor pressure, such as different types of explosives, can cause challenges in selective detection and quantification.</p><p>Unlike sensing paradigms based on immobilized chemoselective interfaces on sensor surfaces, spectroscopic techniques based on unique molecular vibrational transitions in the mid infrared (IR) ''molecular fingerprint'' regime, where many molecules display characteristic vibrational peaks free from overtone, are highly selective 8 . Spectroscopic signal from a mixture of molecules follows the superposition principle, unless there are intermolecular interactions. This is different from sensing based on partially selective chemoselective interfaces, which fails in mixtures due to the lack of orthogonality in sensor responses. Although mid-IR absorption spectroscopy based chemical sensing offers high selectivity, it lacks sensitivity when used for detection of surface adsorbed chemicals.</p><p>Photothermal cantilever deflection spectroscopy (PCDS), which combines the extreme thermal sensitivity of a bi-material microcantilever with the high selectivity of mid-IR spectroscopy, is capable of obtaining molecular signatures of trace amounts of adsorbed molecules on the cantilever surface [9][10][11][12][13][14][15] . In the PCDS technique, the target molecules are first allowed to adsorb on a bi-material cantilever. During resonant excitation of target molecules using IR light, the bi-material cantilever undergoes deflection, the amplitude of cantilever deflection as a function of IR wavelength resembles the infrared absorption spectra of the adsorbed molecules. Unlike conventional IR absorption spectroscopy, in which a small intensity change is collected by cryogenically cooled mid-IR detectors in a large background with inherent laser source noise, PCDS, an ''action spectroscopy'', measures the photothermal effect of a small number of adsorbed molecules with a high photon flux taking full advantage of the high brightness of a quantum cascade laser (QCL) light source. Therefore, the PCDS signal strength scales with the intensity of incident photons while the noise in PCDS mainly comes from thermomechanical noise of a microcantilever. Using a high power tunable QCL, we have been able to increase the sensitivity of detection by one order of magnitude. This, therefore, lays the foundation for the enhancement of the sensitivity of detection by increasing the intensity of the excitation source.</p><p>In this report, we demonstrate the successful implementation of PCDS for selective detection and quantification of the ternary mixtures of similar explosive molecules (trinitrotoluene (TNT), cyclotrimethylene trinitramine (RDX), and pentaerythritol tetranitrate (PETN)), which represent the three most commonly found explosive classes (nitro-aromatics, nitramines, and nitrate esters) with tens of picogram resolution in ambient condition. In addition, we improve the limit of recognition (LOR), the maximum recognizable mixture composition range, by an order of magnitude (,3251) with a 100% recognition rate, by calibrating and analyzing PCDS spectra compared to the LOR of immobilized chemoselective interfaces-based multi-transducer array microsensors. In contrast to an immobilized chemoselective interfaces-based microsensor array, which has a relatively short operation-life due to the degradation of coating in ambient condition, resulting in the loss of selectivity and capability of the quantitative detection, the PCDS technique utilizes a very robust single microstructure without any chemoselective interfaces. Significant field applications potential is demonstrated by the selective detection and quantification of ternary mixtures of explosive molecules in ambient condition.</p><!><p>The PCDS setup used in this study is shown in Fig. 1a. Inherently, the PCDS provides two orthogonal signals in a single transducer platform. The nanomechanical IR spectrum, a differential plot of the amplitude of the cantilever deflection as a function of impinging IR wavelength with and without target molecules, represents molecular signatures of the target molecules adsorbed on the cantilever surface while the resonance frequency change of the microcantilever gives real time information of adsorbed mass (Fig. 1b). The bi-material microcantilever serves as an extremely sensitive thermal sensor as well as a microresonator for the detection, speciation, and quantification of ternary mixtures of the explosive molecules. When IR photons are absorbed by the explosive molecules on the cantilever surface, the explosive molecules undergo transitions from the n50 ground vibrational states to the n51 excited states (Fig. 1c). The high frequency low density normal modes (represented on a sharp Morse potential) is coupled to the high n excited states of other low frequency high density normal modes (one of which represented on a flat Morse potential) via intramolecular vibrational energy redistribution. Eventually, the energy is released to the phonon bath of the bi-material cantilever surface through multiple steps of vibrational energy relaxation. These nonradiative decay processes result in heating up the bi-material cantilever, generating the deflection of the cantilever.</p><p>Within certain dynamic ranges, the normalized peak amplitudes of the nanomechanical IR spectrum can be utilized to estimate the relative mass ratio of each target molecule in a mixture since the IR spectrum of a mixture is a linear superposition of individual spectra. Fig. 2 shows the normalized nanomechanical IR absorption spectra of TNT (black), RDX (green), PETN (red), 15151 mixture of these three explosives (blue), a weighted linear superposition of individual explosive spectra (sky blue), and the Fourier transform infrared (FTIR) spectrum of the ternary mixture (orange). The PCDS spectra of the individual explosives were taken separately as references and agreed quite well with our previous report 15 . These spectra were acquired with a monochromatic IR source and normalized by the adsorbed mass of explosive molecules for calibration purposes. The adsorbed mass of TNT, RDX, PETN, and the ternary mixture on the cantilever was 6.78 ng, 6.44 ng, 6.99 ng, and 9.8 ng respectively, as calculated using Eq. 2. The relative mass ratio of the ternary mixture (TNT5RDX5PETN) adsorbed on the cantilever surface was estimated to be 0.3650.150.54 from the mathematical fitting of PCDS spectrum of the ternary mixture with normalized PCDS spectra of individual explosive molecules. Therefore, the actual mass of TNT, RDX, and PETN on the cantilever surface was determined to be 3.53 ng, 0.98 ng, and 5.29 ng respectively. Even though the same volume for each explosive solution with same concentration was mixed, the actual mass of each explosive adsorbed on the cantilever surface was quite different due to different molecular affinity to the silicon oxide surface. Since their vapor pressures are different, desorption rates from the surface are also different 7 .</p><p>Several distinct peaks and shoulders appeared in the ternary mixture spectrum since the mixture spectrum is a linear superposition of individual spectra (note that spectral resolution of our IR monochromator in this range is approximately 0.12 mm). The peaks at 6.06, 6.38, and 6.49 mm are due to the asymmetric stretching of the NO 2 (nitro) group bonds while the peaks at 7.27, 7.46, and 7.82 mm are from the symmetric stretching of the same group bonds. Comparing these peaks with those of individual TNT, RDX, and PETN spectra, it is apparent that the peaks at 6.49 and 7.46 mm are from TNT, the peaks at 6.38 and 7.27 mm are from RDX, and the peaks at 6.06 and 7.82 mm are from PETN molecules [16][17][18][19][20] . It is interesting to note that the prominent RDX peak at 7.57 mm is not clear in the PCDS spectrum and the prominent PETN peak at 7.82 mm is missing in the FTIR spectrum due to the difference in the relative mass ratio between RDX and PETN. Other than these two differences, all of the characteristic peaks are separate and distinct, matching closely with each other. This demonstrates the capability of PCDS to distinguish between these closely related explosive molecular species. Since the peaks and shoulders are highly distinguishable, we can surmise that PCDS can detect differences between such closely related molecular species and anticipate that PCDS can distinguish between other interfering compounds and target molecules while sensing in ''real world'' environments.</p><p>We have improved the limit of detection (LOD) and explored the LOR of this PCDS setup by employing a tunable QCL as a powerful IR source. Fig. 3a presents the normalized peak amplitude of RDX at 7.57 mm (green squares) and the standard deviation of spectrum noise in a non-absorbing region (violet circles) as a function of the incident laser power. The inset shows the magnified view of the standard deviation of noise. The straight lines are the linear fit of the normalized peak amplitudes and the standard deviation of noise. Although the noise increased when increasing the incident laser power, the PCDS signal enhancement dominated and consequently signal-to-noise ratio (SNR) increased in our tested power range. Fig. 3b shows normalized PCDS spectra of TNT (black), RDX (green), and PETN (red) acquired with the same cantilever using the maximum power of QCL in our tested range with a 5 nm spectral resolution. The peaks between 7.1 and 8.0 mm are from the symmetric stretching vibration of the NO 2 (nitro) group bonds with carbon (C-NO 2 ) in TNT (7.46 mm); nitrogen (N-NO 2 ) in RDX (7.57 mm); and oxygen (O-NO 2 ) in PETN (7.82 mm), respectively. The peak amplitudes at 7.46, 7.57, and 7.82 mm for TNT (black squares), RDX (green triangles), and PETN (red circles) were plotted as a function of adsorbed mass of each explosive molecule in Fig. 3c to explore the LOD of the PCDS setup. The straight lines are the linear fit of the peak amplitudes for TNT (black), RDX (green), and PETN (red) respectively. It was estimated that the limit of detection for TNT, RDX, and PETN is 39 pg, 28 pg, and 79 pg, respectively with an SNR of 3.</p><p>The concept of a LOR was introduced more than a decade ago and is well established as an additional criterion for evaluating the performance of a vapor sensor array 21 . Originally, LOR was defined as the maximum recognizable mixture composition range which can be reliably determined from the response pattern of a sensor array. This is especially important in the speciation of vapor mixture components. To estimate the LOR of our PCDS setup, 15 ternary mixtures of standard explosives samples were prepared using varying volume ratios of each standard sample solution. Fig. 4a shows the normalized PCDS spectra of ternary mixtures (TNT5RDX5PETN) of explosives with the volume ratio of the TNT sample solution increasing from 15151 to 155151. The intensity of a peak is directly proportional to the adsorbed mass. The actual masses of TNT, RDX, and PETN on the cantilever surface were determined with the total adsorbed mass from the resonance frequency shift measurements and the estimated relative mass ratios obtained from the mathematical fitting of the PCDS spectra of the ternary mixtures with linear superpositions of normalized PCDS spectra of individual explosive molecules. A representative fitting result is provided in Supplementary Information. In a similar manner, the normalized PCDS spectra of ternary mixtures of explosives with increasing RDX and PETN concentrations (Fig. 4b and 4c) were analyzed and the relative mass ratios were plotted with respect to the relative mass ratio of increasing explosives as shown in Fig. 4d. It was estimated that the LOR based on the LOD of our PCDS setup for TNT to RDX is 3251 and RDX to TNT is 3151 with an SNR of 3 which ensures there are no false positives or negatives. The LOR for TNT to PETN is 2351 and PETN to TNT is 3251; the LOR for RDX to PETN is 3051 and PETN to RDX is 2651 with the same SNR.</p><p>The results demonstrate that the PCDS technique using a very robust single microcantilever transducer without any chemical interfaces overcomes the LOR of immobilized chemoselective interfacesbased multi-transducer array microsensors up to an order of magnitude 6 and achieves room temperature reversibility without leading to unacceptable levels of false positives or negatives.</p><!><p>The relationship between adsorbed mass and resonant frequency shift is given by 22 Dm~m</p><p>where Dm is the mass of the adsorbate and m 0 is the mass of the clean cantilever. E 0 , t 0 , and f 0 are the initial values of the Young's modulus, thickness and resonance frequency of the cantilever, respectively. DE, Dt, and Df are the changes in the Young's modulus, thickness, and resonance frequency of the cantilever, respectively. In this study, if the changes in thickness and Young's modulus are negligible and explosive molecules are considered uniformly adsorbed on the surface, then Eq. 1 can be simplified to:</p><p>Using this equation, the adsorbed mass of explosives on the cantilever is determined.</p><p>In PCDS, the amplitude of cantilever deflection depends on the impinging power of IR, the IR absorption mode and the amount of adsorbed molecules as well as the thermal sensitivity of the cantilever 15 . Although the PCDS signal can be further increased by enhancing the thermal sensitivity of the microcantilever as well as by increasing the impinging power of IR, we should take thermomechanical noise, a dominant noise source for PCDS, into account in order to evaluate SNR which determines the limit of detection 23,24 . The root mean square amplitude of cantilever deflection from thermomechanical noise at well below the resonance frequency is given by 25</p><p>where k B is the Boltzmann constant, T is the absolute temperature, B is the measurement bandwidth, Q is the quality factor, k is the spring constant, and v 0 is the angular resonance frequency of the cantilever. Although the thermomechanical noise increases when increasing the incident IR laser power due to heating effect, the maximum temperature rise in the cantilever is linearly proportional to the incident laser power as detailed in Supplementary Information. Therefore, thermomechanical noise grows slower than the PCDS signal shown in Fig. 3a and consequently SNR increases.</p><p>The LOR for the PCDS setup also can be further extended by using broadly tunable QCLs ranging from 5.5 mm to 8.0 mm which covers the asymmetric and symmetric stretching vibrations of the NO 2 (nitro) group bonds in explosive molecules. The more characteristic peaks of analytes we have, the wider the range of recognizable mixture composition we can expect to achieve. With the advent of miniature IR sources, it is possible to decrease the size of the device into a handheld one. Therefore, significant field application potential is anticipated by miniaturizing a broadly tunable IR source and readout electronics which are integrated into a microcantilever sensor system.</p><p>We have observed that, although the positions of the absorption peaks in the PCDS spectra agree closely with those in conventional IR spectra, the relative intensities of the observed PCDS peaks do not 15 . We reasoned that, unlike conventional IR absorption spectroscopy which measures the molecular IR absorption following the Beer-Lambert law, PCDS signals come from the nonradiative decayinduced thermal variation of the cantilever as a result of molecular IR absorption and multi-step coupling of the vibrational excited states of surface adsorbed molecules to the phonon bath of the cantilever through intramolecular vibrational energy redistribution and vibrational energy relaxation. Therefore, the differences between the relative peak intensities of PCDS and conventional IR spectra could be used for quantifying the vibrational dynamics of the adsorbed molecules and the energy transfer processes on the surface.</p><!><p>Chemicals. Three standard explosive samples (TNT, RDX, and PETN) were purchased from AccuStandard, Inc. (New Haven, CT) and used without further purification. As indicated by the manufacturer, the standard concentration of each explosive is 1 mg/mL.</p><p>Preparation of a microcantilever. Rectangular silicon cantilevers (CSC12-E) were obtained from MikroMasch USA (San Jose, CA). The dimension of each cantilever was 350 mm in length, 35 mm in width, and 1 mm in thickness. The microcantilevers were cleaned by rinsing with acetone, ethanol, and a UV ozone treatment then coated with 10 nm of chromium (adhesion layer) followed by 200 nm of gold using an e-beam evaporator. The individual explosive and ternary mixtures (by volume of standard sample solution) of explosive molecules (TNT, RDX, and PETN) were deposited on a microcantilever sequentially using micro glass capillaries and the cantilever was completely regenerated by UV ozone cleaning following each measurement.</p><p>The PCDS experimental setup. For the PCDS experiments, the explosives-deposited cantilever was mounted on a stainless steel cantilever holder which was attached to the head unit of MultiMode atomic force microscope (AFM) (Bruker, Santa Barbara, CA). The deflection and resonance frequency of the microcantilever were measured using the optical beam deflection method with a laser diode and a position sensitive detector. The IR radiation from the monochromator (Foxboro Miran 1A-CVF) was mechanically chopped at 80 Hz and focused on the cantilever. The IR wavelength was scanned from 2.5 mm to 14.5 mm (4000 cm 21 to 690 cm 21 in wavenumber) and has a resolution of 0.05 mm at 3 mm, 0.12 mm at 6 mm, and 0.25 mm at 11 mm according to the manufacturer. The 200 kHz pulsed IR radiation with 10% duty cycle from the QCL (Daylight Solutions, UT-8) was electrically burst at 80 Hz using a function generator DS345 (Stanford Research Systems, Sunnyvale, CA) and directed to the cantilever. The laser power was measured with a FieldMax II laser power meter (Coherent Inc., Santa Clara, CA). The IR wavelength was scanned from 7.1 mm to 8.3 mm (1408 cm 21 to 1204 cm 21 in wavenumber) with a spectral resolution of 5 nm. The nanomechanical IR spectra were taken using a SR850 lock-in amplifier (Stanford Research Systems, Sunnyvale, CA) and the resonance frequencies of the microcantilever were measured with a SR760 spectrum analyzer (Stanford Research Systems, Sunnyvale, CA).</p><p>FTIR microscope. The ternary mixture of explosive molecules on the microcantilever chip was characterized using a standard FTIR technique as a reference. Two (2) microliters of 15151 mixture solution was drop-cast onto the microcantilever chip, and the FTIR spectra were obtained using an FTIR microscope (Nicolet Continumm FTIR microscope) in reflection mode. The number of registered scans was 200 with resolution of 4 cm 21 .</p>

Scientific Reports - Nature

Ultrafast Realization of Ionic Liquids with Excellent CO 2 Absorption: A trinity study of machine learning, synthesis, and precision measurement

Efficient CO2 capture is indispensable for achieving a carbon-neutral society while maintaining a high quality of life. Since the discovery that ionic liquids (ILs) can absorb CO2, various solvents composed of molecular ions have been developed and their CO2 solubility has been studied. However, it is challenging to optimize these materials to realize targeted properties as the number of candidate ion combinations for designing novel ILs is of the order of 10 18 . In this study, electronicstructure informatics was applied as an interdisciplinary approach to quantum chemistry calculations, and combined with machine learning to search 402,114 IL candidates to identify those with better CO2 solubility than known materials. Guided by the machine-learning results, trihexyl(tetradecyl)phosphonium perfluorooctanesulfonate was synthesized and it was experimentally confirmed that this IL has higher CO2 solubility than trihexyl(tetradecyl)phosphonium bis(trifluoromethanesulfonyl)amide, which is the previous best IL for CO2 absorption. The method developed in this study could be transferable to gas-absorbing liquids in general, such as deep eutectic solvents (hydrogen-bonded mixed organic solvents in a broad sense), which also have numerous practical applications. Therefore, we believe that our method for developing functional liquids will significantly contribute to the development of a carbon-neutral society.

ultrafast_realization_of_ionic_liquids_with_excellent_co_2_absorption:_a_trinity_study_of_machine_le

INTRODUCTION<!>METHODS<!>Class<!>Fluorineorganic<!>RESULTS AND DISCUSSION

<p>Developing CO2-absorbing materials with better performance is a critical step toward achieving efficient CCUS (carbon dioxide capture, utilization, and storage), which has been proposed as one strategy to address global warming. 1 While there are several types of CO2 absorbers, the intensive study of ionic liquids (ILs: room temperature molten salts) would give basic science contributing to environmental engineering toward the development of a safe and sustainable society.</p><p>ILs are highly stable (low volatility and highly heat resistant) and can be chemically designed with a wide variety of ion combinations. [2][3][4][5] ILs have been applied to various gas-fixing technologies, such as temperature/pressure swing, chemical/physical absorption, and cryogenics/membrane-based separation. 6,7 A major limitation of the development of ILs for applications is the combinatorial explosion of the ion species, i.e., there are 10 18 ILs in theory. 2 Even when ILs are limited to physical absorbents (those not leading the CO2 chemisorption), the optimization of the CO2 absorption capacity is experimentally challenging. The synthesis of ILs and characterization of their physical properties are more labor intensive than for electrically neutral molecular liquids due to their high viscosities.</p><p>Despite the vast number of candidates, the record for the maximum amount of CO2 physically absorbed by an IL has not been broken for over a decade. 8 To date, performing a series of targeted syntheses followed by high-precision experimental measurements have been necessary to obtain practical CO2 absorbers.</p><p>Machine learning has proven to be an excellent approach for such combinatorial problems. 9-13 However, to date, machine learning has not been applied to identify better ILs for CO2 absorption in a comprehensive manner including molecular design, synthesis, and CO2 solubility observations. One of the simple reasons is that the chemical structure of ionic species cannot be uniquely defined due to charge delocalization. In addition, slight differences in the electronic states of ionic species, which can have critical effects on the properties of ILs, are challenging to describe using fingerprints.</p><p>Considering this background, this study focused on a materials informatics approach based on quantum chemistry calculations to construct a sizeable electronic structure database for ILs (Figure 1). This study aimed to develop a method to search for better CO2-absorbing ILs with high accuracy and a much shorter development time by using quantum-chemical features and machinelearning techniques (electronic structure informatics). The prediction model is proposed for quick identification of ILs with better CO2 absorption properties which is applicable for large group of materials. To prove the effectiveness of the method, the synthesis of two promising candidates and the measuring of their CO2 solubilities are performed.</p><!><p>First, a theoretical screening was performed based on quantum chemistry calculations and machine learning. Following our previous studies, 14,15 6,991 stable ion structures (6,933 cations and 58 anions in Figure 2 and Table 1) were explored by density functional theory calculations at the BP/TZVP-D3 level. [16][17][18][19][20] Using the surface-charge-density distributions (σ-profiles) [21][22][23][24] and the optimal structures, the geometric and electronic features given in Table 2 were calculated for all of the ions. Then, a set of Henry's law constant (! !" ! ) values at 298.15 K was evaluated by COSMO-RS theory [21][22][23]25 for 20,000 ILs (randomly selected from the total 402,114 candidates). Half of this set were used to train a machine learning model to predict ! !" ! for the other IL candidates in this study. Using a cycle of feature selection, model creation, and performance evaluation, the essential molecular (ion) features for the CO2 absorption problem were systematically selected (wrapper method). 26 For model creation, the Gaussian process regression method with the ARDMatern 5/2 kernel 27 and 5-fold cross-validation were applied to the standardized data. The performance of the created model was evaluated with 10,000 sets of test data from the value of the coefficient of determination (R 2 ), the root mean squared error (RMSE), and the mean absolute error (MAE). Using the well-trained model, superior ILs for CO2 absorption were predicted within a minute. The quantum chemistry and statistical thermodynamics calculations and machine learning were performed with TURBOMOLE 7.0. 28 , COSMOtherm C30_1705 29 , and MATLAB 30 packages.</p><!><p>Structures Fluorineinorganic Nonfluorineinorganic</p><!><p>Nonfluorineorganic respectively. These ILs were synthesized according to methods in the literature. 31,32 The density (ρ), viscosity (η), and CO2 solubility (3 +, ! ) under atmospheric to high pressure conditions were measured for the ILs using a vibrating tube densimeter (Anton Paar, DMA 5000M), a rotating-cylinder viscometer (Anton Paar, Stabinger SVM 3000), and a magnetic suspension balance (Rubotherm GmbH) (see Supporting Information and references [33][34][35][36][37][38][39] ). 4 +, ! was evaluated for the ILs as the solubility gradients ( -.</p><p>-/ "# !</p><p>) in the region of 3 +, ! < 0.1. Finally, the Gibbs energy (DabsG ∞ ), enthalpy (DabsH ∞ ), and entropy (DabsS ∞ ) of CO2 absorption for the ILs were experimentally obtained using the following thermodynamic relations (1)-( 3) as a function of ln 4 +, ! , 40</p><p>where DabsH ∞ and DabsS ∞ are closely related to the solute-solvent interaction strength and the free volume size of the solvent.</p><!><p>The accuracy of the machine-learning was confirmed by the learning curves (Figure 3). It was shown that highly accurate HCO2 predictions (R 2 > 0.90, RMSE< 1.5 MPa, MAE< 0.13 MPa)</p><p>were achieved when 12 features were applied (Figure 3(a)). The wrapper method clarified that the first-and second-most important features were geometric V and electronic M2. This is considered reasonable because CO2 molecules are physically absorbed in the polar voids that consist of ions in the ILs. It was also confirmed that the 10,000 sets of training data (2% of the candidates) were sufficient for obtaining an accurate model (Figure 3(b)). S2 and S4). Both the mole fraction and molarity-scaled CO2 solubilities were also measured (Figure 4 and Table S6). The solubilities proportional to pressure means that the ILs absorb CO2 by a physisorption mechanism.</p><p>The CO2 solubility increased in the anion order of PF6 − < TFSA − < PFOS − . The large numbers of fluorine atoms and S=O bonds in PFOS − can develop strong interactions between the anion and CO2, which leads to the largest anion-CO2 contact probability for PFOS − (PFOS − : 0.318, TFSA − : 0.232, PF6 − : 0.151) in the COSMO-RS results. The absorption Gibbs energy of [P66614][PFOS] was the most stable among the three ILs due to the large negative enthalpy (Table 3).</p><p>In this study, we focused on the predictivity of the CO2 solubility only by assuming that all cation/anion pairs would produce liquids instead of solids. Nevertheless, the fact that we have</p>

ChemRxiv

Atypical Protein Kinase C\xce\xb9 as a human oncogene and therapeutic target

Protein kinase inhibitors represent a major class of targeted therapeutics that has made a positive impact on treatment of cancer and other disease indications. Among the promising kinase targets for further therapeutic development are members of the Protein Kinase C (PKC) family.The PKCs are central components of many signaling pathways that regulate diverse cellular functions including proliferation, cell cycle, differentiation, survival, cell migration, and polarity. Genetic manipulation of individual PKC isozymes has demonstrated that they often fulfill distinct, nonredundant cellular functions.11 Participation of PKC members in different intracellular signaling pathways reflects responses to varying extracellular stimuli, intracellular localization, tissue distribution, phosphorylation status, and intermolecular interactions. PKC activity, localization, phosphorylation, and/or expression are often altered in human tumors, and PKC isozymes have been implicated in various aspects of transformation, including uncontrolled proliferation, migration, invasion, metastasis, angiogenesis, and resistance to apoptosis. Despite the strong relationship between PKC isozymes and cancer, to date only atypical PKCiota has been shown to function as a bona fide oncogene, and as such is a particularly attractive therapeutic target for cancer treatment. In this review, we discuss the role of PKCiota in transformation and describe mechanism-based approaches to therapeutically target oncogenic PKCiota signaling in cancer.

atypical_protein_kinase_c\xce\xb9_as_a_human_oncogene_and_therapeutic_target

Introduction<!>General PKC Activation Mechanisms<!>aPKC Activation Mechanisms<!>Protein-protein interactions regulate PKC\xce\xb9 localization and activity<!>The PKC\xce\xb9 gene (PRKCI) is frequently amplified in human cancers<!>PKC\xce\xb9 is over-express and mislocalized in human tumors<!>PKC\xce\xb9 is required for the cancer cell phenotype<!>PKC\xce\xb9 expression predicts survival and is associated with tumor aggressiveness<!>Upstream activators of PKC\xce\xb9<!>Effectors of PKC\xce\xb9 signaling<!>Therapeutic Targeting of aPKC\xce\xb9<!>Priming processes<!>Partner interactions<!>PB1 domain inhibitors<!>Substrate interactions<!>Perspectives<!><!>PRKCI copy number gain is a major genetic mechanism driving PRKCI overexpression in human tumors<!>Potential modes of PRKCI-directed therapeutic intervention

<p>Protein kinase inhibitors represent a major class of targeted therapeutics that has made a positive impact on treatment of cancer and other disease indications. There are currently 23 FDA-approved kinase inhibitors in use for various cancer and immune indications (see http://www.brimr.org/PKI/PKIs.htm). In addition, there is a rich pipeline of novel agents targeting disease-associated kinase targets in the clinic or at various stages of pre-clinical development (http://www.insightpharmareports.com/uploadedFiles/Executive_Summary%282%29.pdf). Among the promising kinase targets for further therapeutic development are members of the Protein Kinase C (PKC) family.</p><p>PKCs are a family of lipid-dependent serine/threonine kinases that represent a branch of the ACG kinase group.1 The PKC family is subdivided into 4 classes based on unique structural and regulatory properties. The conventional PKCs (cPKCs) are comprised of the PKCα, alternatively spliced PKCβI and βII, and PKCγ isozymes. These PKC isozymes bind diacylglycerol (DAG) and are activated by phosphatidylserine (PS) in a Ca2+-dependent manner, properties consistent with the initial description of PKC regulation.2 The novel PKCs (nPKCs) include the PKCδ, ε, η, θ, and μ isozymes. These PKC isozymes require DAG and PS for activation but are insensitive to Ca2+.3 The atypical PKCs (aPKCs), comprised of ζ and ι (also known as λ in rodents), are Ca2+-independent and do not respond to DAG.4, 5 Finally, the three PRK members (PRK 1–3) are also insensitive to Ca2+ and DAG.6, 7</p><p>The PKCs are central components of many signaling pathways that regulate diverse cellular functions including proliferation, cell cycle, differentiation, survival, cell migration, and polarity (recently reviewed in8–10). Genetic manipulation of individual PKC isozymes has demonstrated that they often fulfill distinct, non-redundant cellular functions.11 Participation of PKC members in different intracellular signaling pathways reflects responses to varying extracellular stimuli, intracellular localization, tissue distribution, phosphorylation status, and intermolecular interactions (recently reviewed in12). PKC activity, localization, phosphorylation, and/or expression are often altered in human tumors, and PKC isozymes have been implicated in various aspects of transformation, including uncontrolled proliferation, migration, invasion, metastasis, angiogenesis, and resistance to apoptosis (reviewed in10, 13). Despite the strong relationship between PKC isozymes and cancer, to date only atypical PKCι has been shown to function as a bona fide oncogene,14, 15 and as such is a particularly attractive therapeutic target for cancer treatment. In this review, we discuss the role of PKCι in transformation and describe mechanism-based approaches to therapeutically target oncogenic PKCι signaling in cancer.</p><!><p>Since Nishizuka's initial report of a proteolytically-activated protein kinase,16 it has been apparent that PKCs are present in cells in an inhibited basal state. Subsequent studies have identified upstream signaling pathways that allow PKCs to assume a fully “primed”, catalytically competent, inhibited basal state, which can be acutely activated to trigger PKC-dependent processes (reviewed in17, 18). These upstream signaling pathways lead to multi-site PKC phosphorylation on three conserved kinase domain serine/threonine phosphorylation sites – the activation loop, C-terminal turn, and hydrophobic motif sites - which in combination act to stabilize a fully active catalytic domain. In aPKC isoforms, the hydrophobic motif phosphorylation site acceptor amino acid is replaced with the partial phospho-mimetic glutamic acid. In the phosphorylated, primed state, the kinase and regulatory domains bind in an auto-inhibited conformation. Auto-inhibition appears to involve both a pseudosubstrate site present in all PKC family members, and other regulatory-kinase domain packing interactions19, 20 creating a `closed' inactive conformation.</p><p>Though no structures have been determined for an inactive full length PKC, a structure for a partially activated PKCβ structure has been described.21 A key feature of this structure is the orientation of the “NFD” motif within the kinase domain such that the Phe side chain makes contact with the ATP adenine ring22 to form a structure incompatible with an active, nucleotide-bound kinase domain. This structure likely reflects the fully-inhibited state, which is probably not nucleotide-bound, although the protein substrate binding pocket is expected to be occupied by the pseudosubstrate motif.</p><p>From this primed, inhibited state, acute activation involves loss of inhibitory regulatory-catalytic domain interactions and subsequent binding of Mg-ATP and protein substrates in the nucleotide and substrate binding pockets, respectively. For the classical PKC isoforms (cPKCα, β, γ), activation involves Ca2+-dependent recruitment of the kinase to anionic phospholipids, typically at the plasma membrane, through the Ca2+ binding C2 domains. Once at the membrane, the now proximal tandem C1A–C1B domain, is able to sample the membrane for DAG, a process that involves a conformation change exposing the otherwise obscured, intrinsically rather rigid DAG binding sites. The K/R-rich pseudosubstrate site, which is contiguous with the N-terminus of the C1A domain, interacts directly with anionic phospholipids to further stabilize membrane association. The nPKC isoforms are also activated by DAG binding to their C1A–C1B domains, but they do not display Ca2+ dependence for membrane recruitment. The C2 domains of certain nPKCs also have been shown to possess phosphotyrosine binding activity involved in localizing PKCδ to CDCP1, and in activation of PKCθ.23</p><p>Following activation, several PKC isoforms can bind to RACKs – receptors for activated C-kinases (reviewed in24). It has been proposed that RACK interactions can lock the kinase in an open conformation thereby contributing to maintenance of the active state. Studies on PKC-RACK interactions have led to the idea that expression of short synthetic forms of the domains responsible for these interactions can disrupt RACK-PKC interactions and serve as isoform-selective PKC inhibitors. Such inhibitors have been reported for many PKC isoforms, but to date not for the aPKC family. Similarly, protein scaffolds that direct localization of PKC isoforms prior to activation have been identified, among which are members of the A-Kinase Activating Proteins (AKAPs) (recently reviewed in25). AKAPs can bind specific PKC isoforms and link them to particular pathway outputs (eg. PKCη>PKD). Inhibitors of AKAP function in the form of short oligopeptides that compete for PKC-AKAP interactions have been described, however there is little evidence for a role of AKAPs in aPKC action.</p><!><p>The aPKCs (PKCζ and PKCι) are structurally and functionally distinct from other PKCs. The catalytic activity of the aPKCs does not require DAG, PS, or calcium, and the aPKCs do not serve as cellular receptors for phorbol esters.4, 26 Rather, PKCι can be activated by various acidic phospholipids including phosphatidylinositol 3,4,5 trisphosphate,7, 27 through specific protein–protein interactions mediated by a Phox Bem1 (PB1) domain within the N-terminal regulatory domain of the aPKCs (reviewed in28;discussed in detail below), and potentially through phosphorylation events that can regulate PKCι and its localization.</p><p>In addition to the priming phosphorylation events described above, PKCι has been reported to be phosphorylated by Src at tyrosines 256, 271, 325 in response to NGF stimulation.29 Site-directed mutagenesis indicates that Y325 phosphorylation is required for NGF-mediated PKCι activation and subsequent NFκB activation,29 whereas T256 phosphorylation regulates nuclear-cytoplasmic PKCι localization by modulating a canonical nuclear localization sequence (NLS).30 Src-mediated phosphorylation is also necessary for PKCι-dependent recruitment of β-COP to pre-golgi intermediates31 and the movement of PKCι into endosomes where it binds p62.32 Mass spectrometry analysis has revealed several other potential phosphorylation sites on PKCι,33 however, to date, neither the responsible kinase(s), nor the functional importance of these phosphorylation events, have been elucidated.</p><!><p>The aPKCs possess a region within their regulatory domain that is unique to the atypical PKCs, the Phox-Bem (PB)1 domain. The PB1 domain is a structurally conserved motif found on a family of signaling molecules (reviewed in28) that mediates their homo- and heterotypic interactions through specific interaction codes. The PB1 domain interactions formed between aPKCs and other PB1 domain containing proteins such as ZIP/p62, Par6 and MEK5 [MAPK(mitogen-activated protein kinase)/ ERK(extracellular-signal-regulated kinase)kinase 5] are critical for aPKC activation, localization and function in several contexts, including cell polarity, cell proliferation, cell survival, and more recently cell transformation.14, 34–38</p><p>Perhaps the best studied PB1-PB1 domain interaction involving PKCι is that between PKCι and the polarity protein Par6, which is essential for epithelial cell polarity (recently reviewed in39). PKCι interacts with Par6 to form a heterodimeric complex that is activated when Par6 binds to GTP-loaded cdc42/Rac1. Par6 binds PKCι through a characteristic PB1-PB1 interaction, for which a crystal structure has been solved.40 Par3 subsequently binds the PKCι-Par6 complex through a Par3 PDZ domain interaction with the kinase domain of PKCι, and a PDZ:PDZ domain interaction between Par3 and Par6. Par3 functions to localize PKCι/Par6 to the apical compartment of polarized cell membranes through a network of interactions involving nectin-willin-afadin (recently reviewed in39). The isoprenylated small GTPases cdc42/Rac1 are typically membrane-associated in their active GTP-bound states, contributing to the association of the cdc42/Rac-Par6-PKCι-Par3 complex with the apical membrane. Interestingly, a frequent mechanism by which human tumors lose polarity, a hallmark of cancer, is through loss of Par3 and/or other components of tight junctions such as E-cadherin.41, 42 As a consequence, PKCι is frequently mis-localized within the cytoplasm and nucleus of transformed cells and human tumors14, 15, 43, 44 Despite the loss of membrane restricted localization, recent studies have demonstrated that PKCι retains its association with Par6 in tumor cells;35, 36, 45 and this complex, and PKCι activity, is required for maintenance of the transformed phenotype.34–36, 45–47</p><!><p>The PKCι gene, PRKCI, resides on chromosome 3q26, one of the most frequently amplified genomic regions in human cancers. Compelling evidence across multiple tumor types demonstrate that PRKCI is a relevant target of 3q26 amplification. PRKCI copy number gains are observed in ~80% of human primary lung squamous cell carcinomas (LSCC),14 ~70% of serous epithelial ovarian cancer15 and ~53% of ESCC tumors.48 Consistent with these published findings, analysis of human tumor genomic datasets from the Cancer Genome Atlas, and other large scale sequencing projects (compiled at http://www.cbioportal.org/public-portal/), demonstrates that PRKCI copy number gains are prominent in many major forms of human cancer, being most prevalent in cervical, head and neck, lung squamous and ovarian serous cancers (Figure 1A). Surprisingly however, other major tumor types such as bladder, breast, kidney, lung adenocarcinoma, stomach and uterine cancers also harbor frequent PRKCI copy number gains, albeit less frequently than the tumor types mentioned above. Interestingly, gene expression data from these same tumor datasets reveal a strong positive correlation between PRKCI copy number gains and elevated PKCι mRNA expression across these tumor types (Figure 1B). Thus, tumor-specific gene copy number gain in PRKCI is a major genetic mechanism driving PKCι expression in human tumors. In contrast, PRKCI is rarely mutated with frequencies ranging from 0–3% across tumor types. Of approximately 9,000 human tumor samples derived from different tissues analyzed for PRKCI somatic mutations in the COSMIC database (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/), only 0.81% of tumors harbor a PRKCI mutation. Although of low frequency, one mutated site (R471), which has been reported on multiple occasions, maps to a substrate docking domain required for PKCι to support cellular polarization, while not influencing catalytic capacity per se.49 Thus, R471 mutation mediates a change of function wherein one aspect of PKCι output potential is selectively compromised.</p><!><p>PKCι is frequently overexpressed in the majority of tumor types examined (recently reviewed in50 (Figure 1). PKCι is overexpressed at the mRNA and protein level in NSCLC, ovarian, brain, breast, rhabdomyosarcomas,51 melanomas, esophageal, gastric, colon, liver, pancreas, and bile duct tumors. Interestingly, immunohistochemical analyses have reveal PKCι overexpression in tumor cells, with little to no staining in tumor-associated stroma and adjacent matched normal lung epithelium, indicating that PKCι overexpression is largely restricted to tumor cells. PKCι is frequently mislocalized in several tumor types. Whereas PKCι is localized to the apical and not the basal membrane of normal ovarian surface epithelial cells and benign serous and mucinous cysts, apical membrane staining was lost in 85% of serous low malignant samples and in all serous epithelial ovarian cancers analyzed where staining was diffuse throughout the tumor cells.15 NSCLC tumors reveal intense PKCι cytoplasmic and nuclear staining whereas adjacent normal lung epithelial cells exhibited membrane staining.14 Similarly, PKCι localized throughout the cytoplasm of primary breast tumor cells, whereas it localized to the apical membrane of normal breast epithelial cells.44 Cytoplasmic PKCι in hepatocellular carcinoma (HCC) correlated with reduced cell-cell contact, loss of both adherens and tight junction formation, reduced E-cadherin expression, and an increase in cytoplasmic beta-catenin.43 Taken together, these findings indicate that PKCι overexpression and intracellular mislocalization is a frequent event in cancer cells that is associated with loss of cellular polarity.</p><p>Although PRKCI gene copy gain is a major mechanism that drives PKCι overexpression in some human tumor types, alternative mechanisms may also promote PKCι overexpression. PKCι is frequently overexpressed in tumor types that harbor infrequent 3q26 amplification including colon cancer,37 pancreatic cancer,52, 53 and chronic myelogenous leukemia (CML).54 In CML cells, a Bcr-Abl/Mek/Erk signaling axis transcriptionally activates PKCι expression through an Elk1 element within the promoter region of PRKCI.54 Whether this or similar transcriptional mechanisms are relevant in other tumor types remains undetermined. Furthermore, PKCι activity has been shown to be primed by phosphorylation and, at least in the related PKCα it is documented that dephosphorylation at these sites can trigger PKC degradation.55 However, it is presently unclear whether PKCι phosphorylation and other post-translational mechanisms serve to regulate PKCι stability in tumor cells.</p><!><p>PKCι is required for maintenance of the transformed phenotype of cancer cells, regulating transformed growth, survival and chemoresistance of several tumor types including CML, prostate cancer, NSCLC, glioma, and ESCC (reviewed in50). Genetic or pharmacologic inhibition of PKCι blocks transformed growth of NSCLC, ovarian cancer, ESCC,56 colon carcinoma, PDAC,53 and rhabdomyosarcoma51 cells. In addition, PKCι promotes cellular migration and or invasion of NSCLC, ESCC,56 PDAC, and glioma cells. PKCι is also critical for tumorigenesis in vivo. Orthotopic or subcutaneous implantation of NSCLC, ESCC,56 and PDAC cells in which PKCι has been genetically-depleted into immune deficient mice results in impaired tumor initiation, growth and metastatic potential. Transgenic mice expressing constitutively active PKCι (caPKCι) in the colonic epithelium exhibit a higher incidence of colon tumors when compared to transgenic mice expressing kdPKCι or non-transgenic littermates after exposure to the colon-specific carcinogen azoxymethane.37 Furthermore, transgenic caPKCι mice develop mostly malignant carcinomas whereas non-transgenic mice develop mainly benign tubular adenomas.37 Consistent with the requirements of PKCι for cellular invasion, ESCC56 and PDAC53 cells with reduced PKCι expression exhibit a decrease in metastasis. These findings demonstrate that PKCι is functionally required for the transformed behavior of cancer cells and indicate roles for PKCι in tumor initiation, progression and metastatic behavior.</p><!><p>Reflecting its functional role in cellular transformation, PKCι expression profiling may be of prognostic value to predict survival in patients with various tumors types (reviewed in50). Elevated PKCι expression is associated with decreased survival in patients with NSCLC, cholangiocarcinoma, ovarian, bile duct, and prostate tumors. In other cancer types such as ESCC and breast, PKCι expression is greater in later stage tumors suggesting a role for PKCι in tumor aggressiveness, progression and metastasis. Thus, PKCι expression may be useful for identifying patients that are more likely to relapse and those who may benefit from more aggressive adjuvant chemotherapeutic treatment. In this regard, patients with high PKCι expressing gastric tumors are more likely to experience relapse,57 and elevated PKCι in early stage NSCLC tumors is associated with poor outcome.14</p><!><p>PKCι participates in a number of oncogenic signaling pathways that promote transformation (reviewed in10, 50, 58). PKCι plays a critical role in oncogenic Ras-mediated transformation. Exogenous over-expression of PKCι increases the size and number of soft agar colonies formed by oncogenic Ras-transformed ovarian surface epithelial cells.59 Likewise, PKCι activity is elevated in Ras-transformed intestinal epithelial cells, and is required for invasion and anchorage independent growth of these cells in vitro. The importance of PKCι as a downstream effector of oncogenic Rasin vivo has been demonstrated in several mouse models of K-Ras-mediated tumorgenesis.37, 60 In a mouse model of K-Ras-mediated colon cancer, K-RasLA2 transgenic mice, which contain a latent oncogenic K-Ras G12D allele that is activated by spontaneous recombination, develop oncogenic K-Ras-dependent aberrant crypt foci (ACF) in the colonic epithelium.37 ACFs are likely precursors to colon cancer61 and harbor many of the same genetic and biochemical alterations found in colon tumors, including activating K-Ras mutations. KrasLA/kdPKCι mice, which express kinase deficient PKCι in the colonic epithelium, develop significantly fewer ACF than KrasLA mice.62 A mouse model in which oncogenic KrasG12D is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCι gene (Prkci) was used to assess whether PKCι is involved in lung tumor development.60 Genetic disruption of Prkci blocked KRas-mediated lung tumor formation in vivo, a phenotype that was traced to a defect in KRas-mediated expansion of bronchioalveolar stem cells (BASCs) in Prkci deficient mice.60 Interestingly, Kras induced uncontrolled proliferation and morphological transformation of BASCs maintained in three dimensional culture.60 Whereas non-transformed BASCs form acinar structures characterized by a single large central lumen and a single layer of polarized epithelial cells, Kras-mediated transformation is characterized by disruption of cellular polarity, loss of the central lumen and uncontrolled growth of BASC cells as an amorphous mass.60 These morphological and proliferative changes induced by oncogenic Kras are strictly dependent upon PKCι expression.60 These data provide direct evidence that PKCι-dependent, Kras-mediated transformation involves both disruption of cellular polarity and stimulation of transformed growth.60 BASCs exhibit stem-like properties and are thought to be the tumor-initiating cells in KRas-mediated lung adenocarcinoma formation.63 Thus, Prkci is required for the earliest identifiable events in KRas mediated lung tumorigenesis in vivo. PKCι is also required for maintenance of the transformed phenotype and in vivo tumorigenic potential of established human cancer cells harboring oncogenic KRAS mutations.36 Expression of kdPKCι or RNAi knockdown of PKCι in NSCLC cells that harbor an oncogenic KRas mutation resulted in reduced formation and growth of xenograft tumors in immune-deficient mice.36 Greater than 90% of pancreatic tumors harbor oncogenic KRas mutation, and orthotopic implantation of KRas mutant pancreatic cancer cells depleted of PKCι into the pancreas of nude mice led to decreased tumor burden, reduced markers of angiogenesis and fewer metastases.53</p><p>Several studies have implicated aPKCs in Src-mediated transformation.30, 64 Src is reported to directly bind and phosphorylate aPKCs at multiple sites to promote their activation.30, 64 As noted above, Src-mediated phosphorylation of tyrosine 325 was shown to be functionally required for Src activation of PKCι in response to NGF.29 In NSCLC cells, NNK has been reported to increase c-Src-activated PKCι, which in turn phosphorylates Bad resulting in reduced Bad/Bcl-XL interaction.64 PKCι-mediated phosphorylation abrogates the pro-apoptotic function of Bad, and enhances cell survival and decreased sensitivity of NNK treated NSCLC cells to VP-16 and cisplatin.64 Genetic or pharmacological inhibition of Src led to decreased aPKC activation loop phosphorylation and reduced proliferation of androgen dependent prostate cancer cells.65 Additionally, inhibition of aPKC decreased v-Src-mediated morphological transformation, migration, matrix degradation and invasion of transformed NIH3T3 cells.66</p><p>Phosphoinositide 3-kinase (PI3K) has been implicated in the activation of aPKC. The PI3K lipid metabolite, PIP3, may promote aPKC activation through 3-phosphoinositide dependent protein kinase-1 (PDK-1) T403 loop phosphorylation.67 Consistently, RNAi-mediated knockdown of PDK1 inhibited priming phosphorylation of PKCι at T555 and reduced PKCι expression in glioma cells.68 Recently PI3K/PKCι signaling has been linked to the alternative splicing of Bcl-x pre-mRNA to promote cell survival in NSCLC cells.69 NSCLC, breast, cervical, and esophageal tumors exhibit increased expression of anti-apoptotic Bcl-x(L) when compared to their respective matched normal tissues. Interestingly, treatment of NSCLC cells with PI3K or pan-PKC inhibitors resulted in a reduced ratio of Bcl-x(L)/(s) splice variants as did knockdown of PKCι.69 Furthermore, over-expression of Bcl-x(L) could rescue PKCι knockdown mediated inhibition of NSCLC cell growth in soft agar. Thus, PI3K/PKCι promote an antiapoptotic/prosurvival phenotype in NSCLC cells through modulation of Bcl-x(L)/(s) splice variant ratios.</p><p>PKCι is a downstream effector of Brc-Abl-mediated chronic myelogenous leukemia (CML) cell survival and resistance to chemotherapeutic drugs.38, 70 In K562 CML cells, Brc-Abl activates a Ras/Mek/Erk signaling pathway that stimulates PKCι expression through an Elk1 transcription site within the PKCι promoter.54 PKCι is activated upon treating K562 cells with taxol,38 and overexpression of PKCι leads to enhanced resistance of K562 cells to taxol-induced apoptosis, whereas inhibition of PKCι expression enhances taxol mediated apoptotic cell death of K562 cells.70 Furthermore, inhibition of Bcr-Abl blocked taxol-induced PKCι activation and sensitized K562 cells to taxol mediated apoptosis. In contrast, expression of caPKCι protects K562 cells from chemotherapeutic drug-mediated apoptosis.70 Interestingly, in Bcr-Abl negative HL60 promyelocytic leukemia cells, taxol did not induce sustained PKCι activation.38 Thus activation of PKCι is an important downstream mediator of Bcr-Abl-induced chemotherapeutic resistance.</p><!><p>PKCι promotes cell survival in some tumor cells through activation of NFκB signaling. PKCι associates with IKKαβ and IκBα in TNFα -treated DU-145 prostate carcinoma cells where it is thought to phosphorylate IKKαβ, which subsequently phosphorylates IκBα resulting in NFκB/p65 translocation into the nucleus and transcriptional activation of targets.71 Interestingly, PKCι does not associate with IKKαβ and IκBα in transformed non-tumorigenic RWPE-1 prostate cells. Rather, TNFα treatment of RWPE-1 induces association of PKCζ with both IKKαβ and IκBα suggesting that PKCι may be preferentially used to activate NFκB pathways in tumorigenic prostate cancer cells.71 As described above, CML cells expressing Bcr-Abl are resistant to chemotherapy-induced apoptosis as a result of increased PKCι activation.38, 70, 72 Treatment of CML cells with taxol induces IκBα phosphorylation and NFκB nuclear translocation and transcriptional activation and disruption of PKCι function sensitizes K562 cells to taxol-induced apoptosis and inhibits RelA transcriptional activity.72 Overexpression of NFκB in K562 cells with disrupted PKCι function, rescues taxol-induced apoptosis. In addition, overexpression of constitutively active PKCι further upregulates NFκB transcriptional activity. Thus, PKCι induction of NFκB transactivation is important for Bcr-Abl–dependent resistance to taxol-induced apoptosis.70, 72 Glioblastoma multiforme are highly resistant to most standard cancer chemotherapeutics.73 RNAi-mediated depletion of PKCι results in sensitization of U87MG glioblastoma cells to cisplatin.73 Interestingly, unlike CML and prostate cancer cells, activation of the NFκB pathway does not appear to be a major mechanism driving PKCι dependent chemoresistance in glioblastoma. Rather, PKCι-mediated survival appears to result from PKCι-mediated attenuation of p38 mitogen-activated protein kinase signaling that protects these cells against cytotoxicity to chemotherapeutic agents.73 Thus, PKCι appears to function in several signaling pathways that promote cell survival in different tumor cell types.</p><p>Given the crucial role that polarity plays in maintaining the highly organized epithelial architecture, it is perhaps not surprising that several components of the polarity complex, particularly PKCι, Par6, Par3 and Rac1 have been directly implicated in oncogenesis. Aberrant expression of genes associated with cellular transformation such as ErbB2, Ras, NOTCH and TGFβ disrupt cell polarity and cause mislocalization of polarity complex components from the apical-lateral membrane. Activation of ErbB2 disrupts cell polarity through a mechanism in which ErbB2 causes mislocalization of Par6 from the apical-lateral border and disassociates Par3 from the aPKC/Par6 complex.46 Likewise, in rat proximal epithelial cells, TGFβ disrupts cell polarity through downregulation of Par3 and redistribution of aPKC/Par6 complex from the cell membrane to the cytoplasm.74 A recent report demonstrated that loss of Par3 cooperates with the intracellular domain of NOTCH (NICD) or H-Ras to induce breast tumorigenesis, tumor invasion and metastasis.75 A model was proposed in which the loss of Par3 in transformed cells triggers mislocalization and activation of aPKC.75 aPKC in turn activates a JAK/Stat3/MMP signaling cascade that induces changes in the ECM that promote tumor cell invasion and metastasis.75</p><p>Rac1 is a key downstream intermediate in oncogenic PKCι signal in multiple tumor types including the colon,37 lung,35, 36 and pancreas.53 In NSCLC cells, the PB1:PB1 interaction between PKCι and Par6 is required for the transformed phenotype and Rac1 activation.34, 36, 53 RNAi mediated depletion of PKCι, Par6 or Rac1 blocks transformation.35 Expression of PB1 domain mutants of PKCι or Par6 that inhibit the PKCι-Par6 interaction fail to restore transformation in PKCι and Par6 knockdown cells respectively,35 and either decreased PKCι or Par6 expression inhibits activation of Rac1, whereas expression of a constitutively active Rac1 allele (RacV12) in either PKCι- or Par6-depleted NSCLC cells restores transformation.35 Thus, Rac1 is a key effector of PKCι-mediated transformation in NSCLC. Proteomics analysis of proteins that associate with the PKCι-Par6 complex in NSCLC cells identified ECT2, a Rho family GTPase guanine nucleotide exchange factor (GEF), as a component of the PKCι-Par6 complex.34, 47 RNAi-mediated knock down of ECT2 inhibits Rac1 activity and blocks transformed growth, invasion, and tumorigenicity of NSCLC cells.34 Interestingly, the role of ECT2 in NSCLC transformation is distinct from its well established role in cytokinesis.34 In NSCLC cells ECT2 is mislocalized to the cytoplasm where it binds the PKCι-Par6 complex. Knock down of either PKCι or Par6 causes redistribution of ECT2 to the nucleus and loss of transformed growth and invasion.34 PKCι directly phosphorylates ECT2 at a single site, T328, in vitro and modulates T328 phosphorylation in NSCLC cells.47 T328 ECT2 phosphorylation is required for efficient binding of ECT2 to the PKCι-Par6 complex, activation of Rac1, and the transformed phenotype of NSCLC cells.47 Interestingly, the ECT2 gene resides on chromosome 3q26 in close proximity to PRKCI. Expression analysis in primary NSCLC tumors demonstrated that PRKCI and ECT2 are co-amplified and overexpressed in NSCLC.34 Thus, ECT2 and PKCι are genetically linked through coordinate gene amplification in NSCLC tumors, and biochemically and functionally linked in NSCLC transformation through formation of an oncogenic PKCι-Par6- ECT2 complex that drives NSCLC cell transformation.</p><p>The PKCι/Par6 complex has also been implicated in transforming growth factor β (TGF-β) induced epithelial-to-mesenchymal transition (EMT).76 TGF-β induces PKCι activation and TβRII and PKCι cooperate to phosphorylate Par6 at S345, a phosphorylation event required for TGF-β EMT and subsequent migration and invasion of NSCLC cells.76</p><p>In addition to Rac1, RhoB, another small GTPase, has been implicated in oncogenic PKCι signaling. Microarray analysis of PKCι-regulated genes in glioblastoma cells identified RhoB as being up-regulated in PKCι knockdown cells.77 Overexpression on RhoB in glioblastoma cells led to decreased proliferation, migration and invasion.77 Interestingly, constitutive expression of RhoB can repress PKCι activation implying a model whereby PKCι represses RhoB to promote an invasive phenotype, and RhoB repression of PKCι activity may lead to a noninvasive phenotype in glioblastoma cells.</p><p>In order to identify potential transcriptional targets of PKCι required for transformation, a genome-wide gene expression analysis has been carried out in H1703 NSCLC cells expressing either control or PKCι RNAi.35 The matrix metalloproteinase 10/stromolysin 2 (MMP10) emerged from this analysis as a major PKCι target.35 RNAi mediated depletion of PKCι, Par6, or Rac1 inhibits MMP10 expression in NSCLC cells,35 and similar to PKCι and Par6 knockdown, RNAi mediated knockdown of MMP10 blocks anchorage-independent growth and cell invasion. in NSCLC cells35 In addition, loss of transformed growth and invasion in PKCι KD or Par6 KD NSCLC cells is rescued by addition of catalytically-active MMP10.35 MMP10 is also a critical downstream effector of PKCι in Kras-transformed BASCs.60 Similar to Prkci, Mmp10 is required for BASC transformation and tumor initiation in vivo.78 Analysis of primary tumors revealed that MMP10 is overexpressed in NSCLC and MMP10 expression correlates positively with PKCι expression.35, 78 Gene set enhancement analysis of publicly-available gene expression profiles showed that elevated Mmp10 expression correlates strongly with both cancer stem cell and tumor metastasis gene signatures.78 Thus, expression of MMP10 is regulated through the PKCι-Par6-Rac1 signaling axis, and represents a key downstream effector in PKCι-mediated transformation in lung cancer cells. In addition to MMP10, a gene expression meta-analysis in primary lung adenocarcinomas identified the genes COPB2, ELF3, RFC4, and PLS1 as PKCι transcriptional targets.79 RNAi-mediated knock down of PKCι in lung adenocarcinoma (LAC) cell lines led to a significant reduction in expression of each of the four target genes, indicating that PKCι regulates the expression of these four genes in LAC cells.79 RNAi-mediated knock down of each of these genes led to significant inhibition of anchorage independent growth and cellular invasion demonstrating that each of them play a role in LAC tranformation.79 Several of these PKCι-regulated genes are coordinately overexpressed with PKCι in other major tumor types including lung squamous cell carcinoma, breast, colon prostate, pancreatic, and glioblastomas.79 suggesting that these PKCι regulated genes may serve as useful biomarkers in determining the effectiveness of PKCι-directed therapies and may themselves serve as targets for the development of novel prognostic markers and/or therapeutic agents.</p><!><p>As described above there is compelling evidence for aPKC as a target for intervention in cancer. In principle, interventions in aPKC could be afforded by: (i) modulating transcription/translation, for instance through an anti-sense approach as has been pursued for PKCα (ii) blocking upstream priming events; (iii) interfering with acute activation processes; (iv) modifying protein substrate/scaffold interactions; (v) inhibiting catalytic activity (Figure 2). We will comment here on those avenues where progress has been made in aPKC intervention.</p><!><p>The priming pathways have been reported to involve P13kinase, PDK1 and TORC2 (see above) and all these have been the object of drug development programs. For the class I P13kinases (α, β, γ, δ) there are multiple inhibitors of varying isoform selectivity under investigation clinically see80. However it remains moot as to whether the product of this class I PI3 kinase pathway, phosphatidylinositol 3,4,5 trisphosphate, is generally rate limiting for the priming of aPKC (either directed at aPKC itself or through the PDK1 PH domain). By contrast the inhibition of PDK1 or mTOR catalytic activity might be expected to influence aPKC priming directly, albeit as a chronic and not an acute effect.17 However as discussed above, the turnover of these sites in PKCs is generally much slower than found in for example Akt/PKB. Thus, it is unclear whether even chronic inhibition will greatly influence the steady state phosphorylation of aPKC which will tend to ratchet towards an occupied state.17 Multiple agents have been characterized retrospectively as targeting PDK1 (eg UCN-01 a broad specificity kinase inhibitor which potently inhibits PDK181 or targeted to PDK1 by design (eg AR-12, GSK2334470).82 A number of these agents have been in, are in, or are approved for clincial testing. Whether any of these agents influence the aPKC pathway in patients has not been reported. A more selective approach to intervention in the PDK1-aPKC cascade has come from targeting the aPKC PIF pocket which is involved in the PDK1-PKCι interaction (see19, 83). Several small molecule inhibitors that act at the PKCι PIF pocket have been identified.19, 84 The application and efficacy of such approaches to PKCι awaits further developments. The immunosuppressive agent rapamycin, acting via TOR85 has been used to target mammalian TOR in the context of TORC1. Rapamycin and derivatives with improved PK properties have been in clinical trials and some single agent activity has been documented. However the evidence indicates that PKC isoforms are targeted directly by TORC2 and not TORC1 and it is evident that TORC2 itself can contribute to the transformed phenotype.86 Agents which block both TORC1 and TORC2 activity have been developed and, interestingly, show greater activity in vitro and in vivo than TORC1 selective agents.87 It will be of interest to monitor whether biomarkers of aPKC action are modulated in patient studies with these agents, although as noted above the kinetics of steady state change in aPKC priming might not afford much leverage through these upstream pathways.</p><!><p>As noted above there are no RACK-interaction directed inhibitors of the aPKCs and indeed there are no RACKs formally described for this branch of the family. However there are agents that suppress partner interactions. Interference of one such aPKC partner interaction has been proposed as a mechanisms of action of the triterpenoid ursolic acid,88 a compound present in many fruits, enriched in apple peel and touted as a body-building supplement. Ursolic acid has been reported to block aPKCζ-sequestosome 1 interaction resulting in and associated with this is a net priming dephosphorylation of aPKCζ;88 this drug may well influence both aPKC isoforms, since antibodies detecting these priming site phosphorylations not distinguish aPKCι and aPKCζ. Derivatives of ursolic acid have been reported to display increased cytotoxicity in a range of tumor cell lines,89 however the structure activity relationship for these derivatives in relation to aPKC-sequestosome1 interaction have not been determined, nor have they been shown to modulate any of the other pathways affected by ursolic acid (for example NFκB, STAT3; reviewed in90). It remains to be seen whether ursolic is the basis of an aPKC intervention.</p><!><p>The PKCι PB1 domain is uniquely present in atypical PKCs, and the PB1 domain interaction formed between PKCι and Par6 is required for the oncogenic PKCι-Par6-Rac1-MMP10 signaling axis that promotes anchorage-independent growth and invasion of human NSCLC cells in vitro and tumorigenicity in vivo.35 Therefore, compounds that can disrupt the PB1-PB1 domain interaction between PKCι and Par6 may serve as a novel mechanism-based therapeutic intervention. A high throughput screen for small molecular weight compounds that can disrupt the PB1-PB1 domain interaction between PKCι and Par6 identified gold-containing compounds such as aurothioglucose (ATG), aurothiomalate (ATM) and auranofin (ANF) as selective and relatively potent inhibitors of PKCι and Par6 binding.91 These compounds are FDA-approved for treatment of rheumatoid arthritis patients,92 and ANF is still used clinically. These compounds exhibit dose dependent inhibition of PKCι-Par6 binding with IC50s of ~1μM, good antitumor activity, and they inhibit PKCι-mediated Rac1 activation and anchorage independent growth of NSCLC cells in vitro and tumorigenicity in vivo.91 The inhibitory efficacy of ATM has been tested on a panel of cell lines that represent the major subtypes of lung cancer including lung adenocarcinoma (LAC), lung squamous cell carcinoma (LSCC), large cell carcinoma (LCC), and small cell lung carcinoma (SCLC).93 ATM inhibited transformed growth in all cell lines tested with IC50s ranging from ~300 nM – 100 μM.93 Interestingly, ATM sensitivity among this group of cell lines did not correlate with tumor sub-type, KRas status, or sensitivity to a panel of standard chemotherapeutic agents frequently used to treat lung cancer patients, including cis-platin, placitaxel, and gemcitabine.93 Rather, PKCι expression (measured by mRNA or protein) was the major molecular characteristic of lung cancer cells corresponding to ATM responsiveness.93 ATM inhibits tumorigenicity of lung cell tumors in vivo at plasma drug concentrations consistent with the IC50 of the cell lines to ATM in vitro and ATM plasma drug concentrations routinely achieved in RA patients.93 Given the correlation between PKCι expression and ATM sensitivity, PKCι expression profiling in lung tumor samples may be useful in identifying lung cancer patients most likely to respond to these agents. In addition to NSCLC, the use of ATM and other gold-compound PKCι targeted therapy has also shown promise in other tumor types. Treatment of primary rhabdomyosarcoma cell cultures with ATM resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations.51 Combination therapy studies of ATM and vincristine (VCR), a microtubule inhibitor currently used in treatment regimens of rhabdomyosarcoma, exhibited synergistic anti-proliferative effects in cells and a trend towards further sensitizing tumors to VCR treatment in vivo.51 ATM mediated inhibition of PKCι in the prostate cancer cell line, PC3U, induced apoptosis, but ATM treatment did not affect the growth of human primary epithelial prostate cells, PrEC.94 A phase I dose escalation trial to determine an appropriate dosing regimen and describe toxicities associated with ATM in patients with advanced NSCLC, ovarian cancer, and pancreatic cancer was recently completed.95 ATM was well tolerated at the MTD of 50mg IM/weekly, and exhibited modest signs of tumor response (stable disease in 2/15 patients while on therapy) in this heavily pretreated patient population. Further trials are ongoing with the related compound auranofin (ANF), which exhibits higher potency for PKCι-Par6 inhibition (unpublished observations).</p><p>The precise mechanism by which ATG, ATM and ANF elicit their inflammatory activities in RA is unknown. A proposed mechanism of action is the formation of gold-cysteine adducts with target cellular proteins (reviewed in50). The PB1 domain of the aPKCs contains a cysteine residue, (Cys69) that is not present in other PB1 domain-containing proteins. The crystal structure of the PKCι-Par6 complex reveals that Cys69 is located within the conserved OPR, PC, and AID (OPCA) motif of PKCι at the binding interface between PKCι and Par6 in the complex.45 Molecular modeling of the interaction between ATM and the PKCι PB1 domain, predicts formation of an adduct between Cys69 and ATM that would cause steric hindrance to Par6 binding.45 Mutation of PKCι Cys69 to isoleucine (C69I) or valine (C69V), amino acids that frequently exist at this position in other PB1 domains, does not alter Par6 binding but makes PKCι resistant to ATM inhibitory effects on PKCι-Par6 binding in vitro.45 Consistent with these findings, expression of the C69I PKCι mutant in NSCLC cells supports transformed growth and renders these cells resistant to the inhibitory effects of ATM on transformed growth.45</p><!><p>The regulatory domain pseudosubstrate sites from individual isoforms have been exploited as natural inhibitors to generate short oligopeptide inhibitors.96 Thus cell permeable forms of synthetic pseudosubstrate site sequences have been employed to block function of their cognate target eg PKCα derived pseudosubstrate site targeting PKCα. A critical issue for use of these peptide inhibitors is specificity. Typically these peptides are employed in the 10–100µM range and it is questionable as to whether they display specificity at these concentrations. Binding of one PKC isoform to a non-cognate pseudosubstrate site sequence is well established, as evidenced by the use of synthetic pseudosubstrate site oligopeptides with A>S changes that converts them to good substrates.97 These oligopeptides exhibit poor differentiating ability between PKC isoforms. A more specific inhibitor that is protein substrate competitive has been identified for aPKCι and that is [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate.68 This drug was shown to inhibit PKCι competitively with respect to a protein substrate (myelin basic protein) in vitro and to block proliferation of neuroblastoma cells potentially via CDK7.68 The specific site of action will be of interest for this class of compounds, moreover their selectivity for aPKCι and potentially for a subset of its substrates offers some interesting opportunities for fine tuning interventions.</p><p>The majority of PKC inhibitors are typical protein kinase small molecule inhibitors that compete for the ATP binding pocket. Notably, until very recently, no compounds had shown any selectivity for the aPKC isoforms and much pharmacological dissection of the PKC family had exploited differences between compounds of decreasing specificity: Gö6976 (cPKC specific;98), bis-indolyl maleimide I (BIMI; cPKC and nPKC selective;99) and Gö6983 (cPKC, nPKC and aPKC selective);100 so, for example, a process inhibited by Gö6983 but not BIMI provides consistent evidence on aPKC involvement (certainly not proof given the lack of absolute specificity for these kinases).</p><p>More recently aPKC selective ATP-competitive inhibitors have been described.101 The compound CRT0066854 is a thieno[2`,3-d] pyrimidine compound, which has been shown to display good kinase selectivity across a broad kinase panel (selectivity score S(80) at 1µM is ~0.02) with excellent selectivity for aPKCι/ζ within the PKC family. This aPKC inhibitor selectively blocks aPKCι substrate phosphorylation in cells and inhibits proliferation in 2D and 3D cultures, suppresses polarized morphogenesis and directional migration – effects all mimicking aPKC intervention through siRNA.102 The basis of specificity for CRT0066854 has been established through a high resolution inhibitor bound complex of the aPKCι kinase domain. Here it has been shown that the fused tricyclic scaffold forms hydrophobic contacts with the glycine loop, while the substituents at the 2 and 3 positions, respectively contribute hinge-binding and a combination of charge and hydrophobic interactions. Notably the CRT0066854 benzyl group forming part of the substituent in the 3 position displaces Phe543 of the PKCι “NFD motif”. As seen for the domain interaction dependent displacement of the NFD motif in the PKCβ structure (discussed above), the benzyl group confers an inactive conformation on the PKCι kinase domain contributing to its specificity. CRT0066854 as noted above inhibits growth in soft agar and suppresses migration, and can suppress also the transformed morphology associated with mutant Ras expression.49 This compound provides clear evidence of an ability to selectively target the aPKC isoform catalytic activity with consequences consistent with characterized behaviors of aPKC pathways. How such agents play out in the clinical setting remains to be seen.</p><!><p>Studies to date provide compelling evidence that PKCι is oncogenic in a variety of human cancers. Functionally, PKCι is required for multiple aspects of the transformed phenotype and appears to participate in the initiation, progression and metastatic stages of cancer. Furthermore, PKCι participates downstream of other key oncogenes that have proven difficult to target therapeutically such as oncogenic Kras. Acquisition of tumor-specific mutations in targeted molecules has become a recurrent theme and presents a major hurdle in achieving curative therapy in cancer patients. Interestingly, PKCι has been shown to promote chemoresistance in a number of cancer types and inhibition of PKCι sensitizes these tumor cells to chemotherapeutics. There is growing evidence that human tumors contain a population of cells that exhibit properties of tumor-initiating or cancer stem cells (CSCs) that are thought to be responsible for lung tumor initiation, maintenance, relapse and chemoresistance. These findings have led to the hypothesis that CSCs must be effectively targeted to elicit effective and long lasting therapeutic treatment of cancer patients. Genetic ablation of PKCι in a lung tumorigenesis model indicates that PKCι plays a critical role in transformation of lung CSCs. Despite the critical requirement for PKCι in tumor cell function, normal cells as well as tumor cells grown in adherent non-transformed growth conditions can better tolerate PKCι inhibition, thus providing a therapeutic window. For these reasons PKCι is an attractive therapeutic target for novel cancer treatment.</p><p>As discussed above, several approaches have been taken to modulate oncogenic PKCι function for cancer therapeutics. The use of non-selective PKC inhibitors in the clinic have given mixed results, and have not been very effective in treating tumors. This in part is due to the high homology between the PKC isozymes and the relative lack of specificity in previously designed PKC inhibitors. In this regard, PKCι and PKCζ are highly related, exhibiting 72% overall amino acid sequence homology and 86% identity within the kinase domain. While PKCι appears to consistently play a promotive role in transformation PKCζ is reported to have tumor suppressive functions in some contexts. Therefore, PKCι isozyme specific inhibitors may need to be utilized in order to circumvent the possible cross target effects on aPKCζ Future work is also required to identify distinct targets of PKCι signaling that can be developed as robust, sensitive markers of therapeutic response to PKCι inhibition in clinical trials.</p><!><p>This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.</p><!><p>A. The percentage of each listed tumor type harboring copy number gains, somatic mutation or multiple alterations in the indicated tumor types is shown. Data were compiled from the cBioPortal for Cancer Genomics (http://www.cbioportal.org/public-portal/). B. PRKCI copy number gain correlates with PRKCI overexpression across human tumor types indicating that copy number gain is a major causative genetic mechanism driving PKCι expression in these tumors. For each of the tumor types shown in A, the % of tumors harboring PRKCI gene copy number gains was plotted against the % of tumors exhibiting PRKCI overexpression. A direct relationship exists between PRKCI copy number gain and overexpression across tumor types (correlation co-efficient R2=0.7756).</p><!><p>Five potential modes of therapeutic intervention are outlined: (I) control of PRKCI transcription, (II) disruption of PB1 domain protein-protein interactions between PKCι and its oncogenic partner Par6; (III) inhibition of upstream kinase cascades that regulate PKCι priming; (IV) inhibition of PKCι-scaffold/protein substrate interactions; (V) inhibition of PKCι catalytic activity through competitive inhibition of ATP binding. See text for discussion and details.</p>

PubMed Author Manuscript

Generalized Optimized Effective Potential for Orbital Functionals and Self-Consistent Calculation of Random Phase Approximations

A new self-consistent procedure for calculating the total energy with an orbital-dependent density functional approximation (DFA), the generalized optimized effective potential (GOEP), is developed in the present work. The GOEP is a nonlocal Hermitian potential that delivers the sets of occupied and virtual orbitals and minimizes the total energy. The GOEP optimization leads to the same minimum as does the orbital optimization. The GOEP method is promising as an effective optimization approach for orbital-dependent functionals, as demonstrated for the self-consistent calculations of the random phase approximation (RPA) to the correlation functionals in the particle\xe2\x80\x94hole (ph) and particle\xe2\x80\x94particle (pp) channels. The results show that the accuracy in describing the weakly interacting van der Waals systems is significantly improved in the self-consistent calculations. In particular, the important single excitations contribution in non-self-consistent RPA calculations can be captured self-consistently through the GOEP optimization, leading to orbital renormalization, without using the single excitations in the energy functional.

generalized_optimized_effective_potential_for_orbital_functionals_and_self-consistent_calculation_of

<p>Density functional theory (DFT)1–5 has achieved much success in electronic structure theory. It has been widely implemented in modern quantum chemistry softwares and has made significant impacts in many fields. However, challenges remain for DFT in describing van der Waals interaction,6 strongly correlated systems,7 and systems having features of fractional charges and fractional spins because of the delocalization and static correlation errors.8 In general, the accuracy of density functionals can be improved by introducing the orbital dependence into the exchange-correlation energy expression.9 Hybrid functionals such as B3LYP10–13 are the simplest type of orbital functionals depending on the occupied orbitals through the one-electron density matrix. For such functionals, self-consistent calculations are carried out normally by solving the generalized Kohn—Sham (GKS) equations. Virtual orbitals can be used in the correlation energy functionals as in the second-order Møller—Plesset (MP2),14 double hybrid (DH) functionals,15–20 and random-phase approximations (RPA).21–32 Orbital dependent functionals can also be more complicated because of the lack of invariance with respect to unitary rotations, such as the self-interaction correction (SIC)33 and Koopmans-compliant (KC) functionals.34</p><p>The calculations with functionals depending on virtual orbitals are usually performed in a post-SCF procedure, which will lead to nonvariational total energies and unrelaxed orbitals.18 A self-consistent field (SCF) calculation is desired for higher accuracy. The optimized effective potential (OEP)35–39 and orbital optimization (OO)40 are widely used for SCF calculations. For the OEP, it can provide accurate local exchange-correlation potentials and has been used extensively for functionals of occupied orbitals, such the exact exchange and hybrid functionals. However, the ground-state energies are not improved for functionals depending on virtual orbitals.41–43 The reason for this failure is that the orbitals are only optimized in the space of υ-representable densities by a local potential, the OEP, rather than the whole space. In contrast, the orbitals from OO are optimized in the whole space. Both MP2 and DH functionals have been self-consistently calculated with OO,44,45 but no OO has been developed for the RPA functionals.</p><p>Here we developed a new optimization method, the generalized optimized effective potential (GOEP) method, to achieve the SCF calculations for general orbital-dependent functionals. The GOEP is a nonlocal potential in space (1)vsGOEP(r,r′)=∑pq〈r|ϕp〉(vsGOEP)pq〈ϕq|r′〉 where p and q are GOEP orbital indices. The orbitals {ϕp} are defined to be eigenvectors of the GOEP nonlocal Hamiltonian hsGOEP=t+vsGOEP. Thus, unlike the local OEP method, the density matrix obtained from the GOEP is fully flexible because there is no restriction of the locality in real space of the potential within the GOEP. Note that the mapping of hsGOEP to the noninteracting density matrix, or the set of occupied and virtual orbitals, is many-to-one. For example, within the occupied space, the diagonal elements of hsGOEP can vary arbitrarily as long as they are less than all the diagonal elements in the virtual space. Therefore, the eigenvalues of the GOEP do not have physical meanings. They only partition the space into occupied and virtual spaces during the optimization to ensure the occupied and virtual spaces will not cross. This many-to-one mapping does not pose any issue because the unique noninteracting density matrix or the set of GOEP orbitals is what determines the total energy. We have proved that the optimization of an orbital functional with respect to the GOEP Hamiltonian is equivalent to OO (see Section 1 in the Supporting Information (SI)). Computationally, it is convenient to perform the GOEP in the eigenvector or GOEP orbital space. Because all orbitals are determined uniquely by hsGOEP, a total energy orbital functional, E, is thus a functional of hsGOEP. The energy derivatives with respect to GOEP off-diagonal elements can be expressed as follows (see the SI) (2)∂E∂(vsGOEP)pq=〈ϕq|δEδϕp*〉−〈(δEδϕq)*|ϕp〉ϵp−ϵq We use the index i/a/p to represent occupied/virtual/general orbitals. Notice that this expression assumes nondegenerate orbitals. Because the GOEP is Hermitian, ∂E/∂(vsGOEP)qp will be the complex conjugate of ∂E/∂(vsGOEP)pq. At the stationary point, we have (3)∂E∂(vsGOEP)pq=0 The change in the GOEP, δvsGOEP, is equivalent to the change in the Hamiltonian δhsGOEP. For orbital functionals with invariance to unitary rotations within both occupied and virtual spaces, we only need to consider the variation of the occupied—virtual block because the variation of the occupied—occupied and virtual—virtual blocks only leads to unitary rotations within the occupied and virtual spaces. In this case, degeneracy within the occupied or virtual space in eq 2 does not matter because the denominator in eq 2, ϵa − ϵi, cannot be zero if the occupied and virtual spaces are not mixed.</p><p>Now we apply the GOEP method to the RPA for the correlation energy functional. The RPA can be developed in two channels: particle—hole (ph) channel leads to ph-RPA;21–28 particle—particle (pp) and hole—hole (hh) channels lead to pp-RPA.29,30 The ph-RPA is a fully nonlocal functional of density and can describe van der Waals interactions, crystalline solids, and surface adsorption;27 furthermore, the pp-RPA meets the flat-plane condition for systems with fractional charges and spins.29 RPA calculations are usually performed in a post-SCF fashion. Despite the correct dissociation limit of diatomic systems with ph-RPA, the lack of SCF will lead to problems in the intermediate distance.46 Although RPA has been self-consistently calculated with the local OEP, the binding energy curves are not improved compared with those from post-SCF calculations.41,42 In this work, we aim to explore the SCF calculations of RPA correlation functionals in both the ph-channel and the pp-channel with self-consistent calculations and without the local OEP restriction. The total energy expression is (4)Etotal=EHF[ρs]+EcRPA[{ϕp}] where {ϕp} are the canonical orbitals of the GOEP and ρs is the reference density matrix consisting of the occupied GOEP orbitals (5)ρs=∑i|ϕi〉〈ϕi| The total energy is calculated by combining the HF energy and RPA correlation energy, which are both evaluated with the GOEP orbitals. The RPA correlation energy can be formulated from the solution of the generalized eigenvalue problem (see SI eqs 32 and 34). The ph-RPA correlation energy can be expressed as (6)Ecph-RPA=12(∑n>0ωnN−TrA) and that of the pp-RPA is (7)Ecpp-RPA=∑nωnN+2−TrA where ωn are the neutral excitation energies, ωnN+2 are two-electron addition energies, and A represents matrices in the RPA eigenvalue equations.25,29 Note that the noncanonical form of the generalized ph- or pp-RPA eigenvalue problem is used.47 Because this expression is invariant with rotations, as we have shown (see the SI), we can perform the optimization in the occupied-virtual space only. The ph-RPA can be derived within the framework of DFT via the adiabatic-connection fluctuation—dissipation (ACFD) theorem;23,48–50 the pp-RPA can also be derived in the equivalence of ACFD in the pairing channel.29 In all applications, a density functional approximation (DFA) is adopted as the reference DFA functional, the KS or GKS orbitals of which are used for constructing the RPA correlation energy.</p><p>It is important to realize that in the eigenvalue equations for either ph-RPA or pp-RPA, as shown in Section 2 of the SI, a DFA is used, explicitly through the KS or GKS Hamiltonians, defined as the functional derivatives of the energy with respect to the density matrix. The use of DFA cannot be replaced with the GOEP because the GOEP does not provide meaningful orbital energies. The second-order functional derivatives of the DFA are also involved in the energy gradient for the GOEP calculations (see Section 3 in the SI). The full process of this optimization is as follows: (1) Carry out an SCF calculation with a certain DFA; (2) build the RPA matrix and calculate the total energy perturbatively; (3) calculate the gradient δE/δhsGOEP and update hsGOEP with the gradient information; (4) diagonalize the Hamiltonian hsGOEP to obtain the canonical orbitals {ϕp}; and (5) go to step 2 until both total energy and gradient converge.</p><p>We first tested van der Waals systems. Figure 1 shows the binding energy curves with both ph and pp-RPA for He2. The post-SCF results have the correct dissociation limit; however, at the intermediate distance it is underbinded. With the Hartree—Fock (HF) reference, the SCF calculation does not change from the post-SCF result. In contrast, both curves of SCF-ph-PBE and SCF-pp-PBE are improved significantly from the post-SCF results and in excellent agreement with the reference. This is a major difference from the SCF-RPA calculations carried out with the local OEP, where the converged total energies are not much changed from the post-SCF calculations,42 highlighting the importance of full optimization with the GOEP.</p><p>Because the total energy contains several energy contributions, we decompose the total energy to investigate which part is improved in the GOEP calculation. In the following notations, E@F refers to the energy E evaluated with the canonical orbitals of the reference DFA functional F and E@ SCF-F refers to the energy E evaluated with the GOEP orbitals, where F is the DFA reference in the RPA functional. In this case, the total energy of a post-SCF calculation is further decomposed into (8)Etotal=ET@F+Eext@F+EJ@F+EEX@F+ERPA@F where ET, Eext, EJ, EEX, and ERPA refer to the kinetic energy, external energy, Coulomb energy, exchange energy, and RPA correlation energy, respectively. In post-SCF RPA calculations, Ren and coworkers53 show that the exchange-correlation part is better described in a hybrid way with two sets of orbitals from HF and PBE54 calculations, (9)Exc=EEX@HF+ERPA@PBE To develop a clear understanding of the effects of full orbital optimization as in the GOEP, we analyzed the exchange and correlation energies with different methods and plotted the results in Figure 2. For post-SCF calculations, HF reference can provide better exchange energy, but the correlation energy has large error. PBE reference provides accurate correlation energy; however, the exchange energy has a 4 meV barrier in the intermediate range. The self-consistent calculation with HF reference does not change the binding energy, as EEX@SCF-HF and ERPA@SCF-HF are almost the same with EEX@HF and ERPA@HF, but for the self-consistent calculation with PBE reference, ERPA@SCF-PBE keeps the accuracy of ERPA@PBE, while the exchange energy, EEX@SCF-PBE, is corrected toward EEX@HF. Therefore, the binding energy curve is significantly improved with the GOEP. The failure of the post-SCF RPA in the intermediate distance has been attributed to the neglect of the contribution from single excitations (SEs).53</p><p>Whether there is a SE contribution to the RPA approximate correlation energy depends on the derivation. In the usual derivation of the ACFD theory, when the electron density is kept constant along the adiabatic connection, the singles contribution does not appear.23,48,50,55 If the density is not kept constant along the adiabatic connection, then the singles contributes to the correlation energy.55 With the commonly used post-SCF RPA scheme, most molecular systems are underbinded systematically. By adding the SE perturbatively, it has been shown that the SE contribution to the energy improves the result significantly.53 However, in our GOEP calculations, the binding energy curves are improved greatly, without adding the SE contribution. We now analyze the SE within the GOEP. The SE contribution to the second-order correlation energy can be expressed as (see Section 4 in the SI)50,56 (10)EcSE=∑iocc∑avir|〈ϕi|fHF−hsGOEP|ϕa〉|2ϵ˜i−ϵ˜a where {ϕi} are the orbitals of the nonlocal GOEP Hamiltonian hsGOEP. The orbital energies, ϵ˜i and ϵ˜a, are from the rotated GOEP orbitals that diagonalize the occupied and virtual subspaces of the reference KS or GKS Hamiltonian hs from the DFA (11)(hs)ij=δijϵ˜i(hs)ab=δabϵ˜a Because {ϕi} are the eigenvectors of hsGOEP, 〈ϕi|hsGOEP|ϕa〉 will be zero. The only existing term on the numerator is thus the single-particle HF Fock operator, |〈ϕi|fHF|ϕa〉|2.</p><p>The absolute values of the second-order SE contribution were plotted with and without the SCF in Figure 3c. In the intermediate range, the second-order SE contribution from the PBE is −0.5 meV, while that from the GOEP is only −0.008 meV. Moreover, the second-order SE contribution from the GOEP is almost 0 along all distances. A test set of atoms also shows a significant reduction of the second-order SE with the GOEP (Table 1). This reduction is caused by the orbital renormalization. Because the HF energy is the predominant term in the total energy expression (eq 4), the optimized orbitals within the GOEP bear great similarity to HF orbitals and the term |〈ϕi|fHF|ϕa〉|2 will approach zero. Therefore, at the stationary point, the effect of second-order SE is mostly brought into the total energy. As a consequence, the optimal orbitals provide the much improved description for binding energy curves. A diagrammatic representation of this renormalization process is shown in Figure 3, from (a) to (b).</p><p>While the second-order SE was shown to be important for post-SCF calculations with a semilocal functional (e.g., PBE) as reference,53,55 it will be ill-behaved for systems with small energy gaps, such as the dissociation limit, because the gap of the molecule will approach zero. Thus higher order diagrams are required in this situation, and the correction to the correlation energy is named renormalized SE (rSE).53 However, in the SCF calculations with GOEP method, no ill behavior is found at the dissociation limit because the second-order SE is not included. It is absorbed into the total energy through the orbital renormalization in the self-consistent calculation. This is a key result from present SCF calculations of RPA functionals; we can have the RPA total energy functional with good accuracy and without the SE contribution, just as given in the derivation from ACFD with density being kept constant.</p><p>A more challenging case, Be2, was also tested. The commonly used CCSD(T) method overbinds at the equilibrium region, and both ph and pp-RPA not only underbind but also show an unphysical local maximum beyond the binding region. Previous work suggested that this behavior may be caused by the lack of self-consistency in the calculation.46 Moreover, this unphysical maximum was also observed in the SCF calculations with the local OEP method.42 This now can be explained from eq 10: Orbitals from the local OEP are not fully optimized because of the local potential restriction, and thus the second-order SE correction is still required for RPA. Therefore, the SCF-RPA calculations with the GOEP is desirable for systems like Be2 because of the orbital renormalization. The result shows that although the GOEP does not improve the binding length with the pp-RPA significantly, it completely eliminates the unphysical local maximum and corrects the binding energy-(Figure 4). From the exchange energy decomposition, we notice that different from He2, the barrier of EEX@PBE curve emerges beyond the equilibrium bond length, which is exactly the region where the unphysical maximum (marked in the shaded area) occurs. The SCF calculation brings down this barrier so that the binding energy curve is corrected.</p><p>We finally consider the H + H2 exchange reaction with the GOEP SCF calculations, along with the post-SCF pp-RPA. The reaction energy curve is shown in Figure 5. The pp-RPA overestimates the reaction barrier by 4 kcal/mol. The SCF calculation brings down this barrier by 0.5 kcal/mol, and the reaction energy profile is closer to the reference with the SCF procedure. In addition, several bonded systems, such as He2+, H2, and LiH, were tested. These systems do not show significant improvement with the SCF in comparison with the post-SCF results. In all cases, SE contributions are reduced with the SCF calculation. For these bonded systems, the error of the RPA originates mostly from the inherent error of the RPA functional itself. One explanation is that for bonded systems the correlation energy is mainly contributed from double excitations.56 It has been proved that the ph-RPA is equivalent to ring couple-cluster doubles (CCD)24 and the pp-RPA is equivalent to ladder CCD.30,59 Neither includes full diagrams of the doubles.</p><p>To summarize, we have developed a new optimization method, GOEP, to achieve SCF calculations for general orbital-dependent functionals. We applied the GOEP method to phand pp-RPA correlation energy functionals. We have shown that for van der Waals systems, SCF is of great importance and the SCF-RPA using the GOEP performs well. It significantly improves the accuracy of the binding energy curves: The underbinded feature of He2 is corrected and the unphysical local maximum of Be2 is totally eliminated. The energy decomposition reveals that the exchange energy, EEX@SCF-PBE, is much improved from EEX@PBE, and the effect of second-order SE contribution is brought into the total energy functional with the SCF calculation through the orbital renormalization without using the single excitations in the energy functional. This is significant because the SE part of the functional can become singular for systems with zero or small gaps. Our results strongly support using the RPA correlation energy functional without the SE term in SCF calculations to achieve good accuracy and eliminate the possible singularity of the SE term. We conclude that the GOEP is a promising tool to achieve SCF calculation for orbital-dependent functionals.</p>

PubMed Author Manuscript

Toward miniaturized analysis of chemical identity and purity of\nradiopharmaceuticals via microchip electrophoresis

Miniaturized synthesis of positron emission tomography (PET) tracers is poised to offer numerous advantages including reduced tracer production costs and increased availability of diverse tracers. While many steps of the tracer production process have been miniaturized, there has been relatively little development of microscale systems for the quality control (QC) testing process that is required by regulatory agencies to ensure purity, identity, and biological safety of the radiotracer before use in human subjects. Every batch must be tested, and in contrast with ordinary pharmaceuticals, the whole set of tests of radiopharmaceuticals must be completed within a short-period of time to minimize losses due to radioactive decay. By replacing conventional techniques with microscale analytical ones, it may be possible to significantly reduce instrument cost, conserve lab space, shorten analysis times, and streamline this aspect of PET tracer production. We focus in this work on miniaturizing the subset of QC tests for chemical identity and purity. These tests generally require high-resolution chromatographic separation prior to detection to enable the approach to be applied to many different tracers (and their impurities), and have not yet, to the best of our knowledge, been tackled in microfluidic systems. Toward this end, we previously explored the feasibility of using the technique of capillary electrophoresis (CE) as a replacement for the \xe2\x80\x98gold standard\xe2\x80\x99 approach of using high-performance liquid chromatography (HPLC) since CE offers similar separating power, flexibility and sensitivity, but can readily be implemented in a microchip format. Using a conventional CE system, we previously demonstrated the successful separation of non-radioactive version of a clinical PET tracer, 3\xe2\x80\x99-deoxy-3\xe2\x80\x99-fluorothymidine (FLT), from its known byproducts, and the separation of the PET tracer 1-(2\xe2\x80\x99-deoxy-2\xe2\x80\x99-fluoro-\xce\xb2-D-arabinofuranosyl)-cytosine (D-FAC) from its \xce\xb1-isomer, with sensitivity nearly as good as HPLC. Building on this feasibility study, in this paper, we describe the first effort to miniaturize the chemical identity and purity tests by using microchip electrophoresis (MCE). The fully-automated proof-of-concept system comprises a chip for sample injection, a separation capillary, and an optical detection chip. Using the same model compound (FLT and its known byproducts), we demonstrate that samples can be injected, separated, and detected, and show the potential to match the performance of HPLC. Addition of a radiation detector in the future would enable analysis of radiochemical identity and purity in the same device. We envision that eventually this MCE method could be combined with other miniaturized QC tests into a compact integrated system for automated routine QC testing of radiopharmaceuticals in the future.

toward_miniaturized_analysis_of_chemical_identity_and_purity_of\nradiopharmaceuticals_via_microchip_

Introduction<!>Reagents<!>Miniaturized CE system<!>Injection chip<!>Detection chip<!>Conditioning<!>Separation<!>UV absorbance measurements<!>Characterization of injection chip<!>Characterization of detection chip<!>Evaluating separation efficiency<!>Benchmark comparisons<!>Sample injection<!>Sample detection<!>Separation of samples<!>Conclusions

<p>Microscale capillary-electrophoresis (CE)-based devices are increasingly being used for high-resolution separations where portability, ease of integration, or small sample size are of particular importance. Recent examples include environmental analysis [1], biomolecular separations [2, 3], and mobile heath diagnostics [4].</p><p>Another field that can benefit from the advantages of such devices is nuclear medicine, particularly in assessing patient safety of freshly-prepared batches of short-lived radiolabeled imaging tracers for positron-emission tomography (PET) or single photon emission computed tomography (SPECT). PET and SPECT are real-time, 3D medical imaging techniques with exquisite specificity and sensitivity for visualizing particular biological/biochemical processes depending on the tracer used. The information from a PET or SPECT scan is used clinically in the diagnosis of many diseases, prediction of response to therapy, and monitoring response to therapy [5–8]. Imaging is also an indispensable research tool for uncovering mechanisms of disease initiation and progression, developing new therapies, and measuring and optimizing the pharmacokinetic properties of new therapeutic compounds [9]. In the case of PET, the majority of scans are currently performed using the glucose analog 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG) since a wide range of conditions that can be detected via altered metabolism [10–12], but there is a growing interest in visualizing a wide range of biological processes and receptors using other tracers [13, 14].</p><p>Since PET tracers are classified as drug products by regulatory agencies, they must pass stringent quality control (QC) tests after their production for safety of the patient prior to injection. Unlike ordinary pharmaceuticals, the short lifetime of radiopharmaceuticals requires that they be produced in relatively small batches close to the geographical location where the patient is scanned. As described in regulatory documents (e.g. U.S. Pharmacopeia General Chapter <823> [15] and U.S. Food and Drug Administration 21 CFR Part 212 [16]) and several review articles [17, 18], each radiopharmaceutical batch must be evaluated for color and clarity, pH, radioactivity, radioisotope identity, chemical/radiochemical identity, radiochemical purity, residual solvents, chemical purity, pyrogenicity, and sterility. Performing and documenting the tests is cumbersome and time-consuming, and requires an array of expensive analytical chemistry equipment and significant dedicated lab space, and there is considerable interest in the development of automated and lower-cost approaches. Several efforts are underway to develop integrated QC testing instruments that automatically perform and document all of the required tests and calibrations, e.g. QC-1 [19] (Munster, Germany), Trace-ability [20] (Culver City, CA USA), and ABT Molecular Imaging Inc. [21, 22] (Louisville, TN USA). While potentially alleviating the labor burden, these systems are still based on conventional, macroscale instruments linked into an integrated system along with a sample distribution mechanism.</p><p>By replacing conventional analysis techniques with lab-on-a-chip technologies, it may be possible to achieve significant reductions in the size, cost, and complexity of automated QC testing platforms, and potentially to increase sensitivity [2, 23]. Commercial microscale devices already exist for testing of endotoxins [24], and there have been recent efforts to miniaturize some of the other tests, including radioactivity measurement [25], radioisotope identity (half-life) test [25], pH test [26], color and clarity test [26], and Kryptofix 2.2.2 test [27]. While these results represent an impressive step forward, high-resolution miniaturized chromatographic methods, suitable for assessment of chemical or radiochemical identity and purity across a wide range of tracers, are notably missing. Due to the potential presence of several impurities in each batch of PET tracer, and due to the wide variety of tracers and synthesis methods, performing these tests will likely require some kind of chromatographic separation followed by a radiation detector (e.g. gamma rays or positrons) and additional detectors for non-radioactive species (e.g. UV absorbance, refractive index, or pulsed amperometric detectors) to quantify each compound and ensure it is below permitted limits. The identity of each peak can be determined by matching the retention time to a reference standard (or by co-injection of the standard), or, in rare cases, via a mass detector.</p><p>In this paper, we focus on the development of a microscale CE-based device to replace the gold standard approach of high-performance liquid chromatography (HPLC) for this critical and challenging component of QC testing. We have been exploring CE methods due to the possibility of microchip implementation and corresponding reductions in size, cost, and complexity of the overall QC system. Microchip electrophoresis (MCE) has been shown capable of separating a vast range of analytes including large biomolecules (e.g. nucleic acids, proteins), peptides, and inorganic ions and chiral molecules [28, 29] simply by tuning the separation conditions. The versatility and separation power of CE have been noted to be equal to HPLC, or even better in some applications [30]. CE also avoids the use of high pressures, which simplifies the interface with other system components and eliminates the need for bulky and expensive high-pressure valves, pumps and fittings. Additional advantages of CE are the ability to miniaturize the QC system into a microfluidic chip measuring 25 mm × 75 mm or smaller that is operated via a compact electronic control system and power supply, and the extremely tiny sample consumption (typically nanoliters).</p><p>Conventional-scale CE separation of several 99mTc-labeled SPECT species from impurities has been reported [31], and we recently showed that two 18F-labeled PET tracers, namely 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) and 1-(2'-deoxy-2'-[18F]fluoro-β-D-arabinofuranosyl) cytosine ([18F]FAC) can be readily separated from impurities, including Kryptofix 2.2.2 (K222), using MEKC [32]. Compared to traditional HPLC/UV, we observed similar separation resolution and limits of detection (LOD), but reduction in analysis time in some cases, and several orders of magnitude reduction in buffer and sample consumption. (In typical HPLC analysis of radiopharmaceuticals, sample volume is on the order of 10-100 μL, the flow rate is 1-2 mL/min, and the analysis time may be 5-30 min, consuming 5 – 60 mL of mobile phase. On the other hand, in MCE, the buffer consumption can be as low as 100 μL and sample injection volume is typically in the nL range or lower.) However, to the best of our knowledge, there have been no reports on the miniaturization of these approaches to analyze chemical species relevant to the testing of radiopharmaceuticals. Here we describe a proof-of-concept hybrid microfluidic CE device consisting of a hydrodynamic injection chip, a separation capillary, and a microfluidic optical absorbance detection chip to perform chemical identity and purity analysis of FLT and its known impurities. Potentially, with integration of a radiation detector in the future, this approach could also be used for radiochemical identity and purity tests. In addition, this approach could enable the fluid path to be inexpensive and disposable, reducing maintenance and eliminating the need for cleaning, further simplifying the testing process.</p><!><p>Sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic dihydrate (Na2HPO4), boric acid, sodium dodecyl sulfate (SDS), ammonium acetate, ethanol, sodium chloride (NaCl), sodium hydroxide (NaOH), thymine, thymidine, furfuryl alcohol (FA), 2',3'-didehydro-3'-deoxythymidine (stavudine), and 3' deoxy-3'-fluorothymidine (FLT) were purchased from Sigma–Aldrich (Milwaukee, WI, USA). Zidovudine impurity B (chlorothymidine, CLT) was purchased from LGC Standards (Wesel, Germany). Kryptofix2.2.2 (K222), 3-N-Boc-5'-Odimethoxytrityl-3'-O-nosyl-thymidine (Boc-FLT) were purchased from ABX (Radeberg, Germany).</p><p>All samples were prepared with 18 MΩ deionized water using a Milli-Q® Integral Water Purification system (EMD Millipore, Billerica, MA, USA). 30 mM phosphate buffer (PB) was prepared via titration 100 mM solutions of NaH2PO4 and Na2HPO4 and monitored with a pH meter (Mettler, Toledo, Easy five, Columbus, OH, USA). 100 mM SDS in 30 mM phosphate buffer (SDS-PB) was prepared by dissolving SDS in 30 mM PB. All buffers were degassed prior to use.</p><!><p>We combined the three key components (injection, separation, and detection) into a hybrid MCE system (Figure 1). One microfluidic chip, used for sample injection and containing the anode, was connected to the upstream side of a 60 cm long, Teflon-coated fused silica capillary (75 μm I.D., 375 μm O.D; Polymicro, Phoenix, AZ, USA). A second microfluidic chip, used for sample detection and containing the cathode, was connected to the downstream side of the separation capillary. The capillary was connected to each chip via a port perpendicular to the channels within the chip.</p><p>All electronic components were connected to a digital acquisition (DAQ) module (USB 6211, National Instruments Corporation, Austin, TX, USA). A custom-written LabVIEW program (National Instruments Corporation, Austin, TX, USA) was used to coordinate the timing of all functions.</p><!><p>Though the commonly used technique of electrokinetic injection provides a very convenient means to inject samples in CE and MCE, this technique suffers from injection bias, i.e. solutes with higher electrophoretic mobilities are preferentially introduced, resulting in a difference between the composition of the original sample and that injected into the separation channel, as well as changing of the sample composition over time which interferes with repeat measurements [33, 34]. This bias, and other sensitivities of this technique (to voltage, sample conductivity, sample pH, electrolysis, and the possibility of complex formation) [34] could prevent accurate assessment of diverse impurities in PET tracer samples. Thus, pressure-driven injection [34], which avoids the above injection bias was used.</p><p>The design of the microfluidic injection chip, shown in Figure 2A, was adapted from the report of Li et al. [35]. The chip was fabricated from poly(dimethylsiloxane) (PDMS) using multilayer soft lithography [36]. Fabrication details and connection to the upstream end of the capillary are included in the Supporting Information. The chip enables a controlled amount of sample to be loaded from the sample inlet port into the separation channel by momentarily opening a microvalve (v3) for a fixed time. An additional microvalve (v2) enables priming of the sample inlet to eliminate air. The sample was contained in a septum-sealed vial (Fisherbrand™ 2 mL screw thread autosampler vial, Thermo Fisher Scientific, Waltham, MA, USA). Pressurized nitrogen gas was supplied to the vial through an electronic pressure regulator (ITV0010-3BL, SMC Corporation of America, Noblesville, IN, USA). The vial also contained an outlet tubing (#30 PTFE tubing, Cole-Parmer, IL, USA) connected to the sample inlet port of the injector chip. In addition to the sample inlet, the chip also contained an inlet for buffer solution, which was similarly connected to a pressurized vial of the separation buffer (SDS-PB) and controlled via microvalve v1.</p><p>The detailed steps to perform sample injection are illustrated in Figure 2B. Before use, the chip was first primed with buffer by closing v3, opening v1, and pressurizing the buffer vial (6.0 psi) until buffer started flow out of all the buffer wells (and also out the buffer waste well of the detection chip connected to the other end of the capillary). Next, the sample vial was pressurized (1.5 psi) and the sample inlet was primed by closing v1 and v3 and then opening v2 until sample was seen entering the sample waste vial. To load the sample, valve v3 was then opened for a fixed time to allow sample to fill part of the main channel in the chip. After the sample is loaded all valves were closed and electrophoretic potential was applied to separate the sample.</p><p>On-chip microvalves were each controlled by the common port of an electronic solenoid valve (S070B-5DG, SMC Corporation), connected to the chip via #30 PTFE tubing. The solenoid valves switched between two states: (i) supplying pressurized nitrogen (35 psi) to close the on-chip microvalve, and (ii) venting to atmosphere to allow the on-chip microvalve to open via elastic restoration of the PDMS. To avoid the generation of air bubbles inside the sample-containing channels of the chip, the valve control channels were filled with water prior to use as previously described [37].</p><!><p>In typical radio-HPLC systems used in the field of radiochemistry, the flow cell has a path length of ~10 mm (10,000 μm). In the case of capillary electrophoresis in capillaries or microchannels, the optical path length (OPL) is much shorter (e.g. 30-100 μm) if light is directed, via a window, perpendicular to the flow through the capillary of microchannel. Because this short optical path reduces the absorbance 'signal', it typically results in a relatively poor LOD in CE systems compared to HPLC. This problem can be addressed by leveraging the ability to precisely control fluid geometry in microfluidic devices and implementing an increased optical path length. An in-plane Z-shaped detection cell design [38] was selected, due to the simplicity of chip fabrication and interfacing of the illumination and detection optical fibers.</p><p>The chip was fabricated from a single patterned layer of PDMS bonded to a PDMS substrate. Fabrication details, including connection to the downstream end of the capillary, are described in the Supporting Information. The design (Figure 3) includes fiber alignment channels to ensure accurate collinear alignment of both the optical fibers (i.e. to provide illumination via the external light source and detection via the external spectrometer) with a 'jog' in the sample channel representing the extended optical path within the chip [39]. Due to the elastic property of PDMS, the 125 μm OD optical fibers (ThorLabs, Newton, New Jersey, USA) are held stably in these channels by friction forces. The flat ends of the fibers sit flush against the flat end of the fiber channels, providing efficient optical coupling to the sample channel. Since PDMS absorbs strongly in the UV range [40], it was desirable to minimize the thickness of PDMS membrane between the end of the fiber and the sample within the channel. A thickness of 100 μm was chosen as it provides good optical transmission (>85% transmission for wavelengths > 220 nm), sufficient mechanical resistance to deformation, and high electrical breakdown voltage (~2000V [41], sufficient to sustain the CE potential at this point in the separation channel). In addition to the portion of each fiber alignment channel that is collinear with the optical path, there is a continuation that allows the air initially in the channel to be vented. All channels were 125 μm deep and 125 μm wide. Using the same depth for the fiber-aligning channels as for the fluid-containing channels simplifies the chip fabrication, requiring a single thickness of photoresist.</p><p>The performance of the detection chip was compared with two combinations of light sources and detectors, one with lower performance and one with higher performance. Detector 1 comprised a PX-2 pulsed xenon light source (Ocean Optics, Dunedun, FL, USA) and USB-4000 spectrometer (Ocean Optics) and Detector 2 consisted of a DH-2000-BAL continuous deuterium light source (Ocean Optics) and QE-Pro spectrometer (Ocean Optics). The PX-2 is ~5x cheaper than the DH-2000-BAL; however, it has significant noise, which adversely affects LOD. The pulse-to-pulse variation in light intensity is in the range 3-12% depending on pulse frequency [42], compared to an intensity drift of <0.01% per hour [43] for the DH-20000-BAL. Similarly, the USB4000 is ~20x cheaper than the QE-Pro, but has a lower signal-to-noise ratio (275:1 compared to 1000:1) and lower dynamic range.</p><p>While the current work serves as proof of concept for miniaturized analysis of PET radiopharmaceuticals, ultimately it will be necessary to incorporate a radiation detector with good spatial resolution to enable assessment of radiochemical identity and purity in addition to chemical purity. We are currently in the process of developing such an integrated detector for the detection chip and will publish these findings in the future.</p><!><p>After fabrication and assembly of the hybrid chip, it was conditioned prior to use. First, the chips and capillary were filled with water via the buffer inlet port at 10 psi for 30 min to ensure all air was purged from the system. The both ends of the chips were placed in a Petri dish containing a damp Kimwipe and wrapped with parafilm. Next, this procedure was repeated with 1M NaOH to form hydroxyl groups [44] on the inner surfaces of the capillary and PDMS microchannels. The NaOH was removed during the buffer priming step of the sample injection process.</p><!><p>The separation voltage was provided by a 0-30 kV high voltage DC power supply (HV350, Information Unlimited, Amherst, NH, USA). The tip of the high voltage electrode wire was submerged in the separation buffer well of the injection chip and that of the ground electrode wire was submerged in the waste well of the detection chip. Electrodes were held in place by electrically-insulated clamps mounted on a retort stand. 12 kV was supplied to achieve a field of ~200 V/cm along the separation channel. The total length of the separation path from the buffer well to the waste well was 62 cm. The effective separation length, i.e. injection point to the detector, was 61 cm. CE voltage was turned on or off using a solid-state relay in series with the high-voltage side of the circuit. During operation, DC current was monitored in real-time via a digital multimeter (Model 2831E, BK precision, Yorba Linda, CA, USA) to detect any abnormal behavior of the chip. For example, any air/gas bubble formation can lead to interruption of the current with intermittent electrical arcing; if this occurred, the high voltage was immediately interrupted and the fluidic system was reconditioned for ~2 min to purge any bubbles and to re-equilibrate the inner surfaces.</p><!><p>Methods for computing absorbance from the spectrometer signal are described in detail in the Supporting Information. To create an electropherogram, spectrometer output was measured at a rate of 10 samples/s and converted to absorbance, starting at the time of injection.</p><p>Each electropherogram was analyzed using OriginPro 8.5 (OriginLab, Northampton, MA, USA) to determine peak migration times (tm, taken at peak center), peak widths (w1/2, full width at half maximum), as well as other values such as peak areas based on a Gaussian fit to each peak. Peaks were identified based on retention times determined by injecting standard compounds individually.</p><!><p>For the purposes of characterizing the injector, the detection chip was not used; rather, detection of analytes was performed directly in the capillary using a 4-way junction (PEEK Cross, P-729, Idex Health & Science, Oak Harbor, WA, USA) positioned 7 cm away from the downstream end of capillary. A small portion of capillary was covered with a 1/16" OD tubing sleeve (Idex Health & Science, Oak Harbor, WA, USA) and secured via two opposite ports of the junction. The illumination and detection optical fibers were secured in the two perpendicular ports. Note that the effective separation length in this case was 54 cm. The total separation length between the buffer well of the injection chip and the waste vial was 61 cm and the separation voltage applied was +12 kV.</p><p>To assess the sample injection repeatibility, successive injections of 5mM thymidine were performed with a valve opening time of 800 ms (determined as described in the Supporting Information).</p><!><p>Chemical purity tests are performed on radiopharmaceutical preparations to confirm the absence of impurities after the purification and formulation processes. For some impurities (e.g. Kryptofix K222, etc.), there are well-established limits based on toxicity studies that can safely be injected into patients. Unstudied impurities, provided they pose negligible risk of carcinogenicity, are typically limited to 1.5 μg per patient per day (5 nmol for a compound with molar mass of 300 g/mol). A typical radiopharmaceutical preparation has a volume of 1-10 mL (or larger) and contains sufficient material for 1 or more patient scans. In the conservative case (1 mL volume, 1 patient), this gives an upper permitted limit of 5 μM. To establish whether these levels can be detected in our setup, we have characterized the sensitivity of detection chip by measuring the limit of detection (LOD) and limit of quantitation (LOQ) for varying conditions, including varying optical detection path length and varying optical systems).</p><p>To characterize the detection chip, the capillary was connected but the injection chip was not used. Instead, UV absorbance was measured when the detection chip was fully filled with several concentrations of each analyte. The absorbance was measured for each sample at the desired wavelength(s) for ~ 1 min, and then averaged to obtain one data point. This procedure was repeated 3 times while flushing the optical path length with blank solution between each measurement. The three data points were then averaged to obtain an overall absorbance value for the particular concentration of the particular analyte. To minimize the impact of cross-contamination, the most dilute samples were measured prior to more concentrated samples. After performing a linear fit of absorbance versus concentration (i.e. Beer's Law), the LOD and LOQ were determined by calculating the concentration that corresponds to 3x and 10x, respectively, the standard deviation in background absorbance noise. UV absorbance was measured at 256 nm or 224 nm, corresponding to the wavelength of maximum absorbance for the model compounds used (see Supporting Information).</p><!><p>To evaluate separation efficiency, we chose as a model system the PET tracer [18F]3'-fluoro-3'-deoxythimidine ([18F]FLT), for which the impurity profile is well known [45]. The synthesis scheme and the structurally-similar side-products are shown in Figure 4. A mixture of FLT and by-products (5 mM thymidine, 2 mM thymine, 2.5 mM furfuryl alcohol, 5 mM stavudine, 2.6 mM FLT, and 1.4 mM CLT) was injected to assess separation efficiency. Separation was performed with micellar electrokinetic chromatography (MEKC) since the compounds are all neutral.</p><p>Samples were injected via injection chip using a valve opening time of 400 ms (determined as described in the Supporting Information). For each peak in the resulting electropherogram, the number of theoretical plates, N, was calculated as follows [46, 47]: (1)N=5.54(tmw1∕2)2</p><!><p>Performance was compared to separations on an analytical HPLC system as previously described [32]: Knauer Smartline HPLC system using a C18 Luna reverse phase column (4.6 mm × 250 mm, 5 μm; Phenomenex, Torrance, CA, USA). Detection was performed at 224 and 254 nm with an inline UV detector (model 2500, Knauer, Berlin, Germany). The HPLC mobile phase for FLT separations was 10% ethanol in water (v/v), at flow rate of 1 mL/min. All chromatograms were collected by a GinaStar analog to digital converter (Raytest USA Inc., Wilmington, NC, USA) and GinaStar software (Raytest USA Inc., Wilmington, NC, USA). Comparisons were also made to previously reported results using a commercial (macroscale) CE system (PA800, Beckman Coulter, CA, USA) [32].</p><!><p>Injection repeatability of the PDMS injection chip was assessed by determining the consistency of peak area resulting from successive injections of single compound. The relative standard deviation (RSD) of peak area of successive injections of thymidine was 3.9% (n=8). Since this performance was sufficient for remaining experiments to assess the feasibility of the hybrid MCE for chemical purity analysis, further optimization was not performed at the time, and remaining results are performed with this injector.</p><p>However, peak area RSD <2% is generally desired for quantitative analysis [48]. Li et al. reported a peak area RSD as low as 1.77% (n=15) [35], using a similar PDMS injection chip, but with an integrated separation channel rather than external capillary as used here. We suspect that dead volume at the chip to capillary junction in our MCE setup (see discussion below) may be causing the variability.</p><p>Another way to improve performance may be to switch injection methods since the method can have a large impact on the peak area RSD. In HPLC, the amount of sample is measured volumetrically (by the injection loop), resulting in very high injection repeatability. Recently, we explored a novel volumetric injection technique for MCE and showed that a peak area RSD as low as 1.04% (n=4) [37] could be achieved, even using an external capillary for separation. We are thus confident that a next-generation device incorporating a PDMS-based injector will achieve sufficient repeatability for radiopharmaceutical analysis.</p><!><p>Initially, we attempted UV detection directly in the capillary. The LOD and LOQ for several analytes, using both combinations of light source and detector are shown in Table 1. Even when the higher performance setup (Detector 2) was used for the in-capillary detection, LODs were all higher than 20 μM, and significantly worse than values previously measured for HPLC [32]. This is likely due to the much shorter optical path through the sample in the capillary (i.e. ~75 μm, the inner diameter of the capillary) compared to the 10000 μm flow cell in the HPLC system. The values were also significantly worse than those previously measured in a commercial CE system (5 – 11 μM; Beckman Coulter PA800) [32], likely due to differences in the optical system, capillary environment (i.e. temperature-controlled in the commercial CE system), and signal processing.</p><p>To improve the LOD, a PDMS detection chip with a Z-shaped extended optical path (500 μm) was implemented. In combination with the higher performance light source and detector pair (Detector 2), LODs ranged from 2 – 3 μM for the set of compounds with similar chromophore (thymidine, thymine, stavudine, FLT, and CLT), and 7 μM for furfuryl alcohol. Thus, the LOD values of the MCE setup are comparable to the performance of HPLC (i.e., 2 μM for stavudine and FLT, 35 μM for furfuryl alcohol) [32], and are below the typical permitted limit of impurities found in radiopharmaceuticals. Detection performance is summarized in Table 1 for all system configurations. Since the desired performance was achieved, the 500 μm OPL was used in subsequent experiments. For a 500 μm OPL detection chip and Detector Configuration 2, we found the linear range of the various species to be: 2 μM – 5 mM (thymidine), 2 μM – 5 mM (thymine), 7 μM – 3 mM (furfuryl alcohol), 3 μM - 5mM (stavudine), 3 μM – 5 mM (CLT), and 2 μM – 5 mM (FLT).</p><p>We also explored the possibility to achieve similar performance with the lower-performance (and lower cost) light source and detector (Detector 1) by fabricating additional detection chips with different OPL. The LOD and LOQ values for thymidine and furfuryl alcohol in detection chips with different OPL are summarized in Table 2. To more clearly see the effect of OPL, we note that LOD is defined as the concentration of analyte where the absorbance equals 3x the standard deviation of noise (N). Substituting into Beer's law, we can write LOD = 3N/ε/OPL, where ε is the molar absorbtivity. Thus, LOD is inversely proportional to the OPL and the data in Figure 6 were thus fit to this function to extrapolate the OPL necessary to match the performance of HPLC. To achieve LOD = 2 μM for thymidine (and FLT and stavudine, which have an identical chromophore and thus similar absorbance), an OPL of 2500 μm would be required. Similarly, to achieve LOD = 35 μM for furfuryl alcohol, an OPL of 420 μm would be required. Thus, the PDMS detection chip with Detector 1 could match/surpass the detection sensitivity of HPLC by extending the optical path length to 2500 μm.</p><!><p>Previously we showed that mixtures of FLT and its structurally-similar byproducts (thymidine, thymine, furfuryl alcohol, stavudine, and CLT) could be separated by HPLC and by a conventional CE instrument with baseline resolution [49]. We analyzed similar samples to demonstrate the feasibility of injecting, separating, and detecting samples in the hybrid microfluidic system.</p><p>First, we started with the simplest geometry that resembles the commercial CE instrument, i.e., a capillary-only ("0-junction") system without any microchips connected (Figure 5A). For this method, the injection was performed electrokinetically, by inserting the upstream side of the capillary in the sample vial (2 mL, C4013-15A, Thermo Scientific), applying +12 kV for 5 s, then moving the capillary back to the buffer vial prior to separation. Successful baseline separation of the sample mixture (FLT and five impurities) was achieved (Figure 7A).</p><p>Next, the injection microchip was added to the capillary to form a "1-junction" system (Figure 5B). Even though baseline separation was observed for most peaks, the first two peaks were not completely resolved (Figure 7B). Finally, we tested an integrated microfluidic system with injection chip, silica capillary and detection chip (Figure 5C). An electropherogram is shown in Figure 7C. While all expected peaks are discernable, baseline separation was not achieved among the three fastest eluting compounds (thymidine, thymine, and furfuryl alcohol). Qualitatively, it is clear that the peak width using the hybrid MCE device was greater than that for the 0-junction setup, leading to the reduced separation efficiency. This was confirmed by computing the number of theoretical plates, N, for each setup (Table 3): it was found that N is significantly lower for the hybrid MCE device compared to the 0-junction setup.</p><p>To determine where improvements can be made, we analyzed another measure of efficiency: the plate height, H=L/N, where L is the effective separation length. Lower H values indicate more theoretical plates within the separation length meaning a higher separation efficiency. Conveniently, H can be expressed as a sum of contributing factors (injection, detection, diffusion, and geometry) [50]: (2)H=LN=Hinj+Hdet+Hdiff+Hgeo</p><p>The injection and detection components are determined from the length of the injection plug (linj) and the length of the detection cell (i.e. OPL) (ldet), respectively [50]: (3)Hinj=linj212L (4)Hdet=ldet212L where L is the effective separation length.</p><p>The contribution of axial diffusion [47, 50] is given by: (5)Hdiff=2Dav where Da is the diffusion coefficient of the analyte and v is the linear velocity of the analyte.</p><p>The contribution due to the geometry is the most complex [50, 51]: (6)Hgeo=n(ωθ)212L+σni2L+σdv2L</p><p>The first term can be ignored since our system does not currently use a separation channel with a serpentine pattern (n is the number of turns, ω is the width at the top of the channel (peak of the turn), θ is the turn angle). σni represents band broadening from non-ideal behavior of injected sample and Joule heating, and σdv represents the broadening due to dead volume. Both σni and σdv are of unknown form that depends of geometric shape of the channel, channel material, and electric field gradients [51].</p><p>Based on electropherograms, values of N, H, Hinj, and Hdet were computed and are summarized in Table 3 (Detailed calculations can be found in Supporting Information). These results show that the contribution to peak broadening due to the detector (Hdet) in the hybrid MCE (2-junction) device is negligible and broadening due to the injector (Hinj) is ~1% for stavudine and <1% for CLT. Thus, the major contributors to the broadening of peak width are Hdiff or Hgeo.</p><p>For the 0-junction CE system, H (total) was low, i.e. 6.41 μm and 6.38 μm for thymidine and CLT, respectively. Based on the well-defined computed values of Hinj and Hdet, and ignoring Hgeo for the moment, maximum upper bounds on Hdiff for the 0-junction system can be estimated as ~3.5 μm and ~5.2 μm for thymidine and CLT, respectively. It is expected that broadening due to diffusion (Hdiff) would have a similar value for the 1- and 2-junction (hybrid MCE) systems. This is because the analytes, buffer, and temperature were consistent across these systems and thus Da was constant. In addition, the elution velocities were very similar (e.g. for CLT, 0-junction velocity was 0.054 cm/s, 1-junction velocity was 0.038 cm/s, and 2-junction velocity was 0.045 cm/s). Thus, we expect Hdiff to have an upper bound of only a few μm for the 1- and 2-junction cases, and we can deduce that Hgeo must be the dominant factor for both.</p><p>Comparing the CLT peak from the 0- and 1-junction cases, there was a large increase in H (i.e. from 6.4 to 39), and comparing the 1- and 2-junction cases, there was another large increase (i.e. from 39 to 121). Since there are only minor expected differences in the injection, detection, or diffusion components of plate height, these increases must be due to geometric factors. Because of the strong increase in H as the number of junctions increases, the band broadening is likely occurring due to the geometry (e.g. dead volume) at each capillary-to-chip junction.</p><p>The dead-volume could be reduced by various approaches such as precise drilling [51] or molding the capillary port [52], or by tapering the capillary to fit directly in an in-plane microchannel [37, 53].The geometry issue could also be addressed by integration of the separation channel directly into the chip (instead of using a capillary); this would eliminate the junctions altogether and simplify the overall setup, enabling a single integrated microfluidic device for injection, separation, and detection. Separation in PDMS channels has been reported by several groups [54, 55], though some have reported challenges in maintaining stable surface conditioning [56, 57]. An alternative may be to perform separation using an embedded capillary [58, 59]. With an optimized chip, one could expect the total plate height H to be similar to the 0-junction case. Indeed, the elimination of 1 junction shows significant improvement in separation (Figure 5B), and elimination of both junctions shows further improvement (Figure 5A), achieving baseline separation of FLT and five impurities. An optimized hybrid (2-junction) MCE system with improved capillary junction is therefore expected to be capable of similar baseline separation.</p><p>In addition to addressing the dead-volume at the capillary junctions in this manner, optimization of other parameters could also be explored to maximize separation efficiency. For example, applied electrical field can be increased to increase the velocity of analytes, which would reduce diffusive broadening, and either allow reduced separation times or enable the use of increased separation length.</p><!><p>The use of miniaturization to reduce the equipment size and shielding needed for the chemical purity analysis of PET tracers is expected to be a key part of streamlining the QC testing process, and ultimately the overall tracer production process. In this work, we have demonstrated the first proof-of-concept experiments to show the feasibility of microfluidic implementation of chemical identity and purity tests of radiopharmaceuticals.</p><p>The novel hybrid MCE device consists of a PDMS injection chip, a silica capillary, and a PDMS detection chip. Sample injection was based on hydrodynamic injection using microvalves to achieve satisfactory reproducibility while avoiding the known injection bias of conventional electrokinetic injection. The detection chip enabled adjustment of the optical path length to tune the limit of detection. Though an extended path length of 500 μm resulted in LOD comparable to HPLC when the higher performance light source/detector pair was used, we showed that further extension of the optical path (e.g. OPL ~2500 μm) could enable similar sensitivity even with the lower performance light source and detector, without significantly compromising the separation performance. In the integrated hybrid device, mixtures of FLT and impurities were successfully injected, separated, and detected. Even though FLT was successfully separated from all impurities, several impurity peaks were not fully resolved with baseline resolution. While the separation performance of the integrated device was lower than desired, a detailed analysis identified the capillary-chip junctions as the problem. Extrapolating from the performance when junctions are eliminated, we argue that a device with optimized junctions [37] could achieve the requisite performance. Furthermore, the optimized MCE device would be very much smaller than an HPLC system.</p><p>Unlike simple colorimetric tests that have been developed for determination of certain individual impurities (e.g. Kryptofix 2.2.2, a phase transfer catalyst frequently used in the synthesis of 18F-labeled PET tracers), MCE-based testing provides a flexible way to assess different and multiple impurities, possibly by tuning separation conditions and/or adding detectors (e.g. electrochemical, pulsed amperometric, etc.) for detection of species with low UV absorbance. Furthermore, separation prior to detection greatly reduces the chance of false negatives or positives due to non-specific interactions that can occur in colorimetric tests. Due to the flexibility of a chromatographic approach, it is expected that this device could easily be applied to the evaluation of PET tracers other than FLT. Furthermore, integration of a radiation detector would enable assessment of radiochemical identity and purity in the same device.</p><p>In the long term, this device and other microfluidic QC tests could be combined in a unified lab-on-a-chip device for performing fully-automated QC testing of radiopharmaceuticals. In addition to alleviating the burden of performing and documenting QC tests, such a system would reduce the amount of sample consumed for analysis, reduce the radiation exposure to personnel, and potentially reduce the time needed to complete all QC tests.</p>

PubMed Author Manuscript

Two-photon AIE bio-probe with large Stokes shift for specific imaging of lipid droplets

Lipid droplets are dynamic organelles involved in various physiological processes and their detection is thus of high importance to biomedical research. Recent reports show that AIE probes for lipid droplet imaging have the superior advantages of high brightness, large Stokes shift and excellent photostability compared to commercial dyes but suffer from the problem of having a short excitation wavelength. In this work, an AIE probe, namely TPA-BI, was rationally designed and easily prepared from triphenylamine and imidazolone building blocks for the two-photon imaging of lipid droplets. TPA-BI exhibited TICT+AIE features with a large Stokes shift of up to 202 nm and a large two-photon absorption cross-section of up to 213 GM.TPA-BI was more suitable for two-photon imaging of the lipid droplets with the merits of a higher 3D resolution, lesser photobleaching, a reduced autofluorescence and deeper penetration in tissue slices than a commercial probe based on BODIPY 493/503, providing a promising imaging tool for lipid droplet tracking and analysis in biomedical research and clinical diagnosis.

two-photon_aie_bio-probe_with_large_stokes_shift_for_specific_imaging_of_lipid_droplets

Introduction<!>Results and discussion<!>Solvatochromism and twisted intramolecular charge-transfer<!>Aggregation-induced emission<!>Two-photon excited uorescence<!>One-photon LD imaging<!>Two-photon LD imaging<!>Conclusion

<p>Lipid droplets (LDs) that contain mainly diverse neutral lipids such as triacylglycerol and cholesteryl ester are widely found in adipocytes, hepatocytes and the adrenal cortex. For many years, LDs have been regarded as inert reservoirs of neutral lipids for energy storage. However, recent results show that LDs are considered to be dynamic organelles and associated with the storage and metabolism of lipids, signal transduction, apoptosis and so on. 1 The abnormalities of LDs are generally related to some important diseases. 1,2 For example, LDs are found to be critical for the proliferation of the hepatitis C virus, 3 infection of which will lead to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Thus, the localization and analysis of LDs are highly important for biomedical research and clinical diagnosis.</p><p>Techniques based on uorescent materials are emerging as powerful and popular tools for biomedical studies both in vitro and in vivo. 4 They exhibit excellent performances in applications such as localizing subcellular organelles, and monitoring the physiological changes of pH, temperature, viscosity, ions, proteins, and so on with the superior advantages of high resolution and sensitivity, easy operation and low cost. Recently, uorescent probes for the localization of LDs have been developed. Two commercial dyes, namely Nile Red and BODIPY 493/ 503, are widely used but they show some fatal drawbacks, such as strong backgrounds and small Stokes shis. 5 Worse still, these conventional organic uorophores unavoidably face a problem of aggregation-caused quenching (ACQ), where their uorescence is quenched at high concentrations due to the formation of detrimental species such as excimers and exciplexes by strong p-p stacking. 6 The ACQ effect has largely conned their working concentration to a very low nanomolar level, leading to quick photo-bleaching for bioimaging.</p><p>For many years, we and other groups have worked on the development of molecules with aggregation-induced emission (AIE) characteristics that are the exact opposite of the ACQ uorophores. The restriction of intramolecular motion (RIM) has been proposed as the mechanism for the AIE effect. 7 AIE luminogens (AIEgens) are weakly emissive in solutions due to the deactivation of the excited states by active intramolecular motions. However, such motions are suppressed in the aggregate state, thus enabling them to emit intensely upon excitation. AIEgens have found promising biomedical applications due to their superior merits of large Stokes shis, high brightness, good biocompatibility, excellent photostability, etc. 8 Therefore, the development of LD-specic AIE bioprobes could provide a promising approach to solving the problem observed in commercial dyes. Indeed, in our previous work, LD-specic AIE bioprobes, such as TPE-AmAl, FAS, DPAS and TPE-AC (Chart 1), show better performances in terms of brightness, specicity and photostability than their commercial counterparts in both xed and living cell imaging. Meanwhile, these AIE-based bioprobes can be easily synthesized and have good cell permeability. 9 However, most of the LD-specic AIE bioprobes developed so far bear either UV excitation or shortwavelength emission, which is harmful to living cells and suffers from limited penetration depth to tissue and serious auto-uorescence from biosamples. 4,10 Although TPE-AC exhibited a fascinating NIR emission (705 nm), 11 the excitation wavelength was merely 450 nm, which was not long enough to reach the optical window for optimal tissue penetration (750-950 nm). 10b Thus, LD-specic AIE bioprobes with excitation wavelengths in the red and near-infrared (NIR) regions will solve these problems and are in urgent demand. Many efforts have been devoted to designing new LD-specic AIE bioprobes with red and NIR excitations. Unfortunately, such a task is not easy in terms of tedious synthesis and low emission efficiency of the resulting molecules.</p><p>Recently, two-photon uorescence microscopy (2PM) has become popular in biomedical diagnosis and therapy, due to its advantages of a longer-wavelength excitation, lower auto-uorescence, higher 3D resolution and less photobleaching. 12 Luminescent materials with two-photon excitation are crucially determined by a two-photon absorption (2PA) cross section (d 2PA ). Materials bearing higher d 2PA will show stronger twophoton excited uorescence (TPEF) and a less deleterious thermal effect from the strong laser pulse. 13 Therefore, the design of AIE bioprobes with two-photon excitations can provide an easier way to realize red and NIR excitations.</p><p>Benzylidene imidazolone (BI), the analogue of the chromophore of green uorescent protein, has been wildly-studied due to its facile synthesis and excellent biocompatibility. 14 Recently, many of its derivatives have been designed and found to be AIEactive. 15 Compared to TPE, BI possesses a more rigid structure with a less twisted conformation and would be an ideal building block for 2PA materials. 16 However, to the best of our knowledge, BI-based 2PA materials have been rarely reported. Herein, we attempt to integrate the merits of AIE and 2PA into BI. On the other hand, triphenylamine (TPA) is a popular design unit for 2PA 10b,17 and a well-known strong electron donor. The decoration of BI with TPA is thus expected to give a luminogen with a high d 2PA and a longer-wavelength emission. The structural design of the molecule, abbreviated to TPA-BI, is shown in Scheme 1. Indeed, TPA-BI possessed a large d 2PA and exhibited strong TPEF. TPA-BI can specically stain lipid droplets in both xed and live cells with a large Stokes shi and a superior twophoton imaging performance.</p><!><p>Synthesis TPA-BI was readily synthesized in a good yield by the Suzuki coupling of (Z)-5-(4-bromobenzylidene)-2-methyl-3-propyl-3,5dihydro-4H-imidazol-4-one ( 2) and (4-(diphenylamino)phenyl) boronic acid (3) (Scheme 1). Detailed experimental procedures are provided in the Electronic Supplementary Information (ESI †). The structure of TPA-BI was fully characterized and conrmed by NMR and high-resolution mass spectroscopies (ESI, Fig. S1-S3 †).</p><!><p>Molecules with donor (D)-p-acceptor (A) structures are characterized by a prominent solvatochromic effect, where their photophysical properties change by varying the solvent polarity. Hence, the absorption and photoluminescence (PL) spectra of TPA-BI in solvents with different polarities were investigated and the results are shown in Fig. 1 and S4. † In Fig. 1A, under UV light irradiation, the emission colour of the TPA-BI solution could be nely tuned from blue to red when the solvent changed from n-hexane to acetonitrile, nearly covering the full visible spectrum. The emission maximum varied gradually from 447 nm to 619 nm (Fig. 1B). Simultaneously, a pronounced decrease in the emission intensity was observed. On the contrary, the absorption of TPA-BI exhibited little change on changing the solvent polarity (Fig. S4 †). The absorption maximum of TPA-BI only changed from 400 nm to 414 nm by increasing the solvent polarity with an extinction coefficient of $34 000 M À1 cm À1 . All of these results indicate that the photophysical properties of TPA-BI are strongly dependent on the solvent polarity, which is ascribed to the twisted intramolecular charge transfer (TICT) effect from the electron-donating TPA unit to the electron-accepting imidazolone functionality. A large Stokes shi of up to 212 nm was realized, largely avoiding the overlap of the absorption spectrum and emission spectrum. This property is highly demanded for uorescence probes as it prevents the self-absorption or "inner-lter" effect to increase the signal to noise ratio for uorescence imaging.</p><p>To evaluate the effect of the solvents on the PL of TPA-BI, the change in the PL maximum with the solvent polarity parameter (E T (30)) 18 is plotted in Fig. 1C and summarized in Tables S1 and S2. † A linear line with a correlation coefficient of R 2 ¼ 0.992 and a large slope of 11.8 was obtained, indicating the remarkable solvatochromism of TPA-BI. The solvatochromic properties of TPA-BI were also conrmed by the dependence of the uorescence transition energy on the solvent orientation polarizability (Df 0 ) according to the revised Lippert-Mataga equation for TICT molecules (Table S1 and Fig. S5 †). Both results indicate that TPA-BI shows strong solvatochromism resulting from the TICT effect. The TICT effect of TPA-BI can be interpreted by density functional theory (DFT) calculations (Fig. S6 †). The photoexcitation from the S 0 to S 1 state of TPA-BI involves a substantial intramolecular charge transfer (ICT) from TPA to the imidazolone unit. Since the donor and acceptor are linked via a freely rotatable single bond, the activation of the ICT process is likely accompanied by a signicant molecular geometry change and the formation of a TICT state. The TICT state will be largely stabilized and populated in solvents with higher polarity, resulting in a red-shi in the emission band. The TICT effect is responsible for the solvatochromism of TPA-BI and the increase in the Stokes shi from non-polar to polar solvents. The decrease in the PL intensity in a polar solvent should be attributed to the rapid consumption of the energy of the TICT state through non-radiative relaxation pathways. 19</p><!><p>Besides solvatochromism, TPA-BI also shows an aggregationinduced emission (AIE) phenomenon. As shown in Fig. 2, with an increase in the water fraction from 0 to 40% in the dimethylsulfoxide (DMSO)/water mixture, the emission of TPA-BI decreased, accompanied with a slight red-shi in the PL spectrum. This is due to the enhancement of the TICT effect in the presence of the more polar solvent of water in the surrounding environment. Upon further increasing the water fraction from 40% to 70%, an abrupt increase in the emission intensity ($100-fold) was observed along with a blue shi in the PL maximum from 615 nm to 555 nm. To have a more accurate evaluation of the AIE characteristics, we have measured and plotted the quantum yields of TPA-BI in mixtures with different water fractions by an integrating sphere. 20 The plot shows a similar trend with I/I 0 (Fig. S7A †). The uorescence quantum efficiency of TPA-BI in a 70% aqueous mixture was 22%, which was appreciably high for an orange emitter. Due to its poor solubility in water, in solution with high water fractions, aggregates of TPA-BI would be formed. This greatly restricts the intramolecular motion and activates the AIE process. The domination of the AIE effect over the TICT effect results in an increase in the PL intensity. 9a,11 Surprisingly, the emission became weaker and was slightly red-shied again when the water fraction increased from 70% to 90%. This may be attributed to (1) the crystallization-induced emission feature of TPA-BI and ( 2) the effect of the aggregate size. 21 TPA-BI may form crystalline aggregates at low water fractions. At water fractions above 70%, the fast aggregation of the TPA-BI molecules will form less emissive, redder amorphous species with smaller sizes, as conrmed by the DLS results (Fig. S7B †). The emission of small-sized aggregates may be more vulnerable to being affected by the surrounding solvent environment, leading to an emission drop and red-shi at high water fractions.</p><!><p>TPA-BI possesses a conjugated structure with strong electron donating and withdrawing groups and thus it is expected to exhibit strong 2PA. The 2PA of TPA-BI was studied using a TPEF technique with a femtosecond pulsed laser source, and the relative TPEF intensity in different solvents was measured using Rhodamine 6G and uorescein as the standards. 22 The measured wavelength was varied from 720 to 920 nm at an interval of 40 nm and the d 2PA values were obtained. The results are summarized in Fig. 3 and Table S2. † In THF, the maximum d 2PA value (159 GM) was obtained at 840 nm. In various solvents, the highest d 2PA was obtained in diethyl ether and was equal to 213 GM, which was much higher than those of most uorescent proteins (usually < 100 GM, only 39 GM for EGFP), 23 synthetic BI derivatives (<40 GM), 15c and BODIPY dyes (82-128 GM). 24 Thus, TPA-BI may serve as a good two-photon imaging probe to living cells.</p><p>Apart from 2PA, the TPEF of TPA-BI under different laser powers was also studied. The plot of the uorescence intensity against the excitation laser power gave a linear line with a slope of 1.911, conrming the occurrence of two photon absorption (Fig. S8 †). 19c When excited by laser light at 840 nm, TPA-BI emitted intense PL at 447-619 nm in solvents with different polarities, suggesting the TICT feature even under the condition of two-photon excitation (Table S2 †). The spectral patterns resemble the one-photon ones, revealing the same excited state for the radiative decay processes (Fig. 3B). The TPEF cross sections (d 2PEF ) are crucial parameters for biomedical imaging and are provided in Table S2. † The high d 2PEF values in different solvents suggest that TPA-BI possesses a promising potential application in the biomedical eld.</p><!><p>To explore the application of TPA-BI in living cell imaging, its cytotoxicity was rstly evaluated using 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay under different dye concentrations. As suggested in Fig. S9, † no signicant variation in the cell viability was observed even when a high dye concentration of 20 mM was used. This indicates that TPA-BI shows almost no cytotoxicity to living cells and possesses a good cell biocompatibility.</p><p>Cell imaging experiments were then carried out by incubating HeLa cells with 1 mM of TPA-BI for 15 min followed by examination under a uorescence microscope at an excitation wavelength of 400-440 nm. As shown in Fig. 4A, the lipophilic TPA-BI was prone to accumulating in the hydrophobic spherical LDs with bright greenish-blue emission due to the "like-like" interactions. Compared with BODIPY 493/503, a commercial probe for LD imaging, the images stained by TPA-BI showed a lower background signal, thanks to its AIE feature. Colocalization of TPA-BI and BODIPY 493/503 was performed and the same patterns were obtained solely by TPA-BI or BODIPY 493/ 503 with good overlap, demonstrating a good specicity of TPA-BI to LDs (Fig. 4C-E and S10 †).</p><p>Besides a high LD specicity, TPA-BI also showed an excellent resistance to photo-bleaching. More than 80% of its uorescence signal was retained even when it was continuously irradiated by laser light for 50 scans (Fig. S11 †). Such a high photostability is comparable to that of BODIPY 493/503. 25 TPA-BI can also be utilized in LD imaging in other cells lines, such as HepG-2 and A549, and in xed cells (Fig. S12 †). In addition, a negligible emission color change was observed with the increase of the dye concentration, oleic acid concentration or incubation time of oleic acid (Fig. S13 and S14F †). However, more and larger lipid droplets were observed aer increasing the concentration or incubation time of oleic acid, and the uorescence intensity of the whole cell was increased (Fig. S13 and S14A-E †). The statistical results were further conrmed by ow cytometry using BODIPY 493/503 and TPA-BI for staining (Fig. S15 †), suggesting that TPA-BI can be practically applied in the quantitative analysis of LDs by ow cytometry. All of these results demonstrate that TPA-BI indeed acts as a superior probe for LD imaging and analysis bearing wide applications in biomedical research and clinical diagnosis.</p><p>Why does TPA-BI exhibit greenish-blue emission in LDs? To understand this, we measured its uorescence using a confocal microscope in the mode of wavelength scanning. In Fig. 4F, the uorescence spectrum exhibits a peak at 495 nm. The peak value reects the value of E T (30) of the environment and suggests a low polarity inside the LDs. This is understandable as the LDs are surrounded by a phospholipid monolayer and consist of various neutral lipids such as triacylglycerol and cholesteryl ester. 1a To further verify our claim, we carried out the analogue experiment outside cells using the major components in LDs such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and trioleate glycerol (TAG). Without DMPC and TAG, the aggregates of TPA-BI in phosphate buffered saline (PBS) solution emitted orange coloured light at 570 nm, while the emission colour and intensity blue-shied and increased slightly upon the addition of DMPC only. Further addition of TAG resulted in an abrupt increase in the emission intensity and a peak maximum (Fig. 4G). This should be ascribed to the TICT effect of TPA-BI since TAG is more hydrophobic and less polar than DMPC. Under two-photon excitation, the uorescence intensity of TPA-BI increased more than 10-fold upon the addition of TAG, and the extent of this was higher than that achieved by one-photon excitation (Fig. S16 †). This suggests a larger signal to noise ratio for LD imaging by two-photon excitation to allow better contrast. Thus, two-photon excitation clearly outperforms in LD imaging.</p><!><p>As discussed above, TPA-BI shows a large d 2PA of up to 213 GM and its response to the TAG/LDs is largely enhanced under twophoton excitation. To evaluate whether TPA-BI is suitable for two-photon imaging of the LDs, we compared its performance with commercial BODIPY 493/593. As seen in Fig. 5, sufficient signals were obtained for both TPA-BI and BODIPY 493/503 under one-photon excitation. While clear images of the LDs stained by TPA-BI were still observed at an excitation wavelength of 840 nm, almost no signal was obtained for the LDs stained by BODIPY 493/503 under the same conditions. Similar results were obtained even when the excitation wavelength was changed to 900 nm and 980 nm (Fig. S17 †). Thus, TPA-BI is more suitable for two-photon imaging as it can be excited readily by laser light with a low power, thus avoiding photothermal damage to living cells caused by high laser power.</p><p>Several experiments were then conducted to demonstrate the superior advantages of two-photon microscopy (2PM) over onephoton microscopy (1PM) which are better 3D resolution, lesser photobleaching and autouorescence and deeper penetration depth. As shown in Fig. 6A, clustered LDs in HeLa cells were observed with a blurred background by 1PM. The blurred background is believed to be caused by the uorescence of the LDs below and above the focus plane. This problem was solved by 2PM due to the intrinsic sectioning property of 2PM. While a small layer of uorophores was excited at the focus plane in 2PM, all of the uorophores were excited in the light pathway in 1PM. Thus, fewer uorophores were photobleached in 2PM during prolonged observation. To prove this, an experiment was carried at a low concentration of TPA-BI (1 mM) to enable the occurrence of photobleaching. As shown in Fig. 6B, while almost 100% of the signal intensity was retained in 2PM, only half was retained in 1PM.</p><p>Autouorescence is a well-known difficult problem in tissue slices, which oen leads to a low image contrast and is even detrimental to dyes with low emission intensity. Intense auto-uorescence was observed in the xed liver tissue slice by 1PM, which was largely reduced by 2PM (Fig. 7A and B). Aer staining with TPA-BI, clear spherical spots with intense uorescence were observed with a much lower background than with 1PM (Fig. 7C and D). Due to the lesser absorption and scattering of the near-infrared light in the tissue, 26 the longer excitation light (840 nm) in 2PM is believed to have a deeper penetration depth than that of one-photon excitation (442 nm). The uorescent signal of the spherical spot could be detected at a z depth of 45 mm (Fig. 8). Compared to our previous LD-specic AIE bioprobes, 9,11 TPA-BI not only exhibits the merits of AIE probes in 1PM but also performs well in 2PM with a large d 2PA and NIR excitation, exhibiting higher 3D resolution, lower photobleaching rate, reduced auto-uorescence and low damage to living cells. This makes TPA-BI suitable for LD imaging both in cells and tissue slices with two-photon excitation, providing another tool for tissue slice-based disease diagnosis of lipid droplets.</p><!><p>In this work, an AIE probe (TPA-BI) for LD imaging was rationally designed and synthesized. Due to its D-p-A structure, TPA-BI exhibited solvatochromism with a high sensitivity to environmental polarity. TPA-BI exhibited both TICT and AIE features, showing a large Stokes shi of up to 202 nm and a large 2PA cross section of up to 213 GM. TPA-BI demonstrated good cell biocompatibility, high brightness, low background, high selectivity and excellent photostability. The lipid droplet imaging in TPA-BI was applicable for various live cell lines and xed cells. It also allowed LD analysis by ow cytometry. Compared to commercial BODIPY dyes, TPA-BI was more suitable for two-photon imaging of LDs with the merits of higher 3D resolution, lesser photobleaching and autouorescence and deeper penetration in tissue, providing a promising imaging tool for LD tracking and analysis in biomedical research and clinical diagnosis.</p><p>Due to its high sensitivity to polarity and good 2PA cross section, TPA-BI can be further utilized to detect the localized polarity of samples with two-photon excitation in a mixed bulk sample, such as for indicating the phase separation in polymer blends. Because of its synthetic accessibility, further modication of TPA-BI for imaging of other cell organelles or bio-sensing is under investigation in our laboratories.</p>

Royal Society of Chemistry (RSC)

Farnesyl Diphosphate Analogues with Aryl Moieties are Efficient Alternate Substrates for Protein Farnesyltransferase

Farnesylation is an important post-translational modification essential for proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of analogue transfer to peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple turnover activity depends on the analogue structure. Analogues where the first isoprene is replaced by a benzyl group and an analogue where each isoprene is replaced by an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single turnover rate constant for alkylation does depend on the Ca1a2X peptide sequence. These results suggest that the isoprenoid transition state conformation is preferred over the inactive E\xe2\x80\xa2FPP\xe2\x80\xa2 Ca1a2X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for design of novel inhibitors.

farnesyl_diphosphate_analogues_with_aryl_moieties_are_efficient_alternate_substrates_for_protein_far

<!>Materials and Methods<!>General procedure for Mitsunobu coupling (18a\xe2\x80\x93b, 22a\xe2\x80\x93c, 25a\xe2\x80\x93g, 29a\xe2\x80\x93b)<!>(E)-2-((3-methyl-4-(3-phenoxyphenoxy)but-2-en-1-yl)oxy)tetrahydro-2H-pyran 18a<!>(E)-2-((3-methyl-4-(4-phenoxyphenoxy)but-2-en-1-yl)oxy)tetrahydro-2H-pyran 18b<!>(E)-3-((2-methyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)benzaldehyde, 22a<!>(E)-4-((2-methyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)benzaldehyde 22b<!>(E)-4-ethoxy-3-((2-methyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)benzaldehyde 22c<!>(E)-2-((4-(4-((3-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-yl)oxy)tetrahydro-2H-pyran 25a<!>(E)-2-((3-methyl-4-(3-(phenoxymethyl)phenoxy)but-2-en-1-yl)oxy)tetrahydro-2H-pyran 25b<!>(E)-2-((4-(3-((4-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-yl)oxy)tetrahydro-2H-pyran 25c<!>(E)-2-((4-(3-((3-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-yl)oxy)tetrahydro-2H-pyran 25d<!>(E)-2-((4-(2-ethoxy-5-(phenoxymethyl)phenoxy)-3-methylbut-2-en-1-yl)oxy)tetrahydro-2H-pyran 25e<!>(E)-2-((4-(2-ethoxy-5-((2-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-yl)oxy)tetrahydro-2H-pyran 25f<!>(E)-2-((3-methyl-4-(3-((4-phenoxyphenoxy)methyl)phenoxy)but-2-en-1-yl)oxy)tetrahydro-2H-pyran 25g<!>(E)-3-((3,7-dimethylocta-2,6-dien-1-yl)oxy)benzaldehyde 29a<!>(E)-4-((3,7-dimethylocta-2,6-dien-1-yl)oxy)benzaldehyde 29b<!>General Procedure for THP ether removal<!>(E)-3-methyl-4-(3-phenoxyphenoxy)but-2-en-1-ol 19a<!>(E)-3-methyl-4-(4-phenoxyphenoxy)but-2-en-1-ol, 19b<!>(E)-4-(4-((3-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-ol 26a<!>(E)-3-methyl-4-(3-(phenoxymethyl)phenoxy)but-2-en-1-ol 26b<!>(E)-4-(3-((4-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-ol 26c<!>(E)-4-(3-((3-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-ol 26d<!>(E)-4-(2-ethoxy-5-(phenoxymethyl)phenoxy)-3-methylbut-2-en-1-ol 26e<!>(E)-4-(2-ethoxy-5-((2-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-ol 26f<!>(E)-3-methyl-4-(3-((4-phenoxyphenoxy)methyl)phenoxy)but-2-en-1-ol 26g<!>(3-((3-phenoxyphenoxy)methyl)phenyl)methanol 36<!>General procedure for aldehyde reduction<!>(E)-(3-((4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)phenyl)methanol 23a<!>(E)-(4-((2-methyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)phenyl)methanol 23b<!>(E)-(4-ethoxy-3-((2-methyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)phenyl)methanol 23c<!>(E)-(3-((3,7-dimethylocta-2,6-dien-1-yl)oxy)phenyl)methanol 30a<!>(E)-(4-((3,7-dimethylocta-2,6-dien-1-yl)oxy)phenyl)methanol 30b<!>General procedure for diphosphate synthesis<!>(2E,6E)-3,7-dimethyl-8-(2,3,5,6-tetrafluorophenoxy)octa-2,6-dien-1-diphosphate 3<!>(E)-3-methyl-4-(3-phenoxyphenoxy)but-2-en-1-diphosphate 4<!>(E)-3-methyl-4-(4-phenoxyphenoxy)but-2-en-1-diphosphate 5<!>(E)-4-(4-((3-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-diphosphate 6<!>(E)-3-methyl-4-(3-(phenoxymethyl)phenoxy)but-2-en-1-diphosphate 7<!>(E)-4-(3-((4-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-diphosphate 8<!>(E)-4-(3-((3-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-diphosphate 9<!>(E)-4-(2-ethoxy-5-(phenoxymethyl)phenoxy)-3-methylbut-2-en-1-diphosphate 10<!>(E)-4-(2-ethoxy-5-((2-fluorophenoxy)methyl)phenoxy)-3-methylbut-2-en-1-diphosphate 11<!>(E)-3-methyl-4-(3-((4-phenoxyphenoxy)methyl)phenoxy)but-2-en-1-diphosphate 12<!>(E)-(3-((3,7-dimethylocta-2,6-dien-1-yl)oxy)phenyl)methanyl-diphosphate 13<!>(E)-(4-((3,7-dimethylocta-2,6-dien-1-yl)oxy)phenyl)methanyl-diphosphate 14<!>(3-((3-phenoxyphenoxy)methyl)phenyl)methanyl-diphosphate 15<!>2-((3-((3-phenoxyphenoxy)methyl)benzyl)oxy) tetrahydro-2H-pyran 35<!>Preparation of MDCC-PBP<!>Preparation of WT FTase<!>Single Turnover Kinetics<!>Steady-state kinetics<!>Design And Synthesis Of Aryl Substituted Isoprenoid Diphosphate<!>FTase-Catalyzed Alkylation of GCVLS Depends On The Isoprenoid Donor Structure<!>Analogues Where The Alpha Isoprene Is Replaced By A Benzyl Group Are Transferable By FTase<!>The Single Turnover Rate Constant For GCVLS Alkylation Is The Same For Structurally Diver-gent Isoprenoid Diphosphates<!>The Rate Constant For Alkylation Depends On The Ca1a2X Peptide Sequence<!>Increased Isoprenoid Chemical Reactivity Does Not Alter The Rate Constant For Peptide Alkylation<!>Reaction Steps Involved In FTase Isoprenoid Diphosphate Selectivity<!>Increased Isoprenoid Bulk Does Not Alter The Rate Constant For GCVLS Alkylation<!>The Isoprenoid Structure Mainly Affects Product Release<!>Implications For Development Of Alternative FTase Substrates That Block Prenylated Protein Function (PFIs)

<p>Numerous membrane associated proteins require posttranslational farnesylation catalyzed by protein farnesyltransferase (FTase) for proper function. FTase catalyzes transfer of a 15-carbon farnesyl group from farnesyl diphosphate 1 (FPP) to a conserved cysteine in the C-terminal Ca1a2X motif of a range of proteins, including the oncoprotein H-Ras ("C" refers to the cysteine, "a" to any aliphatic amino acid, and "X" to any amino acid).(1–8) The covalently attached isoprenoid increases the protein's hydrophobicity and promotes membrane localization and enhances protein-protein interactions.(9–11) Clinical development of farnesyltransferase inhibitors (FTIs) as anticancer therapeutics has been hampered by alternative prenylation of some FTase substrates by the related prenyltransferase geranylgeranyl transferase type I (GGTase I) when FTase activity is limited.(12) This has spurred development of alternative FTase-transferable lipids incapable of supporting normal prenyl group functions (Prenyl Function Inhibitors, PFIs).(13–16) Defining the isoprenoid chemical features that affect each step of the transferase reaction mechanism may provide useful insights for developing PFIs.(17)</p><p>The FTase kinetic mechanism is complex (Figure 1) and the enzyme rarely exists in the free, unbound form during the catalytic cycle. The FTase kinetic mechanism is thought to be functionally ordered and efficient catalysis occurs when FPP first binds to the enzyme forming the enzyme•FPP complex (E•FPP) followed by Ca1a2X substrate association where the active site zinc ion directly coordinates the cysteine thiolate to form a ternary complex (E•FPP•Ca1a2X) that is inactive based on the crystal structure. (18–21)</p><p>Models based on structural, mutagenesis and computational studies have been proposed where a conformational rearrangement of the first two FPP isoprene units translocates the reactive isoprenoid C1 5.4 Å across the active site into reacting distance (2.4 Å) of the thiolate to form an active substrate conformation (E•FPP•Ca1a2X)*.(22–24) A variety of experiments suggest that farnesyl transfer to Ca1a2X thiol proceeds by a nucleophilic (SN2, associative) mechanism with electrophilic (SN1, dissociative) character, proceeding through a highly polar "exploded" transition state with considerable (C-1)-O bond cleavage and modest (C1)-S bond formation, partial positive charge on C-1, and partial negative charges on the zinc-coordinated sulfur and on the Mg2+ coordinated diphosphate leaving group (Figure 2).(25–27) Recent computational studies suggest that the transition state structure may depend on the structure of the peptide substrate.(28) The final step in the reaction cycle is product release which is stimulated by binding of either a Ca1a2X peptide (path B) or FPP (path A) to form either the E•peptide or E•FPP complex.(29) The product release pathway depends on the sequence of the peptide.(22, 23, 30)</p><p>Since FPP is capable of binding to the free enzyme, the E• Ca1a2X complex and the E•product complex, a better understanding of the binding interactions between the active site and FPP is needed and is of particular interest for designing PFIs. FPP analogues have been employed to study the FTase mechanism and the interactions between the isoprenoid, enzymes and the Ca1a2X peptide as well as the biological function of the post-translational modification. The reactivity of FPP analogues depends both on the isoprenoid structure and the peptide substrate sequence. (15, 31–33)</p><p>We report the synthesis and reactivity of FPP analogues with a range of steric demands and increased intrinsic chemical reactivity to investigate isoprenoid molecular features that contribute to substrate binding, recognition and catalysis by FTase (Figure 3, Table 1). The analogues vary in the size and electronic properties of one, two, or all three isoprene groups. We used analogues that stabilize the car-bocationic character at C1 to further examine the dissociative character of the transition state.</p><p>We have characterized FTase kinetic properties with these analogues and describe unexpected results regarding their effect on catalysis. We measured single turnover (STO) rate constants to examine the role of intrinsic chemical reactivity and steric bulk on the FPP conformational rearrangement and the chemical step (kobs, Figure 1). Surprisingly, the STO rate constant is the same as FPP for all analogues tested indicating that the FTase-catalyzed peptide alkylation is very tolerant of increased steric bulk in all three isoprene positions and that the rate of peptide alkylation is not limited by the chemical reactivity of the first isoprene in FPP. Within the series tested here, the hydrophobicity of the analogues does not limit the observed rate of thioether formation. However, the rate of peptide alkylation is dependent on the sequence of the C-terminal residue in the Ca1a2X motif. The steady-state data show that while some analogues are fairly good FTase substrates, others have kcat/KMisoprenoid values that are decreased up to 475–fold. These data indicate that a step subsequent to farnesylation, such as product dissociation, is sensitive to the structure of the farnesyl moiety.</p><!><p>The peptides TKCVIM, GCVLS and dansylated GCVLS were synthesized and purified by high-pressure liquid chromatography to more than 90% purity by Sigma-Genosys (The Woodlands, TX), and the molecular masses of peptides were confirmed by electrospray mass spectrometry. 7-Diethylamino-3-(((2-maleimidyl)ethyl)amino) carbonyl)coumarin (MDCC) was purchased from Molecular Probes (Eugene, OR). Farnesyl diphosphate (FPP), purine nucleoside phosphorylase (PNPase), 7-methylguanosine (MEG), and inorganic pyrophosphatase from bakers' yeast (PPiase) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals used were reagent grade.</p><p>All synthetic organic reactions except for resin preparation were performed in PTFE tubes using a Quest 210 apparatus manufactured by Argonaut Technologies. All RP-HPLC was performed on an Agilent 1100 HPLC system equipped with a microplate autosampler, diode array and fluorescence detector. N-dansyl-GCVLS was purchased from Peptidogenics (San Jose, CA, USA). Spectrofluorometric analyses were performed in 96-well flat bottom, non-binding surface, black polystyrene plates (Corning, Excitation wavelength, 340 nm; emission wavelength 505 nm with a 10 nm cutoff) with a SpectraMax GEMINI XPS fluorescence well-plate reader. Absorbance readings were determined using a Cary UV/Vis spectrophotometer. All assays were performed at minimum in triplicate where the average values are reported with a one standard of deviation error. Reaction temperature refers to the external bath. All solvents and reagents were purchased from VWR (EM Science-Omnisolv high purity) and Aldrich respectively and used as received. Merrifield-Cl resin was purchased from Argonaut technologies. Synthetic products were purified by silica gel flash chromatography (EtOAc/hexane) unless otherwise noted. RP-HPLC purification of lipid diphosphates was carried out using a Varian Dynamax, 10 μm, 300 Å, C-18 (10mm × 250 mm) column and eluted with a gradient mobile phase and flow rate of 4 mL/min: 0–3 min, 90% A; 3–18 min, 0% A; 18–20 min, 0% A; 20–23 min, 90% A; and monitored at 254 nm & 210 nm. 1H NMR and 13C NMR spectra of alcohols were obtained in CDCl3 and 1H and 31P of diphosphates in D2O with a Varian Inova spectrometer operating at 400 MHz (1H) and 161.8 MHz (31P). Chemical shifts are reported in ppm from CDCl3 internal peak at 7.27 ppm for 1H; D2O (TSP, 0 ppm for 1H; H3PO4 as an external reference, 0 ppm for 31P). ESI-MS were performed at the University of Kentucky Mass Spectra Facility. Positive and negative ion electrospray ionization (ESI) mass spectra were obtained on a Thermofinnigan LCQ with sample introduction by direct infusion.</p><!><p>DEAD (40% in toluene, 1.2 equiv.) was added drop wise to a stirred solution of alcohol (1equiv.), phenol (1.2 equiv.), Ph3P (1.2 equiv.) in THF at 0°C and stirred for 1 hr. After allowing the reaction to warm to room temperature and stir overnight, it was diluted with sat. NaHCO3, concentrated, and extracted with CH2Cl2 (2X). The organic extracts were dried (MgSO4), filtered and concentrated. Silica gel column chromatography of the oily residue gave the desired compounds.</p><!><p>1.0 gm of 16 gave 1.6 gm of 18a (84% yield). 1H NMR (CDCl3) δ 1.43–1.59 (m, 5H), 1.66–1.72 (m, 1H), 1.76 (s, 3H), 1.77–1.85 (m, 1H), 3.47–3.50 (m, 1H), 3.83–3.88 (m, 1H), 4.06–4.12 (m, 1H), 4.26–4.31 (m, 1H), 4.40 (s, 2H), 4.61 (t, J = 3.6 Hz, 1H), 4.75 (s, 2H), 5.74 (t, J = 6.4 Hz, 1H), 6.79–6.81 (m, 2H), 6.88–6.98 (m, 3H), 7.20–7.28 (m, 4H).</p><!><p>1.0 gm of 16 gave 1.72 gm of 18b (91% yield). 1H NMR (CDCl3) δ 1.48–1.60 (m, 4H), 1.67–1.74 (m, 1H), 1.76 (s, 3H), 1.79–1.84 (m, 1H), 3.46–3.51 (m, 1H), 3.83–3.88 (m, 1H), 4.07–4.12 (m, 1H), 4.20–4.32 (m, 1H), 4.38 (s, 2H), 4.60 (t, J = 3.2 Hz, 1H), 5.72–5.75 (m, 1H), 6.84–6.95(m, 6H), 6.99–7.03 (m, 1H).</p><!><p>500 mg of 16 gave 630 mg of 22a (81% yield). 1H NMR (CDCl3) δ 1.46–1.60 (m, 4H), 1.60 –1.72 (m, 1H), 1.76 (s, 3H), 1.77–1.81 (m, 1H), 3.45–3.51 (m, 1H), 3.82–3.87 (m, 1H), 4.06–4.11 (m, 1H), 4.26–4.31 (m, 1H), 4.45 (s, 2H), 4.60 (t, J = 6.8 Hz, 1H), 5.73–5.77 (m, 1H), 7.15–7.18 (m, 1H), 7.36–7.37 (m, 1H), 7.40–7.44 (m, 2H), 9.79 (s, 1H).</p><!><p>500 mg of 16 gave 619 mg of 22b (69% yield). 1H NMR (CDCl3) δ 1.42–1.55 (m, 7H), 1.57–1.70 (m, 1H), 1.75 (s, 3H), 1.77–1.84 (m, 1H), 3.44–3.49 (m, 1H), 3.80–3.86 (m, 1H), 4.08–4.18 (m, 3H), 4.24–4.29 (m, 1H), 4.54 (s, 2H), 4.57 (t, J = 7.6 Hz, 1H), 5.71–5.75 (m, 1H), 6.91–7.00 (m, 1H), 7.34–7.38 (m, 2H), 9.79 (s, 1H).</p><!><p>500 mg of 16 gave 690 mg of 22c (85% yield). 1H NMR (CDCl3) δ 1.46–1.60 (m, 4H), 1.61–1.72 (m, 1H), 1.76 (s, 3H), 1.77–1.84 (m, 1H), 3.45–3.51 (m, 1H), 3.81–3.87 (m, 1H), 4.05–4.10 (m, 1H), 4.27–4.31(m, 1H), 4.47 (s, 2H), 4.59 (t, J = 3.6 Hz, 1H), 5.72–5.76 (m, 1H), 6.98 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 9.84 (s, 1H).</p><!><p>100 mg of 23b gave 104 mg of 25a (82.5% yield). 1H NMR (CDCl3) δ 1.40–1.70 (m, 5H), 1.77–1.81 (m, 1H), 1.78 (s, 3H), 3.47–3.50 (m, 1H), 3.83–8.88 (m, 1H), 4.08–4.12 (m, 1H), 4.30 (m, 1H), 4.41 (s, 2H), 4.61 (t, J = 3.6 Hz, 2H), 4.92 (s, 2H), 5.73 (t, J = 6.0 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 6.95–7.10 (m, 5H), 7.15–7.20(2H).</p><!><p>100 mg of 23a gave 117 mg of 25b (87% yield). 1H NMR (CDCl3) δ 1.49–1.59 (m, 5H), 1.72 (s, 3H), 1.77–1.85 (m, 1H), 3.46–3.52 (m, 1H), 3.83–3.88 (m, 1H), 4.06–4.12 (m, 1H), 4.26–4.31 (m, 1H), 4.40 (s, 2H), 4.60 (t, J = 4.0 Hz, 1H), 5.00 (s, 2H), 5.72–5.75 (m, 1H), 6.79–6.85 (m, 1H), 6.90–6.98 (m, 4H), 7.20–7.28 (m, 4H).</p><!><p>100 mg of 23a gave 117 mg of 25c. (82% yield). 1H NMR (CDCl3) δ 1.48–1.69 (m, 5H), 1.77–1.81(m, 1H), 1.78 (s, 3H), 3.47–3.50 (m, 1H), 3.83–3.88 (m, 1H), 4.08–4.11 (m, 1H), 4.28 (dd, J = 6.4, 12.4 Hz, 1H), 4.41 (s, 2H), 4.60 (t, J = 3.6 Hz, 2H), 4.93 (s, 2H), 5.73 (t, J = 6.0 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 7.14–7.18 (m, 3H), 7.30 (d, J = 8.8 Hz, 2H).</p><!><p>100 mg of 23a gave 117 mg of 25d. (81% yield). 1H NMR (CDCl3) δ 1.48–1.69 (m, 5H), 1.77–1.81(m, 1H), 1.76 (s, 3H), 3.46–3.51 (m, 1H), 3.83–3.88 (m, 1H), 4.06–4.11 (m, 1H), 4.26–4.31 (m, 1H), 4.40 (s, 2H), 4.60 (t, J = 4.0 Hz, 2H), 4.97 (s, 2H), 5.72–5.75 (m, 1H), 6.72–6.75 (m, 1H), 6.83–6.97 (m, 6H), 7.24–7.27 (m, 1H).</p><!><p>100 mg of 23c gave 77 mg of 25e. (77% yield). 1H NMR (CDCl3) δ 1.41 (t, J = 7.2 Hz, 3H), 1.48–1.69 (m, 5H), 1.76 (s, 3H), 1.77–1.81(m, 1H), 3.46–3.51 (m, 1H), 3.82–3.88 (m, 1H), 4.06–4.11 (m, 1H), 4.26 (dd, J = 6.4, 12.4 Hz, 1H), 4.46 (s, 2H), 4.58 (t, J=2.8 Hz, 1H), 4.93 (s, 2H), 5.70–5.73 (m, 1H), 6.84–6.96 (m, 6H), 7.23–7.28 (m, 2H).</p><!><p>100 mg of 23c gave 120 mg of 25f (80% yield). 1H NMR (CDCl3) δ 1.42 (t, J=7.2 Hz, 3H), 1.48–1.69 (m, 5H), 1.76 (s, 3H), 1.77–1.81(m, 1H), 3.46 (dd, J = 4.4, 10.4 Hz, 1H), 3.82–3.87 (m, 1H), 4.05–4.12 (m, 3H), 4.25 (dd, J = 6, 12.4 Hz, 1H), 4.46 (s, 2H), 4.58 (t, J = 2.4 Hz, 1H), 5.02 (s, 2H), 5.71 (t, J = 7.6 Hz, 1H), 6.95–7.10 (m, 4H), 6.82–6.90 (m, 3H).</p><!><p>100 mg of 23c gave 123 mg of 25g (78% yield). 1H NMR (CDCl3) δ 1.45–1.60 (m, 6H), 1.65–1.75 (m, 2H), 1.78 (s, 3H), 1.79–1.82 (m, 1H), 3.44–3.50 (m, 1H), 3.82–3.90 (m, 1H), 4.06–4.12(m, 1H), 4.26–4.34 (m, 1H), 4.41 (s, 2H), 4.58–4.62 (m, 1H), 4.98 (s, 2H), 5.72–5.78 (m, 1H), 6.78–6.80 (m, 1H), 6.83–7.06 (m, 8H), 7.20–7.30 (m, 4H).</p><!><p>1 gm of geraniol gave 1.6 gm of 29a (95% yield). 1H NMR (CDCl3) δ 1.57 (s, 3H), 1.64 (s, 3H), 1.72 (s, 3H), 2.03–2.11(m, 4H), 4.56 (s, 2H), 4.58 (s, 2H), 5.04–5.08 (m, 1H), 5.44–5.48 (m, 1H), 7.15–7.18 (m, 1H), 7.37–7.42 (m, 3H), 9.94 (s, 1H).</p><!><p>1 gm of geraniol gave 1.6 gm of 29b (78% yield). 1H NMR (CDCl3) δ 1.57 (s, 3H), 1.64 (s, 3H), 1.72 (s, 3H), 2.02–2.14 (m, 4H), 4.60 (d, J = 6.8 Hz, 2H), 5.03–5.06 (m, 1H), 5.43–5.46 (m, 1H), 6.97 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 9.85 (s, 1H).</p><!><p>THP ether and 5% w/v PPTS were stirred in dry MeOH overnight. The reaction mixture was evaporated, taken up in ethyl acetate, washed with sat. NaHCO3, brine, dried (MgSO4), filtered and evaporated. Chromatographic purification of the residue gave alcohol in quantitative yield.</p><!><p>1H NMR (CDCl3) δ 1.75 (s, 3H), 4.25 (d, J = 6.8 Hz, 2H), 4.40 (s, 2H), 5.72–5.77 (m, 1H), 6.55–6.60 (m, 2H), 6.64 (ddd, J = 0.8, 2.4, 8.4 Hz, 1H), 7.00–7.03 (m, 2H), 7.08–7.12 (m, 1H), 7.18–7.22 (m, 1H), 7.30–7.35(m, 2H).</p><!><p>1H NMR (CDCl3) δ 1.75 (s, 3H), 4.22(d, J = 6.8 Hz, 2H), 4.36 (s, 2H), 5.72–5.77(m, 1H), 6.55–6.60 (m, 2H), 6.64 (ddd, J = 0.8, 2.4, 8.4 Hz, 1H), 6.85–6.90 (m, 4H), (7.00–7.03 (m, 1H), 7.08–7.12 (m, 1H), 7.30–7.35(m, 2H).</p><!><p>1H NMR (CDCl3) δ 1.78 (s, 3H), 4.20 (d, J=5.6 Hz, 2H), 4.42 (s, 2H), 5.05 (s, 2H), 5.78–5.80 (m, 1H), 6.81–6.90 (m, 1H), 6.90–7.10 (m, 5H), 7.28–7.35 (m, 3H).</p><!><p>1H NMR (CDCl3) δ 1.78 (s, 3H), 4.20 (d, J = 5.6 Hz, 2H), 4.42 (s, 2H), 5.05 (s, 2H), 5.78–5.80 (m, 1H), 6.81–6.90 (m, 1H), 6.90–7.10 (m, 5H), 7.28–7.35 (m, 3H).</p><!><p>1H NMR (CDCl3) δ 1.54 (s, 3H), 1.76 (brs, 1H), 4.22 (d, J = 5.6 Hz, 2H), 4.40 (s, 2H), 4.97 (s, 2H), 5.74–5.79 (m, 1H), 6.83–7.00 (m, 7H), 7.23–7.27 (m, 1H).</p><!><p>1H NMR (CDCl3) δ 1.42 (t, J = 7.2 Hz, 3H), 1.77 (s, 3H), 4.07 (q, J = 6.8, 14 Hz, 2H), 4.21 (t, J = 5.6Hz, 2H), 4.45 (s, 2H), 4.94 (s, 2H), 5.74–5.78 (m, 1H), 6.83–6.96 (m, 6H), 7.23–7.28 (m, 2H).</p><!><p>1H NMR (CDCl3) δ 1.41 (t, J = 7.2 Hz, 3H), 1.76 (s, 3H), 4.07(q, J = 6.8, 14 Hz, 2H), 4.20 (t, J = 6.0 Hz, 2H), 4.45 (s, 2H), 5.02 (s, 2H), 5.75–5.78 (m, 1H), 6.82–6.91 (m, 3H), 6.95–7.08 (m, 4H).</p><!><p>1H NMR (CDCl3) δ 1.40 (t, J =7.2 Hz, 3H), 1.80 (s, 3H), 4.0 (q, J = 6.8, 14 Hz, 2H), 4.10 (t, J = 6.0 Hz, 2H), 4.40 (s, 2H), 5.00 (s, 2H), 5.78 (t, J = 14 Hz, 1H), 6.80–7.05 (m, 7H).</p><!><p>1H NMR (CDCl3) δ 1.76 (s, 3H), 4.22 (t, J=5.6 Hz, 2H), 4.42 (s, 2H), 5.00 (s, 2H), 5.75–5.80 (m, 1H), 6.84–6.87 (m, 1H), 6.91–7.04 (m, 9H), 7.23–7.29 (m, 3H).</p><!><p>1H NMR (CDCl3) δ 4.70 (s, 2H), 5.02 (s, 2H), 6.90–6.96 (m, 5H), 7.00–7.04 (m, 1H), 7.23–7.25 (m, 2H), 7.36–7.42 (m, 4H).</p><!><p>NaBH4 (2equiv.) was added to aldehyde (1equiv.) in EtOH at 0°C and stirred for 3 – 6 h. The mixture was diluted with water and extracted with CH2Cl2 (2X). The organic extracts were dried (MgSO4), filtered and evaporated. The silica gel column chromatographic purification of the oily residue gave the alcohol in quantitative yield.</p><!><p>1H NMR (CDCl3) δ 1.48–1.57 (m, 5H), 1.62–1.72 (m, 2H), 1.76 (s, 3H), 1.79–1.84 (m, 1H), 3.46–3.51 (m, 1H), 3.82–3.87 (m, 1H), 4.06–4.11 (m, 1H), 4.26–4.31 (m, 1H), 4.41 (s, 2H), 4.59 (t, J = 3.6 Hz, 1H), 4.64 (d, J = 4.4 Hz, 2H), 5.72–5.75 (m, 1H), 6.80–6.92 (m, 1H), 6.90–6.92 (m, 2H), 7.21–7.25 (m, 1H).</p><!><p>1H NMR (CDCl3) δ 1.40–1.44 (m, 3H), 1.48–1.56 (m, 5H), 1.65–1.71 (m, 1H), 1.76 (s, 3H), 3.45–3.48 (m, 1H), 3.82–3.87 (m, 1H), 4.02–4.13 (m, 2H), 4.24–4.28 (m, 1H), 4.45 (s, 2H), 4.56–4.58 (m, 3H), 5.71 (t, J = 7.2 Hz, 1H), 6.79–6.90 (m, 4H).</p><!><p>1H NMR (CDCl3) δ 1.48–1.56 (m, 5H), 1.68–1.73 (m, 2H), 1.76 (s, 3H), 1.77–1.85 (m, 1H), 3.46–3.51 (m, 1H), 3.83–3.88 (m, 1H), 4.06–4.12 (m, 1H), 4.27–4.32 (m, 1H), 4.40 (s, 2H), 4.59–4.61 (m, 3H), 5.72–5.75 (m, 1H), 6.87 (d, J = 8.8 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H).</p><!><p>1H NMR (CDCl3) δ 1.58 (s, 3H), 1.65 (s, 3H), 1.71 (s, 3H), 2.05–2.09 (m, 4H), 4.52 (d, J = 6.4 Hz, 2H), 4.64 (s, 2H), 5.07 (t, J = 6.8 Hz, 1H), 5.46 (t, J = 8.0 Hz, 1H), 6.81–6.83 (m, 1H), 6.89–6.92 (m, 2H), 7.22–7.26 (m, 1H).</p><!><p>1H NMR (CDCl3) δ 1.58 (s, 3H), 1.65 (s, 3H), 1.71 (s, 3H), 2.04–2.11 (m, 4H), 4.51(d, J = 7.2 Hz, 2H), 4.59 (d, J = 6.0 Hz, 2H), 5.05–5.08 (m, 1H), 5.44–5.48 (m, 1H), 6.88 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H).</p><!><p>Ph3PCl2 (2 equiv.) in dry CH3CN was added dropwise to a cooled (0°C) solution of the alcohol (1 equiv.) in CH3CN (5 mL) and stirred for 2 h. The reaction mixture was concentrated and filtered through a pad of silica gel. The chlorides were sufficiently pure to proceed to the next step. ((n-Bu)4N)3HP2O7 (5 equiv.) in CH3CN was then added to the solution of chloride in CH3CN at 0°C and the solution was allowed to warm up to room temperature and stirred over-night. The reaction mixture was concentrated and washed with Et2O. The organic extracts were discarded and the residue suspended in 4 mL ion exchange buffer (25 mM NH4HCO3 in 2% (v/v) i-PrOH/water). The resultant white solution was loaded onto a preequlibrated 6×50 cm column of Dowex AG 50W-X8 (100–200 mesh) cation-exchange resin (NH4+ form). The flask was washed with buffer (2 × 2 mL) and loaded onto the column before eluting with 150 mL of ion exchange buffer. The eluent was lyophilized to yield a white solid. This solid was dissolved in 25 mM solution of NH4HCO3 buffer (4 mL), purified by RP-HPLC (retention time about 7 min) and lyophilized to give the diphosphate as a white powder.</p><!><p>(26% in two steps from the Mitsunobu reaction product).(14) 1H NMR (D2O) δ 1.50 (s, 3H), 1.60 (s, 3H), 1.83–1.87 (m, 2H), 1.98–2.04 (m, 2H), 4.27 (t, J = 6.4Hz, 2H), 4.49 (s, 2H), 5.23 (t, J = 7.6Hz, 1H), 5.36 (t, J = 6.8Hz, 1H), 6.87–6.95 (m, 1H). 31P (D2O) δ −5.90 (d, J = 22Hz, 1P), −9.49 (d, J = 22Hz, 1P). LRMS(EI) (M+-H+) 477 (M+) 478</p><!><p>100 mg of 19a gave 42.7 mg of 4, (23% yield). δ 1H NMR (D2O) δ 1.63 (s, 3H), 4.40 (s, 2H), 4.64 (s, 2H), 5.63–5.69 (m, 1H), 6.85–6.89 (m, 1H), 6.97–7.01 (m, 1H), 7.21–7.25 (m, 2H). 31P (D2O) δ −9.7 (d, J = 22 Hz, 1P), 8.76 (d, J = 22 Hz, 1P). LRMS(EI) (M+-H+) 429 (M+) 430.</p><!><p>100 mg of 19b gave 53 mg of 5 (28% yield). 1H NMR (D2O) δ 1.58 (s, 3H), 4.35 (bs, 2H), 4.62 (2H, s), 5.58 (t, J = 5.6Hz, 1H), 6.50–6.54 (m, 2H), 6.65 (ddd, J = 0.8, 2.4, 8.4Hz, 1H), 6.91–6.94 (m, 2H), 7.03–7.07 (m, 1H), 7.14–7.18 (m, 1H), 7.25–7.30 (m, 2H). 31P (D2O) δ −9.7 (d, J = 20.8 Hz, 1P), −8.51(d, J = 20.8 Hz, 1P). LRMS(EI) (M+-H+) 429 (M+) 430.</p><!><p>50 mg of 26a gave 20mg 6 (22% yield). 1H NMR (D2O) δ 1.59 (s, 3H), 4.35 (t, J = 7.2 Hz, 2H), 4.40 (s, 2H), 5.00 (s, 2H), 5.61 (t, J = 6.8 Hz, 1H), 6.83–7.00 (m, 6H), 7.17–7.21 (m, 3H). 31P (D2O) δ −9.35 (d, J = 22.0 Hz, 1P), −9.82 (d, J = 22.0 Hz, 1P). LRMS(EI) (M+-H+) 429 (M+) 430.</p><!><p>50 mg of 26b gave 24mg of 7 (28% yield). 1H NMR (D2O) δ 1.58 (s, 3H), 4.33 (t, J = 5.6Hz, 2H), 4.35 (s, 2H), 4.94 (s, 2H), 5.61 (t, J = 6.4 Hz, 1H), 6.80–6.92 (m, 2H), 7.15–7.19 (m, 2H). 31P (D2O) δ −5.8 (d, J = 20.0 Hz, 1P), −9.39 (d, J = 20.0 Hz, 1P). LRMS(EI) (M+-H+) 443 (M+) 444.</p><!><p>50 mg of 26c gave 21mg of 8 (25% yield). 1H NMR (D2O) δ 1.60 (s, 3H), 4.37 (t, J = 6.8 Hz, 1H), 4.40 (s, 2H), 5.0 (s, 2H), 5.63 (t, J = 7.2 Hz, 1H), 6.84 – 7.14 (m, 7H), 7.18–7.22 (m, 2H). 31P (D2O) δ −7.48 (d, J = 20.0 Hz, 1P), −9.55 (d, J = 20.0 Hz, 1P). LRMS(EI) (M+-H+) 461 (M+) 462.</p><!><p>50mg of 26d gave 22mg of 9 (28% yield). 1H NMR (D2O) δ 1.58 (s, 3H), 4.33–4.35 (m, 4H), 4.94 (s, 2H), 5.61 (t, J = 6.0Hz, 1H), 6.80–6.92 (m, 5H), 7.15–7.19 (m, 3H). 31P (D2O) δ −5.74 (d, J = 19.6 Hz, 1P), −9.40 (d, J = 19.6 Hz, 1P). LRMS(EI) (M+-H+) 461 (M+) 462.</p><!><p>50 mg of 26e gave 31mg of 10 (36% yield) 1H NMR (D2O) δ 1.15 (t, J = 7.2 Hz, 3H), 1.56 (s, 3H), 3.92 (q, 6.8, 14 Hz, 2H), 4.33 (t, J = 6.4 Hz, 2H), 4.37 (s, 2H), 4.86 (s, 2H), 5.56 (t, J = 6.0 Hz, 1H), 6.82–6.93 (m, 6H), 7.13–7.17 (m, 2H). 31P (D2O) δ −7.03 (d, J = 19.5 Hz, 1P), −9.39 (d, J = 19.5 Hz, 1P). LRMS(EI) (M+-H+) 487 (M+) 488.</p><!><p>50 mg of 26f gave 18mg of 11 (22% yield). 1H NMR (D2O) δ 1.16 (t, J = 7.2 Hz, 3H), 1.57 (s, 3H), 3.91 (q, J=6.8, 14.0 Hz, 3H), 4.33 (t, J = 6.4 Hz, 2H), 4.37 (s, 2H), 4.86 (s, 2H), 5.56 (t, J = 6.4 Hz, 1H), 6.82–6.85 (m, 5H), 6.93 (s, 1H), 7.13–7.17 (m, 2H). 31P (D2O) δ −7.03 (brs, 1P), −9.59 (brs, 1P). LRMS(EI) (M+-H+) 505 (M+) 506.</p><!><p>50mg of 26g gave 21mg of 12 (26% yield). 1H NMR (CDCl3) δ 1.20 (t, J = 7.6 Hz, 3), 1.60 (s, 3H), 3.93 (q, J = 7.2, 14 Hz, 2H), 4.37 (t, J = 6.4 Hz, 2H), 4.40 (s, 2H), 4.94 (s, 2H), 5.59 (t, J = 6.0 Hz, 1H), 6.80–6.84 (m, 3H), 6.92–7.10 (m, 4H). 31P (D2O) δ −9.59 (brs, 1P), −7.81 (brs, 1P). LRMS(EI) (M+-H+) 535 (M+) 536.</p><!><p>100 mg of 30a gave 34.5mg of 13 (19% yield). 1H NMR (D2O) δ 1.42 (s, 3H), 1.47(s, 3H), 1.93–1.99 (m, 4H), 4.47 (d, J = 6.8, 2H), 5.31 (t, J = 6.0 Hz, 1H), 4.97 (t, J = 6.4 Hz, 1H), 6.80 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H). 31P (D2O) δ −10.02 (d, J = 22.0 Hz, 1P), −8.00 (d, J = 22.0 Hz, 1P). LRMS(EI) (M+-H+) 419 (M+) 420.</p><!><p>100 mg of 30b gave 32.0 mg of 14 (18% yield). 1H NMR (D2O) δ 1.42 (s, 3H), 1.47 (s, 3H), 1.56 (s, 3H), 1.94–2.02 (m, 4H), 4.49 (d, J = 8.0 Hz, 2H), 4.80 (d, J = 6.0 Hz, 2H), 4.98 (t, J = 7.2Hz, 1H), 5.33 (t, J = 6.0 Hz, 1H), 6.76–6.78 (m, 1H), 6.94–6.96 (m, 2H), 7.16–7.20 (m, 1H). 31P (D2O) δ −9.78 (d, 22.0 Hz, 1P), −5.93 (d, J = 22.0 Hz, 1P). LRMS(EI) (M+-H+) 419 (M+) 420.</p><!><p>50mg of 36 gave 23mg of 15 (27% yield). 1H NMR (D2O) δ 1.73 (s, 3H), 4.97 (s, 2H), 5.82 (d, J = 6.4 Hz, 2H), 4.97 (s, 2H), 6.51–6.53 (m, 2H), 6.68 (dd, J = 2.0, 9.6 Hz, 2H), 6.87–6.89 (m, 2H), 7.05 (t, J = 7.6 Hz, 1H), 7.15–7.34 (m, 7H). 31P (D2O) δ −7.10 (d, J = 22 Hz, 1P), 9.87 (d, J = 22 Hz, 1P). LRMS(EI) (M+-H+) 465 (M+) 466.</p><!><p>To the cooled 0°C solution of m-hydroxyphenol 33 (500 mg, 2.68 mmol) in dry DMF was added 60% sodium hydride in mineral oil (129 mg, 3.2 mmol). The resultant solution was stirred at 0°C for 1hr and 2hr at room temperature. 2-((3-(chloromethyl)benzyl)oxy)tetrahydro-2H-pyran (645 mg, 2.68 mmol) was added dropwise and stirred over night. The reaction mixture was quenched with water, extracted with methylene chloride, and the organic phase washed with water (3X). The combined organic extracts were washed with brine, dried over MgSO4, concentrated and purified by silica gel column chromatography (hexanes, ethyl acetate) to obtain oily residue gave 35 (980 mg 93%). 1H NMR (CDCl3) δ 1.47–1.81 (m, 2H), 1.80–1.88 (m, 1H), 3.49–3.55 (m, 1H), 3.86–3.92 (m, 1H), 4.49 (d, J = 12.0 Hz, 2H), 4.68 (t, J = 3.6 Hz, 1H), 4.78 (d, J = 12.4 Hz, 2H), 5.00 (s, 2H), 5.57–6.62 (m, 2H), 6.70 (dd, J = 2.4, 8.4 Hz, 1H), 6.98–7.01(m, 2H), 7.06–7.10 (m, 1H), 7.17–7.28 (m, 1H), 7.29–7.39 (m, 6H).</p><!><p>The purification and labeling of the His6-tagged A197C phosphate binding protein (PBP) with the coumarin fluorophore MDCC was performed as described.(27, 34) The final MDCC-labeled PBP was dialyzed against 50 mM Hepes, pH 7.8, 2 mM TCEP, 0.5 units mL−1 purine nucleoside phosphorylase, and 15 mM 7-methylguanosine ("phosphate mop") to remove any residual phosphate by forming ribose-1-phosphate. The low molecular weight species of the "Pi mop" are removed by exchanging the buffer to 50 mM Hepes, pH 7.8, and 2 mM TCEP using Amicon Ultra centrifugal filter devices (10,000 MWCO). The purity of the labeled protein was confirmed by SDS-PAGE. Protein concentration and yield are determined by absorbance at 280 nm using a molecular weight of 35276 g mol−1 and a calculated extinction coefficient of 64204 M−1cm−1, and protein stocks were stored at −80 °C.</p><!><p>Recombinant rat protein FTase expression and purification were carried out in E. coli BL21(DE3) FPT/pET23a cells as described previously.(23, 35) The purified FTase was determined by SDS-PAGE to be >90% pure. The protein was dialyzed at 4 °C against 50 mM Hepes, pH 7.8, and 2 mM TCEP, and stored at −80 °C. The concentration of FTase was determined by active site titration as previously described. (23)</p><!><p>The single turnover rate constant was determined by measuring the release of diphosphate (PPi) using a fluorescently labeled phosphate binding protein (MDCC-PBP) coupled with PPi cleavage by inorganic pyrophosphatase (PPiase), as described in Pais et al. FTase was preincubated with FPP or analogue for >15 minutes at room temperature, and then rapidly mixed with a peptide solution containing MDCC-PBP and PPiase. The final concentrations used was 800 nM FTase, 200 nM FPP/analogue, 25 μM peptide, 5 μM MDCC-PBP, 34 units mL−1 PPiase, 50 mM Heppso, pH 7.8, 5 mM MgCl2 and 2 mM TCEP. Experiments were conducted at 25 °C using a KinTek model SF-2001 stopped-flow apparatus (KinTek Corporation, Austin, TX) to detect an increase in fluorescence upon binding of inorganic phosphate to MDCC-PBP (λex = 430 nm, λem = 450 nm Corion LL-450-F cutoff filter). The stopped-flow syringes and mixing chamber were preincubated prior to experiments in buffer containing a "Pi mop" (50 mM Heppso, pH 7.8, 5 mM MgCl2, 2 mM TCEP, 0.5 units mL−1 PNPase and 15 μM MEG). At least five kinetic traces were averaged and the single turnover rate constant (kobs) was determined by fitting Eq. 1 to the data, where Fl is the observed fluorescence at <450 nm at time t, ΔFl is the amplitude, and Flmax is the fluorescence endpoint. The single turnover rate constant (kobs) was derived from a reversible two-step kinetic mechanism proceeding from the E•FPP•CaaX complex shown in the dashed box in Figure 1 using equation 1.</p><!><p>The steady-state kinetic parameters kcat, KMisoprenoid, and kcat/KMisoprenoid, were determined from the dependence of the initial velocity on the concentration of FPP or analogue at saturating dansylated peptide (dns-GCVLS) concentration or kcat was determined from the dependence of the initial velocity on the concentration of dns-GCVLS at saturating isoprenoid concentration. The initial velocity was measured from the time-dependent increase in fluorescence intensity (λex = 340 nm, λem = 520 nm) upon farnesylation of dansylated GCVLS, as described previously. Reactions are initiated by the addition of FTase (25 nM final concentration) to solutions containing 5 μM dns-GCVLS, varying (1–20 μM) FPP/analogue, 50 mM Heppso, pH 7.8, 5 mM MgCl2, and 2 mM TCEP at 25 °C. For measurements at fixed isoprenoid concentration, reactions are initiated by the addition of FTase (25 nM final concentration) to solutions containing varying (1–10 μM) dns-GCVLS, varying 20 μM isoprenoid, 50 mM Heppso, pH 7.8, 5 mM MgCl2, and 2 mM TCEP at 25 °C The fluorescence intensity over time is measured for the first 10% of the reaction, using a Polarstar Galaxy fluorescence plate reader (BMG Laboratory Technologies, Durham, NC). The initial velocity of the reaction in fluorescence units s−1 (R) is converted to the velocity of the product formed in μM s−1 (V) using Eq. 3, where P is the concentration of the limiting substrate and Fmax is the amplitude in fluorescence measured from the endpoint of each experiment.</p><p>The values of the steady-state kinetic parameters kcat, KMisoprenoid, and kcat/KMisoprenoid are calculated from a fit of the Michaelis-Menten equation to the initial V versus [S] data.</p><!><p>FPP analogues 2–15 (Figure 3) were designed to probe aspects of isoprenoid C1 reactivity, the isoprene conformational rearrangement and the steric constraints of the FTase mechanism. We had previously established that an aniline or phenoxy group is an isostere for the terminal isoprene of FPP and that a range of substituent groups are tolerated by FTase to give transferable analogues under steady state conditions. Analogues 2–15 were designed to test the extent that FPP could be altered and still allow transfer catalyzed by FTase. The chemical reactivity of C1 was increased by replacing the first isoprene with a benzyl moiety in analogues 13, 14 and 15 (Table 1, Schemes 1 & 2). Solvolysis studies suggest benzyl isoprenoids 13, 14 and 15 are expected to be respectively, 5 and 20 times more reactive than the corresponding allylic isoprenoids in SN1 and SN2 type reactions.(36, 37) The terminal isoprene was replaced by an aromatic group in analogues 2–12 and 15 but retained in analogues 13 and 14. The second isoprene in analogues 4–12 and 15 was replaced by a series of substituted phenyl groups. In analogues 4–12, the first isoprene unit was retained and the steric demands and number of rotatable bonds relative to FPP were varied. Analogue 12 is longer than the other FPP analogues, and was designed to mimic the 20-carbon geranylgeranyl diphosphate (GGPP). GGPP is an alternative FTase substrate for some peptide sequences, but is a nano-molar inhibitor of the enzyme with others. (38–40)</p><p>Analogues 2 and 3 were prepared as previously reported. (14, 41)Analogues 4 and 5 were prepared by Mitsunobu coupling of either m- or p-phenoxyphenol with alcohol 16 to give THP protected isoprenols 18a–b (Scheme 1). Removal of the THP ether with PPTS in methanol afforded alcohols 19a–b which were converted to the corresponding chlorides 20a–b using Ph3PCl2 in acetonitrile.(14) The allylic chlorides were diphosphorylated with (n-Bu4)3HP2O7 to give diphosphates 4 and 5 in moderate yield.</p><p>Preparation of FPP analogues 6–12 is shown in Scheme 2 and involved Mitsunobu coupling of alcohol 16 with the appropriate hydroxybenzaldehyde to give aldehydes 22a–c which were reduced with NaBH4 to the corresponding benzyl alcohols 23a–c. A second Mitsunobu reaction with the requisite substituted phenols then afforded THP ethers 25a–g. These protected isoprenoid analogues were converted to the desired diphosphates 6–12 as described above.</p><p>Synthesis of FPP analogues 13 and 14 where the first isoprene moiety was replaced by a benzyl group is shown in Scheme 3. Mitsunobu coupling of geraniol with either m- or p-hydroxybenzaldehyde followed by reduction of the aldehydes 29a–b with NaBH4 gave the corresponding alcohols 30a–b. The desired diphosphates 13 and 14 were obtained by conversion of these alcohols as described above.</p><p>Synthesis of analogue 15 where all of the isoprene units are replaced by aromatic moieties is shown in Scheme 4. Previously described chloride 32(42) was coupled with the sodium salt of m-phenoxyphenol followed by removal of the THP protecting group, chlorination and diphosphorylation as described above.</p><!><p>A fluorescent assay was used to determine the effect of the FPP analogue structure on multiple turnover kinetics catalyzed by FTase. The steady-state kinetic parameters kcat for 1–15 and KMisoprenoid of FPP and analogues 2, 3, 6, 7, 9–11, 13–15 with dansylated GCVLS peptide (dns-GCVLS) were measured (Table 1). KMisoprenoid was measured by varying the FPP or analogue concentration in the presence of saturating peptide, and kcat was measured by varying the isoprenoid concentration in the presence of saturating peptide, or by varying the peptide concentration in the presence of saturating analogue.(43) The dns-GCVLS peptide corresponds to the well-characterized Ca1a2X sequence of H-Ras.(21, 39, 44)</p><p>FTase catalyzed transfer of the analogues to dns-GCVLS with equal or lower efficiency than FPP, with the exception of the GGPP mimetic 12 for which no turnover was detected. For the FPP analogues where the third isoprene is replaced by substituted aniline groups, the steady state kinetic parameters depend in a complex manner on the size and ring position of the substituents, and the sequence of the Ca1a2X peptide. For example, analogue 2 is a poor donor for modification of the dansyl-GCVLS peptide but is a good substrate with the dansyl-GCVIM peptide. The steady-state rate constant kcat/KMisoprenoid is termed the "specificity constant" for reaction of a peptide with different isoprenoids and can be used to determine how efficiently the FPP analogue is transferred to the peptide.(15, 30, 31) Relative to FPP, the value of kcat/KMisoprenoid for the analogues decreases up to 475-fold. Analysis of the steady-state parameters reveals that the rate constant kcat decreases for the analogues 1.3- to 220-fold relative to FPP, while the value of KMisoprenoid varies from 0.2–9.2 μM, which is within a 7.5-fold difference from FPP (KMisoprenoid = 1.5 μM). Therefore, the decreases in kcat roughly follow the same trends observed for kcat/KMisoprenoid.</p><p>The efficiency of the multiple turnover reaction depends on analogue structure. Notably, a meta- or para- linkage geometry of the second isoprene substitution does not appear to alter the the value of kcat/KMisoprenoid for modification of peptides catalyzed by FTase. For example, compounds 9 (meta-) and 6 (para-) have very similar efficiencies. Furthermore, the reactivity of analogues with dns-GCVLS does not depend on having the same number of rotatable bonds as FPP. Analogue 15 has seven rotatable bonds compared with eight in FPP. Clearly, the rotatable bonds in these analogues can adopt conformations sufficient to achieve the reactive ternary complex and the transition state. In contrast, the FTase-catalyzed efficiency of alkylation of peptides with analogues 10 and 11 is severely compromised suggesting that increased steric bulk on the lipid reduces either the peptide affinity or the rate constant for product formation.</p><!><p>Benzyl analogues 13, 14 and 15 are surprisingly good substrates, with values of kcat/KMisoprenoid that are within a factor of three of FPP (Table 1). Remarkably, analogue 15 is almost as good a substrate as FPP for FTase despite having no isoprene units in the structure. Aryl analogue 13 shows that most of the reactivity of FPP is retained by substituting a benzyl group for the first isoprene. This is the first instance where FTase has been shown to catalyze transfer of an isoprenoid with a non-allylic diphosphate to Ca1a2X peptide.</p><!><p>We measured the STO rate constant (kobs) for FTase-catalyzed alkylation of GCVLS by FPP and analogues 2–15 using a fluorescence-based assay that detects the release of pyrophosphate following alkyl transfer under conditions of excess FTase and peptide and limiting isoprenoid diphosphate analogue.(45) The observed rate constant for alkylated peptide product formation (kobs), indicated in Figure 1, measures formation of the E•Product complex from the ternary E•FPP•Peptide complex and includes both the chemical farnesylation step (kchem) and the proposed FPP rotational rearrangement (kconf) to form the active substrate conformation (eq 3).(26, 27, 31) Surprisingly, the observed rate constant under STO conditions (kobs) for all of the analogues is very similar to that measured for FPP (Table 1). The discrepancy between the effects of these analogues on the single turnover and steady-state kinetics suggests that different rate-limiting steps are being measured by the two methods; in particular, product dissociation may contribute significantly to the measured values for the steady-state kinetics.</p><!><p>To delineate the dependence of the STO rate constants on the peptide structure, the reactivity of analogues 2–15 was also measured for TKCVIM and TKCVIF (Table 1). TKCVIM is the K-Ras Ca1a2X peptide and the STO rate constant for farnesylation is nearly twice as fast (FPP, kobs = 6.5 s−1) as the value for GCVLS (FPP, kobs = 3.4 s−1). These rate constants are in good agreement with those previously reported.(31, 46) TKCVIF is the Ca1a2X peptide from TC21 and is normally a substrate for both FTase and GGTase I. The STO rate constant for farnesylation of TKCVIF catalyzed by FTase is 25-fold slower (FPP, kobs = 0.27 s−1) than the value for TKCVIM. Once again, the STO rate constants for the structurally dissimilar analogues 2–15 were identical to FPP for each of the three peptides examined (Table 1). Notably, the GGPP mimetic 12 reacts as efficiently as FPP with all three peptides even though modification by this analogue under steady state turnover conditions is undetectable. These results indicate that the conformational rearrangement and the farnesylation step are insensitive to the structural differences between FPP and analogues 2–15 (Table 1). Rather, the STO rate constant for peptide alkylation depends on the structure of the peptide, particularly the C-terminal residue in the Ca1a2X motif. The uniformly high reactivity of FPP and analogues 2–15 suggests a facile conformational change in the analogue hydrocarbon chains to achieve the transition state, consistent with recent computational studies. (28) Furthermore, the conformational rearrangement of the isoprenoid diphosphate in the active site to achieve the reactive conformation is independent of the product release step.</p><!><p>The electronic structure of the first isoprene unit is thought to be critical for FTase catalyzed transfer to peptide. 3-vinyl-FPP is an efficient alternative substrate for FTase and is likely to have increased delocalization of any cationic character in the transition state.(47, 48) The structure of the first isoprenoid is also an important factor in efficient multiple turnovers as both the 2-Z isomer of FPP and 3-allyl-FPP are potent inhibitors of FTase although they are electronically almost identical to FPP. It is unknown whether these two molecules alkylate peptide substrates under STO conditions.(47, 48) The reactivity of C1 in benzyl analogues 13, 14 and 15 is increased relative to FPP. The solvolytic reactivity of p-geranyloxybenzyl diphosphate 13 is higher than the meta substituted 14 and 15 due to enhanced stabilization of the delocalized positive charge that develops on the aromatic ring in the transition state (Figure 2B).(37) The identical STO reactivity for analogues 13–15 indicates that enhanced stabilization of the carbocationic aspects of the transition state does not contribute to an increased observed rate constant for peptide alkylation catalyzed by FTase. These results contrast with the substantial decrease in reactivity that electron withdrawing fluorine substitution at the C3 methyl of FPP have on both steady state turnover and kchem.(26, 27)</p><p>Isoprenoid hydrophobicity is uncorrelated with efficiency in multi-turnover reactions,(14) although hydrophobicity of the peptide substrates affects reactivity. (31, 46) The lack of any isoprenoid structural effect for analogues 2–15 on the STO rates suggest that interactions between the isoprenoid and the amino acid residues lining the walls of the active site are not particularly sensitive to the polarity of the chain.</p><!><p>For FTase, the steady-state parameter kcat/KMisoprenoid normally includes the rate constants for reaction steps for formation of the E•FPP•Ca1a2X complex, including peptide binding to E•FPP, and the first irreversible step of diphos-phate dissociation (Figure 1).(30, 31, 45) However, since FPP also enhances product dissociation, the measured value of kcat/KMisoprenoid likely includes these steps as well.(29) In contrast, peptide alkylation catalyzed by FTase under single-turnover conditions (with saturating enzyme) monitors only steps that occur after peptide binding through diphosphate release (kobs, dashed box, Figure 1).(45) Therefore, FTase-catalyzed single-turnover kinetics in the presence of limiting isoprenoid diphosphate isolates the alkyl transfer step (kchem) and presumed isoprenoid conformational change (kconf) from the dissociation of the alkylated peptide (koffCaaX, Figure 1) which is frequently the rate-limiting step for kcat under multi-ple turnover conditions.(18, 26, 49) The lack of any isoprenoid structural effect, yet strong Ca1a2X pep-tide sequence effect on kobs suggests that the chemical step and/or the proposed conformational rear-rangement of the FPP depends on how the Ca1a2X peptide binds to FTase. The clear differences in kobs for the three peptides suggests that subtle differences in the Ca1a2X-FTase binding interactions alter the rate of the proposed isoprenoid conformational rearrangement or alter the chemical reactivity of the thiolate nucleophile.</p><!><p>Previous measurements demonstrated that the 2° KIE for reaction of FPP with peptides under STO conditions catalyzed by FTase varies with the structure of the peptide; the α-2° 3H KIE observed for TKCVIF is large (1.13 ± 0.01) but decreases to near unity for GCVLS.(27) The original interpretation of this result was that the conformational change became the rate-limiting step for farnesylation of GCVLS under STO conditions. However, recent computational studies from the Merz group suggest that the ΔG‡ for the conformational step is much smaller than that of the alkylation step, even for GCVLS, and that the change in the 2° KIE reflects alterations in the transition state structure due to changes in the peptide binding mode. The similar STO kobs for all of the analogues indicates that substantial conformational freedom to achieve the transition state is available to the lipid in the confines of the active site and that any barriers to achieving the transition state are small. These observations indicate that the decreased steady-state turnover rate for the analogues most likely reflects alterations in the product dissociation step.</p><!><p>The decoupling of isoprenoid structure and intrinsic chemical reactivity from the STO rate constant kobs while retaining a profound isoprenoid structural dependence on the rate of product release, kcat, indicates that the alkylation reaction depends mainly on the structure of the peptide(46) while product release depends on the structure of both the peptide and the isoprenoid.(30, 31) Equation 2 demonstrates the contribution of the conformational rearrangement kconf and the chemical step kchem to the STO rate constant kobs. Inspection of equation 2 suggests that either kconf and k-conf are unchanged by the range of structural modifications explored here, or that kchem is sufficiently large to be the rate-determining step for all of the substrates. This latter conclusion is consistent with computational studies suggesting that the barrier to achieving the transition state going from the E•FPP•Ca1a2X complex to the E•FPP•Ca1a2X* complex is on the order of 1 kcal/mol for CVIM to 2.5 kcal/mol for CVLM compared to ~20 kcal/mol for the chemical step. (24, 28, 50)</p><p>Mg2+ binding accelerates FTase-catalyzed alkylation of GCVLS and TKCVIF by FPP by 700-fold and 100-fold, respectively. The Mg2+ cofactor may stabilize both the active substrate conformation and the developing charge on the pyrophosphate leaving group.(51–53) The FPP pyrophosphate leaving group in the inactive (E•FPP•Ca1a2X) complex is bound in the FTase PPi binding pocket comprised of positively charged residues.(19, 22, 54–57) Conformational rearrangement of the FPP isoprenoid chain moves the pyrophosphate leaving group out of the PPi binding pocket and creates a Mg2+ binding site that stabilizes the active complex (E•FPP•Ca1a2X*) which is then followed by attack of the zinc bound thiolate nucleophile on C1. For each CaaX peptide, the observed rate constant, kobs, for analogues 2–15 were identical to the natural substrate FPP. The insensitivity of kobs to the increased chemical reactivity of analogues 13–15 indicates that the rate of the chemical step kchem for each of the three peptides was not limited by the intrinsic reactivity of the allylic C1 electrophile. Furthermore, the insensitivity of kobs to the structure of the analogues for all three peptides suggests that there is abundant room in the active site for the proposed conformational rearrangement of the isoprenoid to take place. Consistent with this model, the uniform observed values of kobs for FPP and analogues 2–15 could be accommodated by correlated changes in chemical reactivity that compensate for an isoprenoid structure-dependent decrease in the ratio of the equilibrium constant for the conformational change (kconf/k-conf). However, there is no increase in kobs relative to FPP for the more reactive benzyl analogues 13, 14 and 15 suggesting that kchem depends on the CaaX peptide sequence and does not vary with isoprenoid structure.</p><!><p>This study tested the sensitivity of the chemical step to changes in electronic structure of the lipid electrophile, and the sensitivity of the conformational rearrangement required to achieve the reactive conformation to changes in the lipid structure. The primary conclusions are that (1) increases in chemical reactivity of the lipid electrophile do not increase kobs and (2) increased steric bulk in the isoprene chain does not decrease kobs, but (3) kobs depends on the Ca1a2X X-residue and (4) in contrast, the steady state rate constants for turnover are highly dependent on lipid structure. The ability of FTase to transfer analogues with no isoprene units opens the door to a much wider range of alternative structures as potential PFIs. These results suggest that as long as the chemical reactivity of the analogue C1 is at least as reactive as the allylic group of FPP, efficient S-alkylation is possible and largely unaffected by the chemical structure and bulk of the isoprenoid chain. There must be limits to the size of transferrable lipid moieties, as the FTase pocket has finite dimensions. However, the limits were not reached by the analogues investigated in this study. In contrast to the structure-independent rate of cysteine alkylation, product release was highly dependent on the structure of the analogue. Previously, our lab and others have noted a Ca1a2X sequence dependent interplay in the turnover of FPP analogues.(30, 32) Despite investigation of almost 200 analogues with a wide range of chemical structures, no clear structure-activity relationship has emerged. Broadly, large analogues like 10–12 are inefficiently turned over. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for design of novel inhibitors. Alternatively, the conformation of the isoprenoid in the E•product complex may block efficient binding of the new analogue diphosphate in the exit groove, or is inhibited from rearranging to put the alkylated isoprenoid into the exit groove.</p>

PubMed Author Manuscript

Immunopharmacotherapeutic Manifolds and Modulation of Cocaine Overdose

Cocaine achieves its psychostimulant, reinforcing properties through selectively blocking dopamine transporters, and this neurobiological mechanism impedes the use of classical receptor-antagonist pharmacotherapies to outcompete cocaine at CNS sites. Passive immunization with monoclonal antibodies (mAb) specific for cocaine circumvents this problem as drug is sequestered in the periphery prior to entry into the brain. To optimize an immunopharmacotherapeutic strategy for reversing severe cocaine toxicity, the therapeutic properties of mAb GNC92H2 IgG were compared to those of its engineered formats in a mouse overdose model. Whereas the extended half-life of an IgG justifies its application to the prophylactic treatment of addiction, the rapid, thorough biodistribution of mAb-based fragments, including F(ab\')2, Fab and scFv, may correlate to accelerated scavenging of cocaine and reversal of toxicity. To test this hypothesis, mice were administered the anti-cocaine IgG (180 mg/kg, i.v.) or GNC92H2-based agent after receiving an LD50 cocaine dose (93 mg/kg, i.p.), and the timeline of overdose symptoms was recorded. All formats lowered the rate of lethality despite the >100-fold molar excess of drug to antibody binding capacity. However, only F(ab\')2-92H2 and Fab-92H2 significantly attenuated the progression of premorbid behaviors, and Fab-92H2 prevented seizure generation in a percentage of mice. The calculation of serum half-life of each format demonstrated that the pharmacokinetic profile of Fab-92H2 (elimination half-life, t1/2 \xe2\x88\xbc 100 minutes) best approximated that of cocaine. These results not only confirm the importance of highly specific and tight drug binding by the mAb, but also highlight the benefit of aligning the pharmacokinetic and pharmacodynamic properties of the immunopharmacotherapeutic with the targeted drug.

immunopharmacotherapeutic_manifolds_and_modulation_of_cocaine_overdose

1. Introduction1<!>2.1.1 Production and purification of anti-cocaine mAb GNC92H2 scFv<!>2.1.2 Production and purification of anti-cocaine mAb GNC92H2 Fab and F(ab\')2<!>2.1.3 Enzyme-Linked Immunosorbent Assay<!>2.2.1 Sample preparation<!>2.2.2 Half-life determination<!>2.3.1 Subjects<!>2.3.2 Catheter implantation<!>2.3.3 Cocaine treatment<!>2.3.4 Passive immunization<!>2.3.5 Experiment 1: Prophylactic immunopharmacotherapy to prevent of cocaine-related toxicity<!>2.3.6 Experiment 2: Immunopharmacotherapeutic antidote to cocaine overdose<!>2.3.7 Behavioral monitoring<!>2.4 Statistical Analysis<!>3.1 Plasma Pharmacokinetics of the Anti-cocaine mAb GNC92H2 IgG, F(ab\')2, Fab and scFv Formats<!>3.2 Effect of Pretreatment with Anti-cocaine mAb GNC92H2 IgG on the Outcome of Acute Cocaine Toxicity<!>3.3 Antidotal Immunization with mAb GNC92H2-based Agents<!>3.4 Effect of the Anti-cocaine mAb GNC92H2 IgG, F(ab\')2, Fab and scFv Formats on Cocaine-induced Premorbid Behaviors and Seizure Generation<!>4. Discussion<!>

<p>Cocaine attenuates the clearance of dopamine from synapses through binding the dopamine transporter (Ritz et al., 1987). The net increase in dopaminergic transmission conveys the elevated pleasure or reward response, which explains the risk of long-term addiction as well as acute overdose (Withers et al., 1995). Despite a thorough understanding of the neurochemical basis for cocaine's physiological effects, an effective pharmacotherapy to treat cocaine addiction or overdose has yet to successfully advance through clinical trials (Karila et al., 2008). Currently prescribed therapeutics, which include small molecule modulators of dopaminergic signaling, non/specific monoamine reuptake inhibitors, N-methyl D-aspartate receptor antagonists/partial antagonists/blockers, σ receptor antagonists, ATP-sensitive potassium (KATP) channel openers, and specific classes of GABA receptor modulators, target the same neurocircuitry affected by cocaine rather than the drug molecule itself (Maurice et al., 2002; Reyes et al., 2007; Reyes et al., 2005; Sharkey et al., 1988). As a result, these pharmacotherapies come with an array of side-effects and oftentimes are unable to counteract all symptoms of cocaine use. By contrast, anti-cocaine active and passive vaccines suppress or eliminate the drug high without engaging the 'middleman', or the neurocircuitry activated during the drug high (Carrera et al., 1995; Carrera et al., 2001; Carrera et al., 2000; Carrera et al., 2005; Fox et al., 1996; Kantak et al., 2000; Mets et al., 1998; Norman et al., 2009; Norman et al., 2007; Redwan et al., 2003).</p><p>At physiological pH, the lipid solubility of cocaine ensures its prompt tissue penetration and the rapid development of its neuropharmacological effects; in humans, the initial, euphoric rush of a cocaine high stems from the rate at which blood cocaine levels spike and drive cocaine entry into the brain rather than the sheer magnitude of the brain cocaine content (Hearn, 1997). In mice, the widespread biodistribution of cocaine is achieved within minutes upon i.v. administration (distribution half-life, t1/2α = 1.9 min; elimination half-life, t1/2β = 26.1 min; volume of distribution at steady state, Vdss = 6.0 L/kg) (Benuck et al., 1987; Norman et al., 2007). Intraperitoneal injection grants 71% drug bioavailability, with the peak brain concentration attained within 5-15 minutes, and at an LD50 dose, overdose symptoms such as seizures appear within 3.4 min (Azar et al., 1998; Benuck et al., 1987; Carrera et al., 2005). It follows that an immunotherapeutic agent designed for overdose rescue must both function with comparable pharmacokinetics to the targeted drug (i.e. cocaine) and possess high drug affinity in order to interrupt cocaine influx into the CNS. With respect to the latter, mAb GNC92H2 (Kd = 2 nM; by KinExA (Ohmura et al., 2001) and mAb 2E2 (Kd = 4 nM (Paula et al., 2004)) already possess favorable binding kinetics, and thus further refinement of the mAb binding site is not anticipated to substantially improve the therapeutic utility of GNC92H2 for the indication of overdose reversal. With respect to the former, a few studies have explored the therapeutic benefit of short-acting monovalent antibody fragments over the whole IgG for rapid small-molecule detoxification (e.g. exogenous toxins, pharmaceutical and abused drugs) (Covell et al., 1986; Eddleston and Persson, 2003; Holliger and Hudson, 2005; Ochs and Smith, 1977). Of particular relevance, a comparison between digoxin-specific IgG and Fab fragments in the treatment of acute digoxin toxicity depicted the more rapid and less variable reversal of digoxin-induced cardiac arrhythmias and lethality by Fab infusion than by the equivalent dose of IgG (Lloyd and Smith, 1978). While Fab treatment promoted rapid urinary excretion of digoxin after an initial 10-fold spike in serum digoxin levels, IgG administration mediated a protracted increase in serum digoxin levels (∼50-fold) that appeared to slow digoxin metabolism and urinary exretion (Butler et al., 1977). This trend was observed for mAb-based treatment of acute toxicity from digitoxin, a structurally related cardiac glycoside that, unlike digoxin, displays high serum albumin binding and slower elimination via the liver (Ochs and Smith, 1977). From these studies, it was postulated that IgG-based therapies caused the increased sequestration of these small-molecule toxins in tissue stores, and that this event might subsequently lead to the gradual and potentially hazardous release of toxin back into circulation. In contrast, the rapid urinary excretion of protein-bound digitoxin under Fab treatment appeared to counteract the slow redistribution of toxin between brain, blood flow and tissue stores.</p><p>The aforementioned studies endorsed the use of Fab over IgG for immediate detoxification involving small-molecule toxins and, by extension, abused drugs. As a basic pharmacological principal, the free drug concentration in serum drives its biodistribution through different compartments (e.g. brain, blood, tissue). It has been established that the administration of Fab or IgG at an approximate mole-equivalent dose to the steady state drug concentration (Css) triggers serum drug levels to spike 10- to 100-fold above control levels, respectively (Butler et al., 1977; Valentine et al., 1994). In addition, while the serum drug concentration is equally partitioned between protein-bound and unbound states before mAb administration, both IgG and Fab mediate a dramatic shift in this equilibrium to favor nearly 100% drug-protein binding in serum. The disappearance of free drug in circulation upon immunopharmacotherapeutic treatment encourages the exodus of drug from brain and tissue stores. Interestingly, despite the significant drop in brain drug levels, the concentration of free drug in serum does not necessarily increase proportionately. In the case of PCP exposure, it was hypothesized that PCP metabolism may shift from nonrestrictive to restrictive-type metabolism after immunopharmacotherapeutic treatment so that free drug re-entering circulation is immediately cleared. The uncertain and pharmacologically complex effects of immunopharmacotherapy on drug disposition has not hindered the development and study of mAb-based medications for a varied array of exogenous small-molecules, including cardiac glycosides (e.g., digoxin (Butler et al., 1977), digitoxin (Ochs and Smith, 1977)), pharmaceutical drugs (e.g., desipramine (Lin et al., 1996; Pentel and Keyler, 1995), colchicines (Sabouraud et al., 1991; Urtizberea et al., 1991)), toxins (e.g., snake venom (Chippaux and Goyffon, 1998; Gutiérrez et al., 2003; Lomonte et al., 1996), polychlorobiphenyls (Keyler et al., 1994)), and a few abused drugs (e.g., most notably PCP-specific Fab (Owens and Mayersohn, 1986) and IgG (Valentine and Owens, 1996), but also nicotine-specific IgG (Keyler et al., 2005) and methamphetamine-specific IgG (Byrnes-Blake et al., 2003; McMillan et al., 2002) and scFv (Peterson et al., 2008)). Thus, the potential for smaller GNC92H2 formats, specifically F(ab')2-92H2, Fab-92H2 and scFv-92H2, to rapidly sequester cocaine and reverse acute toxicity seemed worthy of investigation.</p><p>It was previously conjectured that antibody-mediated small-molecule (i.e., drug) detoxification required an equivalent molar ratio between mAb binding capacity and the drug dose (Scherrmann et al., 1989). This guideline has been refuted for PCP and cocaine as the antagonism of drug effects by submolar mAb doses was preserved (Pitas et al., 2006). Nevertheless, the smaller size of Fab and scFv fragments lowers the protein load and infusion volume of a given molar mAb dose relative to the IgG (see Table 3, GNC92H2 format doses), an important consideration given the cardiovascular stress during cocaine overdose (Katz et al., 2007; Maraj et al., 2010). IgG-based therapies permit the recruitment of effector functions and long-term protection, but these traits are superfluous to this abuse scenario as well as directly responsible for the potential immunogenicity and limited biodistribution of the large Fc-containing IgG (Bird et al., 1988; Covell et al., 1986; Holton III et al., 1987). Previous research on desipramine toxicity, which involves a high molar dose of the toxin, revealed that mAb-mediated increases in desipramine-serum concentration were inversely correlated with the mAb distribution volume (i.e., svFv > Fab > F(ab')2 > IgG) (Keyler et al., 1995; Shelver et al., 1996). Similarly, immunopharmacotherapy with a PCP-binding Fab increased serum concentrations and renal elimination of PCP, thereby attenuating acute behavioral and physiological toxicity (Hardin et al., 1998; Valentine et al., 1996; Valentine and Owens, 1996). Herein, our study establishes mAb GNC92H2 fragments as more effective antidotes to severe cocaine toxicity relative to the IgG based on their superior capacity to redistribute cocaine from the brain to serum within the restricted timeframe of cocaine overdose.</p><!><p>The preparation of scFv by this laboratory has been described previously (McKenzie et al., 2007; Meijler et al., 2005). Here, Escherichia coli TG1 cells (Stratagene; Santa Clara, CA) were used for over-expression of soluble scFv-92H2 protein with a C-terminal Flag-tag. To summarize, scFv-92H2 gene fragments were digested with Sfi I (Roche; San Francisco, CA), ligated into the expression vector pET-Flag (derived from pET-15b, Novagen; Gibbstown, NJ), and transformed into E. coli TG1 cells by electroporation. SOC medium (2% peptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose) was added immediately after transformation. The cells were allowed to recover at 37 °C for 1 h, then plated onto Luria-Bertani (LB) agar plates containing carbenicillin (100 μg /mL) and incubated at 30 °C overnight. DNA sequencing was used to confirm the correct sequences. For overexpression, the purified pETFlag-scFv DNA from a single clone was again transformed into E. coli TG1 cells to prepare stock clones, and cells from a single clone were used to inoculate 6 L of SB (super broth: 3% peptone, 2% yeast extract, 1% MOPS) containing carbenicillin (100 g/mL). The cultures were incubated on a shaker (250 rpm) at 37 °C until an OD600 between 0.6 and 0.8 was reached. IPTG was added up to a final concentration of 1 mM, and the temperature was adjusted to 30 °C. The cultures were incubated overnight. The Flag-tagged scFvs were purified on anti-Flag M2 affinity agarose (Sigma-Aldrich; St. Louis, MO). After elution from the column (0.1 M glycine, pH 2.3) and neutralization with 1 M Tris (pH 9), the eluate was prepared for use in animal studies. Upon endotoxin removal (Endoclean™ #18603, BioVintage; San Diego, CA), scFv-92H2 protein solution was extensively dialyzed using Thermo Scientific Slide-A-Lyzer dialysis cassettes (MWCO 10-kDa, Pierce; Rockford, IL) into endotoxin-free 50 mM ammonium biocarbonate and lyophilized before storage. The production and purity of scFv-92H2 was verified by SDS-PAGE. Aliquots of the bacterial supernatant from the overexpression culture, FPLC-isolated anti-Flag M2 affinity column eluate, endotoxin-removed protein solution, dialyzed protein solution, and reconstituted protein for animal injection were collected. Both unreduced and reduced (addition of dithiothreitol, DTT) samples were denatured through boiling, and Nupage LDS (4X) sample buffer (Invitrogen; Carlsbad, CA) was added before sample analysis on a Nupage 4–12% Bis–Tris Gel (1.0×10 mm per well) with Benchmark prestained protein standard (Invitrogen). Bands were visualized by staining with Coomassie Blue. For animal studies, the protein was resuspended in an appropriate volume of sterile isotonic saline, and the final concentration measured by reading the absorbance at 280 nm. The cocaine binding-activity of scFv-92H2 was monitored after reconstitution via accessing GNC-BSA binding by enzyme-linked immunoabsorbant assay (ELISA).</p><!><p>GNC92H2 was previously identified as the mAb clone from GNC-KLH immunizations and hybridoma production with the most favorable overall properties of specificity and affinity for cocaine (isotype κγ2a, no cross reactivity with ecgonine or ecgonine methyl ester) (Carrera et al., 2000). The Fab fragments were isolated through papain (Sigma) digestion of the purified 92H2-IgG, followed by isolation of cleaved Fab-92H2 with Protein A chromatography (Thermo Fisher Scientific Inc.; Rockford, Il). Specifically, papain (10 μg per 1 mg IgG) was preactivated in Buffer A (100 mM sodium acetate, 1 mM EDTA, pH 5.5) supplemented with 1 mM cysteine and then added to the prepared IgG-92H2 solution (5 mg/ml dialyzed into Buffer A, prewarmed in 37 °C water bath). The optimal digestion time was determined through SDS-PAGE analysis of 20 μl aliquots, and the reaction was terminated through the addition of iodoacetamide (final concentration, 75 mM) and a 30-min incubation at room temperature. The digested sample was dialyzed into 1 M PBS, pH 7.4 prior to loading onto a Protein A column for removal of the uncut IgG, Fc, and Fab/c fragments. Additional purification steps included Protein G chromatography (Thermo Fisher Scientific Inc.; Rockford, Il) to remove unwanted enzyme, dialysis of Fab-containing fractions into 20 mM PB pH 7.0, and cation exchange (Mono S; Pharmacia, Sweden) chromatography. The progress of digestion reactions and the effectiveness of purification steps were monitored through SDS-PAGE of unreduced and reduced (addition of DTT) samples. The bivalent F(ab')2 fragments were generated in a similar manner except with pepsin digestion of the purified IgG-92H2. To determine the optimal reaction conditions, pilot digestions were performed in 0.2 M acetate buffer, pH 4 and 4.5, and aliquots were removed at multiple time points for monitoring of digestion progress. The reaction was terminated through the addition of 2 M Tris base, followed by dialysis, Protein A chromatography, and ion-exchange chromatography. Once obtained, the isolated Fab-92H2 and F(ab')2-92H2 were concentrated on a microdialysis/concentration unit (Amicon Corp.; Danvers, MA) with final protein concentrations measured via spectrophotometry. Both formats underwent endotoxin removal purification steps (BioVintage; purity confirmed with Limulus amebocyte lysate (LAL) testing) before use in animal studies.</p><!><p>Cocaine-binding activity of scFv-92H2 as well as GNC92H2 IgG, F(ab')2, Fab, and scFv antibody concentration in mouse blood samples were evaluated by ELISA. The cocaine hapten, termed GNC, was coupled to bovine serum albumin (BSA), and the GNC–BSA conjugate was applied to an ELISA plate (CoStar; 96-well, half-volume) at a concentration of 10 μg/ml in PBS at 37 °C for 1 h with shaking, with unmodified BSA serving as a negative control. After washing with distilled water ten times, the wells were blocked for 1 h at 37 °C with 50 μL of Blotto (5% QuikBlot powder in PBS). Mouse serum (25 μL of an initial 1:500 dilution in Blotto) was added to the first row and serially diluted down the plate. A quantification standard of mAb GNC92H2 dilution stock (IgG-92H2, F(ab')2-92H2, Fab-92H2, or scFv-92H2) covering a range of concentrations (0.005 to 5.0 μg/mL per well) was also plated in a column alongside sera samples, and plates were incubated for 1.5 h at 37 °C. After washing, 25 μL of a 1:5,000 dilution of a goat-anti-mouse IgG (heavy and light chain) horseradish peroxidase conjugate (Thermo Fisher Scientific Inc.; Waltham, MA), goat-anti-mouse IgG (Fab-specific) horseradish peroxidase conjugate (Sigma), or anti-Flag M2 horseradish peroxidase conjugate (Sigma) in Blotto was added to wells for a 1-hour incubation at 37 °C. The plate was developed with the colorimetric reagent tetramethyl-benzidine substrate (TMB, 50 μl/well, Pierce), quenched with an equal volume of 2 M H2SO4, and the absorbance at 450 nm measured on a 96-well ELISA plate reader.</p><!><p>Mice (n = 5) were administered mAb GNC92H2 IgG (180 mg/kg), F(ab')2 (132 mg/kg), Fab (120 mg/kg), or scFv (60 mg/kg) via a bolus infusion (< 1 min) through a polyethylene tube attached to the catheter on the animals' back. To obtain blood samples for mAb quantification, the mouse tail tip was amputated with a sterile scalpel blade, permitting 10 μl of blood to be collected at several time points using heparinized capillary pipette tips. Collections were made prior to mAb infusion and at time (t) = 3 or 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 2 h, 3 h, 6 h, 24 h, 48 h, 72 h, 96 h, 7 d, and 10 d for all mAb formats. Additional weekly samples were collected for up to 4 weeks after mAb administration for mice infused with F(ab')2 and up to 8 weeks for mice infused with IgG. The blood was immediately placed in a 0.6-mL polypropylene microcentrifuge tube containing 40 μl of 0.1 M sodium citrate/0.1% sodium azide, pH 4.75. These samples were diluted 1:10 in sterile PBS in preparation for subsequent ELISA analysis of blood antibody concentration. Samples were stored at 4 °C for immediate analysis or -80 °C for long-term storage.</p><!><p>ELISAs were conducted using IgG-92H2, F(ab')2-92H2, Fab-92H2, and scFv-92H2 stock dilutions in order to generate a standard curve for each mAb format and thus determine its concentration in sera samples for all collection time points. To approximate the time course of mAb elimination in vivo, the pharmacokinetic data of each GNC92H2 format were analyzed according to single and two-compartment models using standard volume of steady-state distribution values, followed by the application of goodness-of-fit and regression analysis to antibody concentration versus time data. Whereas the two-compartment model offers a better approximation in theory to the antibody distribution data, the improvement over the single-compartment model was often minimal relative to its additional complexity. Thus, calculations of elimination half-life reflect the application of the single compartment model to the terminal phase of antibody distribution data.</p><!><p>Male CD-1 mice (30 ± 5 g, n ≥ 10 for overdose experiments) purchased from the Scripps Breeding Facility and Charles River Laboratories (Wilmington, MA) were used. Animals were housed four per cage prior to catheter implantation in a 12:12-h light–dark cycle (lights off at 09:00 h). Water and food pellets were available ad libitum in their living cages. All the experiments described in this study were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health, and were approved by the Animal Care and Use Committee at The Scripps Research Institute. Every effort was made to reduce the number of animals used.</p><!><p>One week after arrival, animals were labeled, weighed, and subjected to intravenous catheterization. Methods for the preparation and surgical implantation of jugular vein catheters in mice are described in detail elsewhere (Ritz and George, 1993). Briefly, mice were placed under general anesthesia with isoflurane vapor mixture and then implanted with a silastic indwelling catheter (silastic tubing, id 0.3 mm, od 0.64 mm; 12 mm in length) in the right external jugular. Animals were surgically prepared via shaving the areas for incision and applying a 70% alcohol and povidone/iodine solution, and then incisions were made in the mid-scapular region as well as anteromedial to the right forearm above the external right jugular vein. The jugular catheter was passed subcutaneously to a dental cement encased polyethylene assembly housing a guide cannula (Plastic Products, C313G) mounted on the back of the animals. The other end of the catheter was passed subcutaneously from the dorsal incision to the ventral incision, and the silastic tubing inserted into the jugular vein, then tied gently with suture thread to the vein. The incisions were sutured closed, and the catheter was capped with a tygon stopper. The catheters were kept patent with daily flushings of heparinized saline (20 U/flush), and animals were housed singly during their full recovery (about 1 week) and up through drug administration, immunization, and behavioral testing procedures.</p><!><p>Animals were administered a 50% lethal dose (LD50 = 93 mg/kg) of (-) cocaine hydrochloride (NIDA; Rockville, MD), which was dissolved in sterile 0.9% saline for i.p. injection at a volume of 10 ml/kg (Hearn et al., 1991). Upon receiving this LD50 cocaine injection, all immunized mice and cocaine alone controls were then transferred to individual locomotor activity cages for observation.</p><!><p>The mAb-based agent or vehicle (sterile 0.9% saline) was administered via a bolus infusion through a 10-in.-long polyethylene tube attached to the catheter. This permitted the experimenter to deliver the mAb infusion during active recording of subject in the locomotor activity chamber without disrupting its behavior or full ambulatory range. The 30-min mAb pretreatment time point allowed the subject to recover from any stress related to the administration of a large bolus mAb infusion prior to cocaine injection. The 3-min post-cocaine treatment time point was chosen on the basis of previous work where the average onset of seizures, one predictor of lethality, occurred 3.4 minutes after drug injection (Carrera et al., 2005). The mAb binding capacity for cocaine remained constant across the different GNC92H2 format doses for the 3 min post-cocaine treatment, and this molar mAb dose represents less than 1/100th the molar amount of cocaine administered (LD50 dose = 93 mg/kg). Antibody doses were allotted in a between-subjects design.</p><!><p>The purpose of this experiment was to provide a benchmark of mAb GNC92H2 efficacy in averting the onset of severe symptoms of cocaine toxicity and to which the therapeutic ability of antidotal passive immunization could be compared. The mAb GNC92H2 IgG format was tested at the lower dose level (IgG: 90 mg/kg) in our murine cocaine overdose model. Thirty minutes following i.v. infusion of IgG-92H2 or a vehicle saline solution, the LD50 cocaine dose was administered by i.p. injection, and test subjects were placed directly into locomotor activity cages for activity monitoring across the entire progression of cocaine-related symptoms. The smaller mAb fragments were not tested in this pretreatment model because their rapid elimination would directly counteract their application to prophylactic immunopharmacotherapy. Conversely, the 30-minute delay between immunization with IgG-92H2 and cocaine injection granted the thorough biodistribution of this mAb format prior to drug exposure, and thus this experiment was expected to reflect the maximum efficacy of an IgG-based therapeutic.</p><!><p>To evaluate the different capacity of each mAb GNC92H2 format to rescue mice from lethal overdose, subjects were immediately placed in individual locomotor activity cages after cocaine injection (i.p). Attached to the indwelling catheter, a 10 inch-long polyethylene tube permitted the passive vaccine to be administered (i.v.) 3 minutes after cocaine exposure without interfering with mouse behavioral monitoring. The behavioral response to cocaine toxicity was recorded in the same manner as experiment 1. Antibody formats were tested at the following doses: 180 mg/kg IgG-92H2, 132 mg/kg F(ab')2-92H2, 120 mg/kg Fab-92H2, 60 mg/kg scFv-92H2, all of which deliver 2.4 μmol/kg cocaine recognition sites. The constant molar dose of all GNC92H2 formats was adjusted based on the molecular weight of each format (e.g., IgG = 150 kDa, F(ab')2 = 110 kDa, Fab = 50 kDa, scFv = 25 kDa) and its antigen binding capacity (e.g., IgG, F(ab')2 are bivalent; Fab, scFv are monovalent).</p><!><p>Behavioral monitoring was conducted via the use of Plexiglas locomotor activity cages (42 × 22 × 20 cm). All mice underwent a brief cage habituation session 24 h prior to behavioral testing. On the test day, mice received an intraperitoneal injection (i.p.) of cocaine, immediately before their transfer to individual cages for 60-min observation sessions. Subjects' behavior was recorded by a "blind" observer for the time-of-onset, severity, and duration premorbid behaviors (e.g., hyperlocomotion, ataxia, Straub tail response, writhing, convulsions, or loss of righting posture for at least 5 s and/or clonic movements, agitation), seizures (e.g., tonic, clonic, wild-running clonis, tonic-clonic), and death. Specifically, each mouse received a binary score of present (1) or absent (0) for each symptom category (premorbid behavior, seizures, death) so as to permit the pooling of nominal data by group and its comparison to control group behavior using the χ2 test. From these data, the incidence of each overdose symptom within groups was computed as a percentage for ease of depiction in figures.</p><p>In addition, the severity of premorbid behaviors and of seizure activity was ranked within successive 3-min time intervals during the 60-min observation period. A scale developed by Sturgeon et al. was applied to the scoring of premorbid ataxia in mice (score 0: inactive; score 1 = unusual, awkward, and/or jerky movements with only occasional loss of balance; score 2 = awkward and jerky movements accompanied by a moderate rate of falling onto their sides but not onto their backs; score 3 = significant loss of balance and falling onto their sides and/or backs when attempting to move about the cage; score 4 = loss of righting ability, or severe impairment of the anti-gravitational reflex to the extent that mice are unable to move beyond a small area within the cage; score 5 = absence of overall movement except for twitching, head bobbing or writhing, and body rolling) (Sturgeon et al., 1979). The ataxia scores of mice in each treatment group were averaged (mean ± S.E.M.) within each time interval.</p><p>A similar ranking system was created for the assessment of seizure severity. Subjects were scored across successive 3-min intervals after cocaine injection based on the following scale: score 0 = absence of seizures; score 1 = short-lasting, mild seizures occasionally accompanied by loss of the righting reflex and with repeated bouts of convulsive activity absent or infrequent; score 2 = convulsive seizures accompanied by severe clonus (wild-running clonus) or rearing, and/or bouts of seizure trains that occur repeatedly or that cause fatal respiratory disorders given their violent nature. These ataxia and seizure ranking systems permitted the severity of overdose symptomology within all mAb treatment groups and the cocaine-alone control group to be compared by analysis of variance (ANOVA), repeated measures ANOVA, and Fisher's PLSD, or by the χ2 distribution for nominal data (e.g., the appearance of cocaine overdose symptomology).</p><!><p>To measure the effect of GNC92H2 format treatment on cocaine-induced toxicity, the appearance of each overdose symptomology (premorbid behaviors, seizures, death) was assigned a value of incidence (present = 1, absent = 0) for each mouse regardless of the time of onset, and group data was analyzed via the χ2 distribution (p < 0.05). The theoretical frequencies for premorbid behaviors, seizures, and death were based on the cocaine alone group data. Whereas the χ2 test of goodness-of-fit was employed to examine the therapeutic benefit of mAb treatment, the χ2 test of independence or the Fisher's exact test was then used to illustrate differences between immunopharmacotherapeutic treatments in their capacity to alter cocaine-induced symptomology. Lastly, the times of onset of behavioral observations were used to score ataxia and seizure severity within successive 3-min time intervals (repeated measure for ANOVA), and group mean scores within these intervals were analyzed for parametricity (normal distribution, homogeneity of variance). If these conditions were met, significant differences were determined by analysis of variance (ANOVA) and post-hoc Student's t tests and Fisher's PLSD. Otherwise, data were analyzed using the non-parametric Mann-Whitney U statistic or the Kruskal-Wallis test. In addition, the maximum ataxia score and maximum seizure score attained within the 60-min session was identified for each subject, and group means for these scores were compared via the statistical tests listed above. Serum antibody levels were also compared by ANOVA (data not shown), and mAb format half-life was evaluated using the model-independent method based on the statistical-moment theory.</p><!><p>To determine the pharmacokinetic profiles of mAb GNC92H2 IgG, F(ab')2, Fab, and scFv distribution in mice, tail vein blood samples were collected immediately before immunization and then as early as 3 min after the i.v. infusion of GNC92H2. From the calculations of mean serum-mAb content at every time point, it was observed that the concentrations of each immunopharmacotherapeutic increased slightly across the first blood collection time points, with IgG-92H2 reaching a peak volume of distribution of Vd ∼ 0.126 ± 0.013 L/kg. The IgG-92H2 and F(ab')2-92H2 levels in sera remained elevated for approximately 1 hour before their gradual decline. The Fab-92H2 and scFv-92H2 fragments were rapidly eliminated from circulation. From these serum levels, the half-life of each format was estimated (Table 1). Gratifyingly, the in vivo pharmacokinetic profile of all GNC92H2 formats approximated the biodistribution data of other murine antibodies reported in the literature (Covell et al., 1986; Holliger and Hudson, 2005).</p><!><p>Mice were immunized with IgG-92H2 (90 mg/kg) 30 minutes prior to cocaine exposure in order to confirm our previous results on IgG-92H2-mediated prevention of lethality and to conduct detailed recordings on overdose symptomology. The analysis of behavioral data of immunized and cocaine alone groups revealed that this IgG-92H2 dose had the capacity to attenuate severe cocaine-induced toxicity; lethality was averted in a significant percentage of immunized mice (χ2 = 8.14, p < 0.005). All subjects demonstrated hyperactivity, increased exploratory behavior, and the initial progression into ataxic behaviors that directly follow cocaine injection. The time-of-onset, duration, and severity of seizure generation showed greater inter-subject variation, and thus any treatment-dependent variation between groups lacked statistical significance. Approximately half of the cocaine alone mice succumbed to cocaine toxicity, as expected for an LD50 drug dose. Whereas a higher percentage of immunized mice survived the LD50 cocaine injection, the duration of premorbid behaviors was protracted in the immunized group. This was evidenced by the statistically significant difference in ataxic behavior in cocaine alone versus immunized mice in observation intervals extending past the 36-min time point (Fig. 1, Student's t test: p < 0.05, 36-45 min post-cocaine).</p><!><p>The mAb pretreatment experiment established the capacity of IgG-92H2 to bind cocaine in vivo and prevent cocaine-induced lethality. Of greater clinical interest was the potential for administration of an anti-cocaine immunopharmacotherapeutic to reverse a potentially lethal overdose after the appearance of the outward signs of acute toxicity. The overall therapeutic effect of GNC92H2-based treatment was examined in the mouse overdose model through computing the percentage of mice within each group that developed symptoms of cocaine toxicity (Fig. 2). The appearance and severity of ataxia, seizures, and lethality as an endpoint, were recorded across 3-min observation intervals for statistical analysis (Table 2). Gratifyingly, the efficacy of IgG-92H2 as a therapeutic for cocaine overdose extends beyond its prophylactic benefit. Antidotal delivery of IgG-92H2 (180 mg/kg) conferred a 50% reduction in the incidence of lethality relative to the cocaine alone group. However, neither the appearance of premorbid behaviors nor seizure generation was significantly altered by the IgG format of this immunopharmacotherapy (Fig. 2).</p><p>F(ab')2-92H2, Fab-92H2, and scFv-92H2 were administered at doses equivalent to the full IgG in binding capacity for cocaine (Table 3). It was hypothesized that both the more rapid biodistribution and lower protein dose of mAb fragments would enhance their therapeutic ability relative to the full immunoglobulin in the scenario of overdose rescue. Relative to the 54% lethality mediated by cocaine alone and the 28% lethality within the IgG-92H2 group, the LD50 dose of cocaine was 20%, 9%, and 36% lethal in the F(ab')2-92H2, Fab-92H2 and scFv-92H2 treatment groups, respectively. Both the F(ab')2-92H2 and Fab-92H2 offered significant protection against death from cocaine overdose, as assessed by the χ2 test of goodness-of-fit (Table 2). The different mAb GNC92H2-based treatments were indistinguishable by the χ2 test of independence (IgG, F(ab')2, Fab, and scFv: χ2 = 2.49, p > 0.40), however the pair-wise comparison between the rate of lethality in the cocaine alone group with each mAb-treated group depicted the superior ability of Fab-92H2 to mediate overdose rescue (IgG-92H2: χ2 = 3.92, p < 0.05; F(ab')2-92H2: χ2 = 4.06, p < 0.05; Fab-92H2: χ2 = 7.63, p < 0.01)</p><p>As with the immunopharmacotherapeutic protection against lethality, specific mAb formats showed different capacities to attenuate the progression of overdose symptomology. IgG-92H2 treatment did not significantly alter the incidence of premorbid behaviors in comparison to cocaine alone subjects, while all mice within F(ab')2-92H2 and scFv-92H2 groups displayed premorbid behaviors. Of all GNC92H2-based treatments, Fab-92H2 served as the best antidote. The initial hyperactivity and slight ataxia failed to transition to the severe overdose symptomology in a significant number of mice within this group relative to the cocaine alone group as indicated by the repeated measures ANOVA (time interval) of ataxia severity [F(1, 34) = 4.783, p < 0.05].</p><!><p>To compare the utility of each GNC92H2 format in alleviating the outward signs of cocaine toxicity, both the severity of ataxic symptoms and of seizures were scored (Table 3). The general time course of ataxia includes circling, brief spurts of locomotor stimulation and depression, and the deterioration in motor coordination within the first few minutes post-cocaine. Then, the ataxic behavior may transition to head-weaving, loss of righting, jerky or convulsive movements, full body contortions (head and body rolls, arched back, lying prone, paw peddling) and finally a loss of the anti-gravitational reflex with movements limited to convulsions. At this point, a subset of intoxicated mice has begun to experience seizures, with an average onset at 3.4 min post-cocaine for the LD50 dose. The ataxic behavior often alternated with seizure activity for several minutes before death or gradual recovery. In the latter case, the progression of ataxic symptoms was often repeated in reverse order.</p><p>To detect changes in the severity of premorbid behaviors by immunization, the ataxia scores for all treated mice across successive 3-min intervals were analyzed statistically by ANOVA and by the unpaired Student's t test for comparisons between two groups within specific intervals. A one-way ANOVA of ataxia scores confirmed that there was no significant difference in the onset of cocaine toxicity among different treatment groups during the 3-min time period preceding passive vaccination [F(4,96) = 1.211, p > 0.05] (see Fig. 3). However, the acute development of ataxic symptoms was modulated by treatment, as illustrated by the one-way ANOVA for each 3-min time interval (3-6 min: [F(4,96) = 4.140, p < 0.005], 6-9 min [F(4,73) = 3.552, p < 0.05], 9-12 min: [F(4,62) = 2.478, p = 0.05], 12-15 min [F(4,60) = 2.780, p < 0.05]). The overall severity of ataxia significantly varied based on treatment, as depicted by a one-way ANOVA between groups for the highest ataxia score experienced by each mouse over the entire observation period [F(4,96) = 4.469, p < 0.005]. Fisher's PLSD was implemented for pair-wise comparisons between treatment groups, and it demonstrated that only Fab-92H2 treatment significantly lessoned the profile of cocaine-induced ataxia relative to the ataxic behavior in the cocaine-alone mice (p < 0.0001). Similarly, this pair-wise statistical analysis confirmed the superiority of Fab-92H2 over all other therapeutic treatments in terms of rescue from cocaine-induced ataxia (Fab vs. IgG: p < 0.005, Fab vs. F(ab')2: p < 0.01, and Fab vs. scFv: p < 0.001).</p><p>Passive immunization with Fab-92H2 3 minutes following cocaine injection was the only mAb GNC92H2-based treatment to stem seizure generation (Fig. 4). Whereas 65% of drug alone mice exhibited seizure activity as a result of acute cocaine toxicity, only 27% of Fab-92H2-immunized mice developed seizures (Fig. 2), a level of protection that was statistically significant by χ2 analysis (seizure occurrence: yes/no, χ2 = 6.749, p < 0.01; seizure severity: 0-3, χ2 = 8.866, p < 0.05). This contrasted with the greater than 50% of mice in IgG-92H2, F(ab')2-92H2, and scFv-92H2 treatment groups exhibiting seizures. Moreover, analyses by χ2 tests of goodness-of-fit for the appearance and severity of seizures of each mAb-treated group (Table 3), in which seizure generation within the cocaine-alone group was treated as the expected values, demonstrated that IgG-92H2 administration lessoned seizure severity but failed to block their occurrence (seizure severity: 0-3, χ2 = 19.74, p < 0.0005; seizure appearance: yes = 1, no = 0, χ2 = 0.03, p > 0.5). Administration of F(ab')2-92H2 or scFv-92H2 did not have a statistically relevant effect on seizure symptomology relative to administration of the saline vehicle in cocaine-alone mice.</p><!><p>Our laboratory and others have reported on the generation of cocaine-binding and cocaine-degrading mAbs to prevent lethality upon binge exposure (Briscoe et al., 2001; Carrera et al., 2005; Mets et al., 1998). However, we wished to extend the therapeutic utility of immunopharmacotherapeutic treatments beyond the protection from terminal overdose to the attenuation of other medically hazardous symptoms of acute cocaine toxicity. The time course of cocaine intoxication, during which mice experience a peak brain concentration 5-15 minutes after i.p. injection, implies that a potential pharmacotherapy must dampen and/or reverse the influx of cocaine into the CNS and the resultant central stimulatory effects within a narrow timeframe (Benuck et al., 1987). We hypothesized that the scFv, Fab and F(ab')2 formats derived from the mAb GNC92H2 scaffold might better mirror the rapid kinetic profile (distribution t1/2α = 1.9 min, elimination t1/2β = 26.1 min) and widespread tissue distribution (Vdss = 6.0 L/kg) of cocaine in vivo and thus achieve greater success than the IgG in cocaine detoxification (Norman et al., 2007).</p><p>In this study, the antibody-based manifolds were created using GNC92H2 as a template so as to preserve its pharmacodynamic properties, including its high binding affinity for cocaine (Kd = 2 nM, as determined via KinExA). In previous work, the Kd values of the GNC92H2 IgG and Fab formats for cocaine and its major metabolites were estimated through competition equilibrium dialysis using radiolabeled ligands (cocaine, benzoylecgonine, ecgonine methyl ester, and ecgonine 3H-labeled at the N-methyl position). Both formats exhibited superior affinity and specificity for cocaine (Kd ∼ 10-7 M) relative to the major nonpsychoactive metabolites (benzoylecgonine: Kd ∼ 10-5 M, ecgonine methyl ester: Kd ∼ 10-3 M, ecgonine: Kd ≫ 10-3 M) (Larsen et al., 2001). Likewise, immunoaffinity-purified scFv-92H2 displayed a Kd ∼ 10-7 M (Moss et al., 2003).</p><p>Previously, we conducted a study on cocaine overdose reversal through passive immunization with IgG-92H2, however given the precedence for mouse age, sex, and strain to exert a measurable effect on cocaine pharmacokinetics and behavioral response (Azar et al., 1998; McCarthy et al., 2004; Visalli et al., 2005), the experiments for IgG-92H2 pretreatment (90 mg/kg) and 3-min post-treatment (180 mg/kg) relative to cocaine injection were repeated to establish a benchmark of IgG-92H2 efficacy. Pretreatment results for the percentage of mice exhibiting each symptomology paralleled past data. However, the scoring of ataxic behavior across successive 3-min intervals revealed a statistically significant phenomenon in the recovery of immunized mice. The overall premorbid behaviors of immunized and cocaine alone groups were indistinguishable in the short-term, but the mild ataxia, which follows the acute, severe symptoms of toxicity, subsided more quickly in unimmunized mice. The prolonged ataxia that was observed under IgG-92H2 pretreatment may suggest a slow release of cocaine from tissue or IgG-92H2-bound stores and its subsequent re-distribution between plasma and brain.</p><p>The different GNC92H2 formats were then evaluated under our murine overdose model, and the ability of these immunopharmacotherapeutics to serve as antidotes for acute cocaine toxicity was compared against IgG-92H2. From this current study emerged preclinical evidence that smaller mAb constructs hold the greatest therapeutic potential for this scenario of cocaine abuse. Whereas all GNC92H2 formats except scFv-92H2 prevented lethality in a significant proportion of subjects when the immunopharmacotherapeutic was delivered three minutes after cocaine exposure, Fab-92H2 and to a lesser extent, F(ab')2-92H2 ameliorated other symptoms of severe toxicity.</p><p>The pharmacodynamic properties of smaller mAb fragments pose two distinct advantages over the IgG with respect to counteracting excessive cocaine levels. Their shorter half-lives reflect their immediate but extensive extravascular distribution as well as their rapid elimination via renal catabolism. Whereas IgG distributes to a volume equivalent to the plasma volume, F(ab')2 and Fab antibody formats have been shown to disperse throughout 2× to 4-5× this volume, respectively, highlighting the extent to which either penetrates superficial and deep tissue (Chippaux and Goyffon, 1998). Anti-drug passive vaccines are effective therapeutics because they shift the gradient of drug molecules from the brain to plasma, and as a result, the drug Vdss approaches that of the administered antibody (i.e., the plasma volume for IgG-based vaccines). Antibody fragments sequester their drug antigen in tissue as well as blood, and thus they tend to mediate a lesser rise in plasma drug concentration than their respective full IgG. As a corollary, the rate of cocaine redistribution between brain, blood and tissue, which is expected to vary with mAb format distribution, may contribute to the unique therapeutic properties of each GNC92H2-based vaccine.</p><p>Immunopharmacotherapeutic use of mAbs against PCP, methamphetamine and nicotine has documented this antibody-mediated drug redistribution and attributed it to the equimolar binding of drug molecules by the available antigen-binding sites of the mAb as opposed to antibody-mediated changes in drug metabolism (Byrnes-Blake et al., 2003; Keyler et al., 2005; Laurenzana et al., 2003; Proksch et al., 2000; Roiko et al., 2009; Valentine and Owens, 1996). By contrast, immunization with a smaller antibody fragment may affect the t1/2β of small-molecule drugs when the antigen-bound immunopharmacotherapeutic agent is within the molar weight threshold for renal filtration and elimination through urine. For example, digitoxin urinary excretion increased upon treatment with anti-digitoxin Fab but decreased below control levels upon use of the IgG (Ochs and Smith, 1977). Even though methamphetamine renal clearance remained unchanged by the anti-methamphetamine scFv6H4 and the rate of digoxin elimination slowed with anti-digoxin Fab treatment, the anti-PCP Fab appeared to accelerate urinary excretion of PCP and partially block PCP metabolism in treated rats (Butler et al., 1977; Peterson et al., 2008; Valentine et al., 1994). Thus, we hypothesized that both the greater tissue penetration of Fab-92H2 and F(ab')2-92H2, which augments the peripheral sink for cocaine upon mAb-mediated drug redistribution, and the direct urinary excretion of cocaine-bound Fab-92H2 would favor a greater efflux of cocaine from of the CNS compartment. The heightened pressure on the blood-brain cocaine gradient by mAb-mediated drug sequestration in the blood and periphery provides a pharmacodynamic explanation for the ability of a submolar mAb dose to clear enough circulating drug molecules to mediate overdose rescue and for the superior therapeutic efficacy of Fab-based versus IgG-based cocaine vaccines.</p><p>Although Fab-92H2 was delivered several minutes into the development of acute cocaine intoxication, post-treatment significantly counteracted the standard progression of premorbid symptoms and future seizure generation. Premorbid behaviors, seizures and lethality were present in 82%, 27%, and 9% of Fab-92H2-treated mice, respectively, versus the corresponding rates of 92%, 65%, and 55% observed in cocaine-alone mice. Whereas the suppression of seizures was not significant in treatment groups administered IgG-92H2, F(ab')2-92H2, or scFv-92H2, the percentage of mice displaying seizures in the cocaine-alone group was markedly lower here in comparison to previous studies performed in this laboratory using different mouse strains (Carrera et al., 2005). This seemingly higher seizure threshold in CD-1 mice would offset any vaccine-mediated percent reduction in seizure activity reported in this study and thus diminish the perceived antidotal value of these GNC92H2-based therapeutics.</p><p>Even though the biodistribution and elimination of the F(ab')2 format is more akin to the IgG than smaller fragments, F(ab')2-92H2 profits over Fab-92H2 and scFv-92H2 in that its bivalency guarantees the preservation of its stability and antigen affinity. It is crucial that any vaccine retains its full binding activity at high concentrations prior to use and upon its injection as a cocaine antidote. In addition, the longer half-life of F(ab')2-92H2 permits drug scavenging for several hours to days, thus granting continued protection from cocaine exposure as drug reenters circulation from lipid-rich tissue stores. These benefits notwithstanding, F(ab')2-92H2 demonstrated a diminished capacity to reverse cocaine overdose relative to Fab-92H2 under this acute model. The severity of ataxic behavior was significantly attenuated, but the F(ab')2-derived reductions in seizure generation and severity were statistically insignificant (Table 2).</p><p>scFv-92H2 conferred the least protection from cocaine-induced symptomology relative to other mAb GNC92H2 constructs in the overdose rescue paradigm. Its half-life of t1/2 ∼ 18 min represents an extension over those reported in the literature for other murine scFv monomers (t1/2 < 10 min). Multivalent scFv species possess higher residence times in serum and tissue due to their greater stability and decreased first-pass renal elimination, and indeed, both the 15-residue linker connecting the VH and VL domains of scFv-92H2, and the high protein concentration required for passive immunization (> 5 mg/ml) favor the formation of diabodies and higher molecular weight multimers (Kortt et al., 1994; Moss et al., 2003; Redwan et al., 2003; Shelver et al., 1996). For drug detoxification, the multivalent derivatives originating from the monovalent anti-methamphetamine scFv (15-residue linker) promoted the redistribution of methamphetamine to the plasma compartment for several hours (Peterson et al., 2008). This in vivo persistence and antigen binding activity of scFv multimers relative to scFv monomers (monovalent t1/2Z ∼ 5.8 min, multivalent t1/2Z ∼ 228 min) may prove advantageous to their clinical use in immunotherapy.</p><p>However, scFv-92H2 demonstrated suboptimal therapeutic efficacy in this overdose model relative to Fab-92H2 despite the improvement in its pharmacokinetic profile via dimerization, which was depicted in half-life data and in previous in vitro studies (Moss et al., 2003). Several seemingly minor factors may have contributed to the decreased efficacy of scFv-92H2 treatment, including: 1. the formation of aggregates, which may diminish the cocaine binding capacity of a given mAb dose, 2. the documented tendency of scFv fragments to possess a lower affinity for antigen in comparison to the larger, stabilized formats, and 3. the rapid elimination of a large fraction of the original scFv dose. With respect to the latter, monomeric scFv undergoes first-pass renal excretion soon after i.v. infusion. We have hypothesized that the 18-min half-life, which was approximated via a single-exponential rather than a bi-exponential curve, is biased by the longer residence times of multimeric species, and that the initial clearance of monomers may lower the effective scFv dose to sub-therapeutic levels. The steepness of the cocaine dose-response curve in mice (Bedford et al., 1982; Hearn et al., 1991) implies that the elimination or inactivation of a small fraction of the scFv dose could have a detectable consequence on its efficacy. Also, scFv fragments possess the most rapid and extensive distribution out of vascular space into superficial and deep tissue relative to the Fab, F(ab')2, and IgG formats (Milenic et al., 1991; Shelver et al., 1996). Even though this characteristic permits scFv-92H2 to sequester cocaine within tissue, the proportionate decreases in free and mAb-bound cocaine in serum may be accompanied by a slower redistribution of cocaine out of the brain. The re-emergence of ataxic behaviors (Fig. 3) and severe cocaine toxicity (Fig. 2, 4) at approximately 12 min after scFv infusion may suggest that scFv levels dropped precipitously or that scFv differentially modulated cocaine redistribution from vasculature and tissue stores to brain.</p><p>In addition to identifying Fab-92H2 as an optimal antidote to cocaine overdose, the application of engineered mAb constructs to rapid small molecule detoxification illustrates a manner in which to circumvent many of the perceived weaknesses of anti-drug vaccines. First, to attain a molar equivalence between antibody binding capacity and the molar drug dose, the target concentration of circulating antibodies must be exceedingly high. With respect to active vaccination strategies, an anti-cocaine vaccine has yet to elicit consistently high antibody titers in clinical trials (Martell et al., 2009). Likewise, the requisite dose of a passive vaccine carries a risk of adverse effects from the protein load of the IgG infusion. The presence of the Fc constant region in IgG-based agents, which augments their size relative to mAb fragments, is both superfluous and potentially detrimental in anti-drug immunopharmacotherapy. Whereas its physiological purpose consists of initiating immune effector functions upon antigen binding, the Fc component of IgG-based therapies contributes to their extended half-life and heightened stability. Neither of these functions is required in the scenario of overdose reversal, and indeed, the slow elimination of anti-drug IgGs may cause the passive vaccine to become immunogenic.</p><p>As alternatives to the IgG, anti-PCP Fab and anti-methamphetamine scFv have been examined in drug detoxification strategies, but in contrast to overdose reversal, testing was conducted using comparatively low, non-lethal drug doses (Peterson et al., 2008; Valentine et al., 1996). Also, subject survival did not hinge on the time-sensitive scavenging of circulating drug or on the delivery of an exceedingly high mAb dose. However, these studies validated the feasibility of using anti-drug mAb fragments to sequester drug in vivo through confirming the absence of Fab-induced renal toxicity and the preservation of scFv binding activity via multimer formation. Our current investigation establishes the therapeutic utility of different engineered GNC92H2 manifolds within a specific drug abuse scenario. To achieve accelerated drug clearance, smaller mAb constructs possess a pharmacokinetic advantage over the IgG and may prove therapeutically beneficial in the treatment of other time-sensitive indications. In sum, our results endorse the case-specific optimization of mAb formats as a route toward developing superior pharmacotherapies.</p><!><p>Therapeutic benefit of prophylactic immunization against cocaine. A) Cocaine-induced ataxia upon pretreatment with mAb GNC92H2 IgG (90 mg/kg) 30 minutes prior to drug administration. The ataxia ranking system, which was adapted from the 0-5 scale proposed by Sturgeon et al., involved assigning a single score (0, 1, 2, 3, 4, 5) to each mouse based on the highest level of ataxic behavior displayed by that subject within a 3-min observation period. Data (mean ± S.E.M.) for cocaine alone mice (unshaded line) and immunized mice (shaded line) represent the ataxia score average of mice in a treatment group for each successive 3-min time interval after i.p. injection of cocaine at time = 0 min. *p < 0.05, ** p < 0.005 for the Student's t test comparison of control versus immunized groups in designated time intervals.</p><p>Protection against cocaine-induced toxicity by passive immunization with mAb GNC92H2-based immunopharmacotherapeutics. Treatments (Cocaine Alone: 10 mL/kg saline, IgG: 180 mg/kg, F(ab')2: 132 mg/kg, Fab: 120 mg/kg, or scFv: 60 mg/kg; i.v.) were administered 3 minutes after cocaine injection (93 mg/kg, i.p.). Data are expressed as a percentage of mice in each treatment group that experiences seizures or lethality from an LD50 cocaine dose.</p><p>Effect of GNC92H2-based immunopharmacotherapeutics on cocaine-induced ataxia. Subjects were administered an LD50 cocaine dose by i.p. injection at time = 0 min, upon which each subject was transferred to a locomotor activity cage for behavioral monitoring. The assigned mAb treatment (top graph: IgG, 180 mg/kg; middle graphs: F(ab')2, 132 mg/kg and Fab, 120 mg/kg; bottom graph: scFv, 60 mg/kg) or vehicle (sterile 0.9% saline) was infused through the jugular vein catheter at time = 3-4 min. Ataxia (0-5 scale) was scored for each subject within successive 3-min intervals for the 39-min observation period, and group scores were averaged within each 3-min interval (mean ± S.E.M.) for comparison of ataxic symptoms between the cocaine alone control (unshaded line) and immunized groups (shaded line). *p < 0.05, ** p < 0.005 for the Student's t test comparison of cocaine alone versus immunized groups within designated time intervals.</p><p>Time-dependent severity of cocaine-induced seizures in cocaine alone and immunized groups. An LD50 dose cocaine was i.p. injected in mice at the start (time = 0 min) of behavioral monitoring, and 3 minutes into the observation period, the designated mAb treatment (top graph: IgG, 180 mg/kg; middle graphs: F(ab')2, 132 mg/kg; Fab, 120 mg/kg; bottom graph: scFv, 60 mg/kg) or vehicle (Cocaine Alone: sterile 0.9% saline) was infused through the indwelling jugular vein catheter. Seizure activity (0-3 scale) was scored for each mouse across successive 3-min intervals and values for each time interval represent group mean scores ± S.E.M. for cocaine alone control (unshaded line) and immunized (shaded line) groups. *p < 0.05, † p = 0.055 for the Student's t test comparison of control versus immunized groups within specific time intervals.</p><p>The pharmacokinetic profiles of mAb GNC92H2 formats.</p><p>(Norman et al., 2007)</p><p>The binding affinity of GNC92H2 IgG for cocaine was measured via several methods: by equilibrium dialysis (Kd = 200 nM), by competition ELISA for cocaine (Kd,app ∼ 13 nM), and by the KinExA system (Kd = 2 nM). Competition ELISA provides a semi-quantitative estimation of of antibody binding affinity for an antigen, whereas KinExA (Sapidyne Instruments Inc.; (Ohmura et al., 2001)) measures the Kd, Kon, and Koff binding constants in the solution phase to characterize bimolecular binding events. It thereby avoids the mass transport limitations and mobility effects inherent to traditional methods that measure binding events between a solution phase and a solid phase, and thus provides the most accurate Kd measure. This method was not used to characterize the other GNC92H2 fragments, whereas binding affinities of the IgG, Fab, and scFv formats were all estimated via equilibrium dialysis.</p><p>Not determined.</p><p>92H2-scFv, isolated and purified via immobilized metal affinity chromatography and high performance liquid chromatography, or via immunoaffinity purification (Moss et al.,2003).</p><p>Statistical analysis of changes to cocaine-induced symptomology by 3-min post-treatment with mAb GNC92H2 IgG, F(ab')2, Fab, or scFv.</p><p>ns: not significant</p><p>Summary of ataxia and seizure severity scores in cocaine control and immunized mice upon injection of an LD50 cocaine dose. Data represents group mean ± S.E.M. of the maximum ataxia (0-5) or seizure (0-3) score attained by each subject within the 60-min observation period.</p><p>Mole-equivalent dose, mmol/kg</p><p>Data adjusted for IgG and F(ab')2 bivalency: two cocaine binding sites per antibody molecule</p>

PubMed Author Manuscript

Noise in cellular signaling pathways: causes and effects

Noise caused by stochastic fluctuations in genetic circuits (transcription and translation) is now appreciated as a central aspect of cell function and phenotypic behavior. Noise has also been detected in signaling networks, but the origin of this noise and how it shapes cellular outcomes remain poorly understood. Here, we argue that noise in signaling networks results from the intrinsic promiscuity of protein-protein interactions, and that this noise has shaped cellular signal transduction. Features promoted by the presence of this molecular signaling noise include multimerization and clustering of signaling components, pleiotropic effects of gross changes in protein concentration, and a probabilistic rather than linear view of signal propagation.

noise_in_cellular_signaling_pathways:_causes_and_effects

Noise in biological systems<!>Promiscuity in PPI networks<!>Noise arising from promiscuity<!>Multimerization to overcome the noise threshold<!>Functional selectivity through integration of multiple signals<!>Probabilistic rather than linear signaling<!>Does pre-activation and pre-clustering to keep reaction times short?<!>Positive effects of noise on fidelity, robustness, and evolvability of cellular signaling<!>Influence of noise modulation on experimental results<!>Concluding remarks

<p>Noise is increasingly appreciated as a force shaping biology. Recent research has revealed genetic circuits that are subject to stochastic fluctuations, or noise, at the [sta1] level of their components. By stochastically influencing state-switching systems, especially those based on positive feedback loops, genetic noise can cause substantial variations in expression of many genes. Thus, genetic noise can cause genetically identical cells to behave differently 1, 2. This noise-driven genetic regulation allows cell state choice to be probabilistic and can cause phenomena such as resistance to antibiotics or anticancer drugs 3, 4. Genetic noise has therefore emerged as a central factor in how biological systems function and evolve 3, 5.</p><p>The observation that noise in transcriptional regulation shapes biological systems raises the question of whether other physiological processes are also subject to the effects of noise. Recently it has been shown that the information transduced in cellular signaling pathways is significantly limited by noise 6, 7. The molecular basis of this noise and how it shapes cellular pathways is poorly understood 7. Based on the accumulated data on kinase-mediated signaling, we here propose that noise is generated by the interconnected and promiscuous nature of the protein-protein interactions (PPIs) that are required to transduce the signals. We further propose that this noise has fundamentally shaped kinase-mediated signaling and possibly cellular signal transduction in general.</p><!><p>If signaling cascades are assumed to be linear relays of events, then each signal has to be transmitted through several mutually exclusive bimolecular PPIs from its origin (e.g., a plasma membrane-localized receptor tyrosine kinase; RTK) to an effector (e.g., a transcription regulator). The human interactome is believed to contain between 130,000 and 650,000 binary interactions, most of which are currently uncharacterized 8-10(Figure 1, Box 1). Each PPI involved in propagating a specific signal through the cell must therefore compete against the bulk of nonspecific competitors. This is particularly difficult when the specific signals and signal recognition domains are extremely similar to the nonspecific ones, as is the case in kinase-mediated signaling.</p><p>In kinase-mediated signaling, the initial event is often linked to phosphorylation of a substrate. Investigations of the kinase-phosphatase interaction network revealed a highly intricate and degenerate array of collaborative interactions that "creates[sta2] the conditions for indiscriminate chatter" 11 within and outside of the network 12. In other words, a central step in this type of signal transduction was shown to result in arbitrary nonspecific interactions and (de)phosphorylation events, in stark contrast to an orderly specific and linear signaling cascade. Phosphorylation events are typically recognized by small phosphoresidue binding domains, such as Src homology (SH) 2 or phosphotyrosine binding (PTB) domains 13-16. Signal propagation normally also involves other protein-protein recognition domains, such as SH3 domains. Mammalian cells can express more than 100 different types of proteins containing each of these domains. For each specific interaction, the biologically relevant domain must compete against all the homologous nonspecific domains for similar consensus binding motifs. Thus, from a simplistic point of view where these domain-containing proteins were expressed at equivalent concentrations, for a specific interaction to prevail against all the potential nonspecific competitors, the difference in affinity between the specific and nonspecific binding events would have to be significantly greater than 2 orders of magnitude (i.e. 1 specific interaction has to compete against >100 non-specific interactions)[sta3]. In some cases the differences in affinities would have to be even greater. For example, in the human cell, more than 300 SH3 domains compete not only with one another but also with WW, GYF, UEV, and EVH1 domains for similar proline-rich binding motifs (which are found in almost 25% of all human proteins) 15, 17. Thus, in the context of other competing interactions, likely differences in expression levels and fluctuations in local protein concentrations, to ensure that a specific interaction will occur, it is likely that the affinity of the interaction will have to be at least 3 orders of magnitude greater than those of the background interactions.[sta4] Such a large difference in affinity between the specific interaction and all nonspecific interactions is seldom observed; in fact, the interactions of binding domains with different ligands often show only a limited affinity range and in some cases show significant promiscuity 13-15. This promiscuity can in part be linked to the fact that in many cases the binding sites presented to ligands are highly similar (Figure 2). The limited range in observed binding affinities is also a consequence of the fact that PPIs must be short-lived to allow cells to cease signaling in a timely manner. For this, PPI off-rates need to be fairly short, and since on-rates are limited by diffusion, affinities are necessarily found within a limited range. In many cases, high affinities would result in a complex with too long a lifetime. For example, even an affinity in the half-micromolar range is sufficient to allow the HIV-1 Nef protein to activate the transforming potential of certain Src kinases by binding to their SH3 domains 18.</p><p>The promiscuity in PPIs is further increased by another energetic component. Some proteins exhibit auto-inhibitory mechanisms, which help prevent these proteins from exposing promiscuous binding sites to the wrong partners in the wrong spatiotemporal context. However, the naturally occurring fluctuations between the inactive and active dynamic conformations, even in the inactivated protein 19, 20, are expected to produce opportunities for spurious nonspecific associations and basal enzymatic activity.</p><p>Promiscuity in PPIs is also observed in genome-wide interaction mapping.[sta5] For example, large-scale investigations in yeast and higher eukaryotes showed that although some SH3-ligand interactions have evolved a selectivity that allows strict exclusivity, many other SH3 interactions display significant cross-reactivity 21-24. Cellular signaling therefore appears to have evolved to incorporate a degree of promiscuity in the PPIs on which it is based. Such PPI promiscuity is also functional, because it allows the same protein to bind to different interaction partners and hence to participate in a different signaling event. Indeed, many signaling proteins form biologically relevant complexes with multiple distinct partners and need to tolerate some degeneracy in ligand recognition specificity 25.</p><p>Compartmentalization is without doubt important for limiting the number of erroneous protein-protein encounters within the cell 25. However, even if the 130,000 to 650,000 possible binary interactions were spread out over 100 different types of compartments, we would expect over 1000 possible interactions per microenvironment. Moreover compartmentalization can not be strict, because many signaling proteins partake in signaling events localized to different compartments, or need to migrate across large distances for function (for examples see 25, 26). Finally, diffusion between adjacent compartments is expected to result in local leaking of components from one microenvironment into the other. Therefore, while certainly required for functional fidelity in signaling, compartmentalization is unlikely to eliminate low-affinity non-specific protein-protein contacts.</p><!><p>Taken together, the intricacy and multivalency of signal transduction, the lack of sufficient intrinsic specificity in many PPIs (both catalytic and noncatalytic) and the fluctuations in auto-inhibition appear to p[sta6]roduce a significant level of signaling promiscuity within the cell. This promiscuity in PPIs is expected to give rise to noise in the form of frequent spurious nonspecific binary associations. This noise is conceptually very different from the noise resulting from stochastic fluctuations of low-abundance genetic regulators. Whereas genetic noise typically causes stochastic bursts in protein production, with possible sudden dramatic changes in cell behavior, signaling noise causes a constant background. In kinase-mediated signaling, this background can consist of low levels of RTK activity through random transient interactions of receptors 20, phosphorylation of receptors and recruited proteins, binding of signaling proteins to receptors in nonfunctional complexes, and background phosphatase activity. A functional signal has to prevail above this background noise. Because fidelity in signaling is achieved despite this noise, the question arises of how this noise has shaped cellular signal transduction.</p><!><p>The noise resulting from transient nonspecific PPIs in interconnected signaling networks will generate a constant "chatter" that has to be surmounted[sta7] for signals to reach their destination. In terms of cellular signal transduction, signals can be amplified through oligomerization or clustering of the signaling components (Figure 3A). Indeed, dimerization of transcription factors has been shown to diminish noise in the genetic networks they control 27. Furthermore, it has been suggested that protein dimerization could dampen intrinsic and extrinsic noise 27. Also, eukaryotic signaling networks contain a significantly higher number of homologous interactions than expected from calculating the probability for their random occurrence 28, indicating that self-interaction has been evolutionarily favored. It has been suggested that ligand-induced oligomerization of cell surface receptors protects intracellular signaling against noise 29, 30. In the case of most RTKs, kinase signaling is initiated by extracellular ligand binding, which induces dimerization of RTKs. This cytokine-induced dimerization allows kinase domains to become juxtaposed, resulting in transphosphorylation of the RTKs 31. Receptor dimerization and clustering are very common, and the signal outcome is sensitive to changes in the status and lifetime of receptor multimers, with possible pathogenic consequences 31, 32. In addition, responses can be amplified through positive feedback loops and cooperative interactions between multiple partners 25.</p><!><p>The promiscuity in PPIs reduces the fidelity of the signaling process and eliminates mutual exclusivity in the pathways. The cell therefore adopts mechanisms that allow nonspecific interactions but precludes these interactions from providing an appropriate signal. One such mechanism that is prevalent in the early signaling events accompanying cell surface receptor stimulation is the recruitment to a RTK of multiple proteins, which form large multiprotein signaling complexes (early signaling complexes, or signalosomes) 33. Each RTK molecule may adopt a large amount of different phosphorylation states that are associated with potentially different ligands and hence downstream responses 34. This process is constantly counteracted by phosphatase activity 34. Only when the correct complement of proteins in the appropriate stoichiometry are recruited to an adequately phosphorylated receptor is a functional signaling entity formed and a downstream response garnered (Figure 3B). Thus, interactions of nonspecific proteins occur, but nonfunctional signaling complexes will result. Although this functional selectivity ensures that only complexes of the correct composition can initiate downstream signals, it also results in a significant population of nonfunctional complexes. Whereas in a high-fidelity system only one complex (or, realistically, a few complexes) would be required to initiate signaling, in promiscuous systems the number of initial complexes required is much higher. The ephemeral, abortive signals of these complexes may add to the noise level.</p><!><p>Background signaling noise therefore demands cell surface receptor multimerization in two different ways. First, the total number of receptors needs to be large to overcome the number of nonfunctional submembrane signaling complexes formed. Second, receptors need to multimerize so that the signal generated persists above the background noise threshold, even including leaking and erroneous diffusion of the signal during propagation. The cumulative potential for formation of non-functional or redundant complexes suggests RTK signaling is probabilistic. The traditional view of a tightly regulated and highly specific linear signaling cascade from cell surface receptors to cellular response (e.g., gene expression) therefore needs to be supplanted by a probabilistic model. In this model, the probability of a given RTK dimer triggering a cellular outcome is in fact extremely low because of the promiscuous nature of signaling, the low probability of achieving a functional complex, and the noise threshold (Figures 2,3). Signal transduction is therefore a 'numbers game', where enough cell surface receptors must be engaged initially to guarantee formation of a functional signaling complex against a background of redundant events. Indeed, cells express tens or even hundreds of thousands of receptors on their surfaces which are exposed to extracellular stimuli. Because the production of huge numbers of receptor molecules is energetically expensive, such numbers appear to be[sta8] required for successful signal transduction. This view is supported, for example, by the observation that the reduction of HLA molecules on the cell surface from 100,000 to about 10,000 by HIV-1 Nef is enough to protect HIV-infected cells against recognition and killing by cytotoxic T cells 35, 36. In other words, 10,000 HLA molecules are not enough to produce a signal strong enough to trigger a cellular response.</p><!><p>To overcome noise, the receptors, as well as subsequent signaling hubs, must first multimerize, then auto-activate, and then assemble large signaling complexes. This diffusion-limited process is slow and will delay cellular signaling responses. Reaction times can be reduced by pre-clustering signaling components and putting them into a "primed" state: a condition from which they can rapidly assemble into the fully active constellation. Then, rather than having to go from zero to above the noise level, signaling networks only have to transition from just below to just above the noise threshold. It is indeed often observed, although only poorly documented, that even in serum starved cells and in the absence of stimuli a significant percentage of many RTKs are already in a phosphorylated state, which is normally associated with an active signaling function 37-40. Ligand-based activation only tips the balance toward a higher percentage of fully active receptors 20. However, this small increase in the percentage of activated receptors appears to make all the difference in terms of signaling outcome 40. The primed states of signaling networks may be important in pushing cells into a state of criticality or supersensitivity, where a spike of activity above the noise level will be rapidly[sta9] perpetuated throughout the signaling cell 41, 42.</p><!><p>An intriguing question is whether, counterintuitively, background noise could also improve fidelity in cellular signaling networks. The noise generated by the intricacy, multivalency, and promiscuity of PPIs introduces a threshold level for signal propagation. Because signals below this threshold will not be able to trigger a response, this threshold level of white noise makes the cell robust against erroneous endogenous signals, such as those originating from the very source of noise: the erroneous PPIs and basal enzymatic activity. By maintaining an appropriate noise level, cells would also protect themselves from erroneous exogenous signals such as, for example, ectopic growth factors or low doses of pathogenic activators.</p><p>Noise may also render cells more robust to inactivation of a signaling component (e.g., through a deleterious mutation). A certain type of noisy interaction may become significant if the biologically relevant binding partner is depleted. Thus, the PPI promiscuity that accounts for part of the endogenous noise may allow the closest homologous binding partner to ultimately illicit a significant cellular response if the biologically relevant partner is depleted. A homologous protein could take the lead to partially rescue a null mutant. This type of partial rescue has indeed been described (e.g., for Src kinases 43) and makes cells robust against protein deletions.</p><p>The evolution of cellular signaling networks is characterized by gene duplication followed by divergence and gain of new function 44. The signaling noise threshold may allow such duplicated genes to evolve into a new function without significantly perturbing existing communication networks.[sta10]</p><!><p>If signaling has adjusted to, and relies on, a significant noise level, gross alterations in this noise level may, in turn, influence signaling in general. For example, strong overexpression or knockout/knockdown of abundant and promiscuous proteins may significantly alter the noise level inside the cell. As a result, gross changes in a particular protein concentration may pleiotropically affect even those cellular signaling pathways that do not use this protein, by altering the noise level (Figure 3C). According to the effects discussed here, this may lead to premature or delayed initiation of signaling cascades or to signal rerouting through significant involvement of proteins homologous to the one affected. More subtly, changes in the specificity of some signaling domains may affect not the total noise level but the "pressure" that noise exerts on particular interactions. Protein overexpression or knockout/knockdown are commonly used tools to assess the function of a particular protein. Potential pleiotropic effects of noise-level modulation would therefore be an important parameter to assess in experimental investigations to avoid false interpretations of the phenotype caused by gross changes in protein concentration.</p><!><p>Although the causes and effects of noise in genetic circuits have become well established, noise originating from promiscuity in PPIs has so far been widely neglected. Here, we argue that a certain level of PPI noise is unavoidable, because of the complex and multivalent nature of eukaryotic signaling networks, and that this sort of noise has profoundly shaped the way cells transmit signals. If cells have adapted to this noise level, then noise has become an integral part of the correct transmission of signals. If so, then many signaling cascades, especially those involving ubiquitous homology domains, may be indirectly linked through noise. This noise may be important in increasing the robustness of cellular signaling by dampening and aborting single erroneous events. Thus, erroneous endogenous signals, and possibly also a certain level of pathogenic intervention, may be dampened. In turn, the promiscuity giving rise to that noise may help cell function recovery through homologous pathways. Importantly, gross changes in protein expression, such as commonly achieved experimentally, may have pleiotropic effects on other cellular signaling networks. Here, we have discussed signaling noise principally from the perspective of kinase-mediated signaling. Similar noise may affect other signaling networks. For example, the recognition of histone modification has been shown to be influenced by noise 4, 45. Noise could therefore play a role in the fidelity of epigenetic memory. Because noise affects central regulatory switches of cell functions, alterations in noise level may play a role in human disease. Understanding how signaling noise affects cell function may therefore provide novel viewpoints on diseases such as cancer or on propagation of epigenetic memory.</p>

PubMed Author Manuscript

Thienoisoindigo-Based Semiconductor Nanowires Assembled with 2-Bromobenzaldehyde via Both Halogen and Chalcogen Bonding

We fabricated nanowires of a conjugated oligomer and applied them to organic field-effect transistors (OFETs). The supramolecular assemblies of a thienoisoindigo-based small molecular organic semiconductor (TIIG-Bz) were prepared by co-precipitation with 2-bromobenzaldehyde (2-BBA) via a combination of halogen bonding (XB) between the bromide in 2-BBA and electron-donor groups in TIIG-Bz, and chalcogen bonding (CB) between the aldehyde in 2-BBA and sulfur in TIIG-Bz. It was found that 2-BBA could be incorporated into the conjugated planes of TIIG-Bz via XB and CB pairs, thereby increasing the π − π stacking area between the conjugated planes. As a result, the driving force for onedimensional growth of the supramolecular assemblies via π − π stacking was significantly enhanced. TIIG-Bz/2-BBA nanowires were used to fabricate OFETs, showing significantly enhanced charge transfer mobility compared to OFETs based on pure TIIG-Bz thin films and nanowires, which demonstrates the benefit of nanowire fabrication using 2-BBA.

thienoisoindigo-based_semiconductor_nanowires_assembled_with_2-bromobenzaldehyde_via_both_halogen_an

<!>Results and Discussion<!>Conclusion<!>Methods

<p>Organic transistors have been developed over the last decades as next-generation electronic devices owing to their versatility for flexible devices and facile fabrication process compared to rigid inorganic semiconductors 1,2 . In particular, one-dimensional (1D) organic nano/micro-wire transistors have attracted significant attention due to their excellent electronic properties, including high charge-carrier mobility in specific directions [3][4][5][6] . Various strategies have been used to prepare organic semiconducting wires, including an ink-jet method 7,8 , electro-spinning 9 , hard [10][11][12] and soft template tools [13][14][15] , self-assembly in solution [16][17][18][19] and physical vapor transport 20,21 . The excellent charge-carrier mobilities were obtained for self-assembled single crystals of organic semiconductors, because their closely assembled structures by intermolecular interactions enable electrons to move efficiently through a conjugated backbone plane 16,22 .</p><p>Organic semiconductors are prone to be crystallized due to the presence of π − π stacking of the conjugated main backbones, which provides the driving force for 1D growth. In general, the self-assembly of single crystalline based on nano/micro-wires of the organic semiconductors shows the strong dependence of the solubility on recrystallization temperature 17,19,23 and solvent type 18,21,24 , which is usually determined by the molecular structure of the organic semiconductors 16,25,26 . Organic semiconductors usually require flexible alkyl side chains to facilitate solution processing 27 . However, the alkyl side chains increase the tendency for lateral packing of the organic semiconductors and disturb 1D growth in the direction of the π − π stacking of conjugated planes during precipitation or recrystallization, resulting in spherical morphologies due to hydrophobic interactions in the lateral direction of the conjugated planes 28 . Therefore, enhancing the driving force for π − π stacking so that π − π interactions between conjugated planes dominate lateral hydrophobic interactions between alkyl chains is critical for preparing nano/micro-organic semiconductor wires.</p><p>Halogen bonding (XB) is a strong and tunable form of non-covalent bonding between halogen atoms and negative sites, with bonding strengths of 10-150 kJ/mol 29 . Since the XB originates from the attraction between the partially positive charged region of halogen atoms, the σ hole, and its counterpart in electron-rich donor atoms, it has been widely used in supra-molecular assembly and crystal engineering of self-assembled structures [30][31][32][33][34][35] . The halogen atoms have dipolar charges; the π hole is perpendicularly negative, while the σ hole is horizontally positive. These charges simultaneously attract electron-rich functional groups, including lone-pair electrons in the same plane, and electron-poor groups in the vertical direction 36 . Furthermore, the XB should benefit for electronic device applications because it does not involve acidic or basic groups that can trap charges and disturb charge transfer, which is clearly different to hydrogen bonding 37 . Hence, the XB is considered an important intermolecular interaction for co-crystallization with conjugated oligomers or polymers for organic electronic devices [38][39][40][41][42][43][44][45] .</p><p>Chalcogen bonding (CB) is a newly identified type of weak non-covalent interaction, recently described by using various modern characterization techniques, such as nuclear magnetic resonance, X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD). Despite its weak interaction, the CB can play a dominant role in crystal design as it has a controllable binding strength [46][47][48] of a few to hundreds of kJ/mol 49 . The CB involves highly polarizable and electronegative atoms (i.e., chalcogen atoms (O, S, Se, Te) which have σ holes and π holes similar to halogen atoms. In this way, the σ hole, the positive part of chalcogen atoms at the opposite side of the covalent bond, can combine with the electron donor and bind via electrostatic interaction 50 . In most chalcogen-containing organic semiconductors, the CB plays an important role in facilitating charge transport by the improving electronic delocalization of their backbones 41,50 .</p><p>Although it has been proven that CB or XB is a useful interaction for molecular assembly, most approaches using CB or XB for transistor devices based on organic semiconductors have focused on intramolecular interactions in planar conjugated planes and consequently high carrier mobility [38][39][40][41][42][43][44][45][50][51][52] . Semiconducting oligomers or polymers bearing chalcogen or halogen atoms can have enhanced intramolecular electron delocalization and narrow optical band gaps due to the planar structure of their backbones. For example, the thienoisoindigo (TIIG) unit has been used as a promising building block to construct high-mobility organic semiconductors for organic field-effect transistors (OFETs). Advantages of this material include strong π − π stacking with a large overlapping area induced by a combination of the planar backbone structure and intramolecular CB, where improved electronic delocalization is derived from the quinoidal structure 51,52 . At present, it is hard to find molecular assemblies of organic semiconductors utilizing intermolecular XB and CB.</p><p>In this study, we fabricated nanowire assemblies of a TIIG-based organic semiconductor, (E)-2,2′-diphenyl-4,4′-bis(2-ethylhexyl)-[6,6′-bithieno [3,2-b]pyrrolylidene]-5,5′(4 H,4 H′)-dione, TIIG-ethylhexyl benzene (TIIG-Bz), which was able to simultaneously engage in XB and CB interactions with 2-bromobenzealdehyde (2-BBA), and demonstrated their use for organic field-effect transistor (OFET) applications. We examined the TIIG-Bz/2-BBA nanowire assemblies using XPS, powder XRD (PXRD), grazing incidence XRD (GIXD), and XPS, and investigated the influence of 2-BBA on nanowire assemblies and charge transfer mobility of OFETs.</p><!><p>Nanowires of TIIG-Bz and supra-molecular nanowire assemblies of TIIG-Bz and 2-BBA were fabricated using the bisolvent phase transfer method 28,32,53 , shown schematically in Fig. 1. This method is a popular fabrication process for nano-wire assemblies because it optimally accommodates molecules with different structures and sizes. In the process, a solution with organic semiconductors dissolved in a good solvent is placed in a vial, then a poor solvent is carefully poured onto the solution. Despite quite good miscibility between the poor and good solvents, their density difference kept them from immediately mixing with each other. Instead, at the interface of the two miscible solvents, conjugated organic molecules dissolved in the good solvent slowly self-assemble as the two solvents mix and the solubility of the organic semiconductor decreases. Consequently, self-assembled nanowires are formed at the interface 28,32,53 . In this study, for supra-molecular assemblies, TIIG-Bz and 2-BBA were dissolved in chloroform (d = 1.4459 g/cm 3 at 20 °C), a good solvent for both chemicals, and co-precipitated at the interface between chloroform and methanol (d = 0.7914 g/cm 3 at 20 °C) upon the inter-diffusion of chloroform and methanol.</p><p>As shown in Fig. 2(a), nanowires of only TIIG-Bz with a thickness ~1-2 μm (60-200 μm in length) were formed via this bisolvent phase transfer process. The crystals of TIIG-Bz molecules preferentially grow in one direction, even without the use of 2-BBA, due to their molecular rigidity and planarity that enhance intermolecular π − π interactions upon exposure to methanol when dissolved in chloroform. However, it should be noted that some TIIG molecules were aggregated in a bundle of wires and formed much thicker wire groups or particles (Fig. S1), indicating inhomogeneous self-assembly in both planar and vertical direction of conjugated plane due to competition between π − π stacking of conjugated planes and lateral packing of ethylhexyl chains. When TIIG-Bz was assembled with 2-BBA via bi-phase separation, supra-molecular nanowires were successfully assembled with a higher aspect ratio, thickness ~800 nm, and length ~180 µm, than the TIIG-Bz nanowires assembled without 2-BBA, as shown in Fig. 2(b,c). During fabrication of TIIG-Bz and 2-BBA, both chloroform and methanol are good solvents for 2-BBA and we used the same concentration of 2-BBA in both the chloroform and methanol phases to prevent interphase diffusive mass transfer of 2-BBA due to concentration differences. Thus, the diffusive mass transfer of TIIG-Bz from the chloroform phase into the methanol phase was induced by a TIIG-Bz concentration gradient and enabled the formation of supra-molecular assemblies at the interface between the chloroform and methanol phases with intermixing of the solvents, followed by the decreased solubility of TIIG-Bz.</p><p>The optical properties of TIIG-Bz dissolved in chloroform and its nanowires dispersed in methanol were examined by UV-Vis-NIR spectroscopy. When TIIG-Bz was dissolved in the chloroform solution, the maximum absorption band appeared at 616 nm with a shoulder at around 650 nm (Fig. 3). In comparison, the absorption spectra of TIIG-Bz assemblies with and without 2-BBA dispersed in methanol showed increasing absorption intensity in the NIR region of 700-1000 nm. The maximum absorption peaks of the TIIG-Bz solution, TIIG-Bz-only, and TIIG-Bz/2-BBA nanowires were all observed at ~616 nm. However, the shoulder band around 700 nm in the spectrum of the TIIG-Bz solution was significantly red-shifted to near 800 nm for the assembled intermolecular structure as shown in Fig. 3(a). In addition, the absorption intensity of the shoulder band in the TIIG-Bz/2-BBA assembly spectra for wires were strongly enhanced compared to those of the pristine TIIG-Bz samples. Such absorbance enhancement of the TIIG-Bz/2-BBA in the NIR region of the spectra was clearly shown even after excluding any scattering effect by measuring the spectra with an integrating sphere. The higher absorbance of the TIIG-Bz wires assembled with 2-BBA in the NIR region was consistent with our recent results of density functional theory calculations for conjugated polymer nanoparticles 54,55 . Such a red shift in the absorption spectra originates from enhanced intermolecular π − π interactions with energy level adjustments, and has been widely observed in thin films for organic optoelectronic devices or nanomaterials of conjugated polymers [56][57][58][59][60][61] . Furthermore, this enhancement in NIR absorption clearly indicated improved intermolecular π − π interactions in the direction of nanowire growth, which was significantly enhanced with increasing nanowire length, showing better aggregation.</p><p>Molecular assembly structures of TIIG-Bz and TIIG-Bz/2-BBA nanowires characterized by PXRD showed clearly different diffraction patterns depending on the incorporation of 2-BBA into TIIG-Bz assemblies. The 2D PXRD pattern of TIIG-Bz nanomaterials (right inset of Fig. 4(a)) shows ring patterns in the small-angle range and short dashes in the wide-angle range up to 2θ = 70° that were symmetric to the vertical, central line as the measurements were made over 360°. These short dashes are a typical feature of single crystals. On the other hand, the TIIG-Bz/2-BBA nanowires showed powder-like features, i.e., peaks with identical intensities along the Debye ring (right inset of Fig. 4(b)). Reflecting these characteristics of the 2D PXRD patterns, the circular averaged 1D profile of the TIIG-Bz/2-BBA nanowires (Fig. 4(b)) showed weakened peaks in the wide-angle range above 2θ = 30°, while that of TIIG-Bz nanomaterials showed peaks up to 2θ = 70° (Fig. 4(a)). It should be noted that identification of all crystallographic peaks in our PXRD data was difficult as both the TIIG-Bz nanomaterials and TIIG-Bz/2-BBA nanowires may not have been single-phase. As shown in the SEM image of Figure S1, TIIG-Bz-only nanomaterials were a mixture of wires, needles, and particles. In addition, the TIIG-Bz/2-BBA nanowires may have been a mixture of assemblies of TIIG-Bz, TIIG-BZ and one 2-BBA, and TIIG-Bz and two 2-BBA molecules due to inhomogeneous 2-BBA distribution. Detailed peak assignment is being performed by preparing single crystals or supramolecular assemblies with homogeneous morphology (this study will be published separately). However, at present, it is obvious that a new structure is developed upon the addition of 2-BBA into the TIIG-Bz assembly, as shown in the left inset of Fig. 4. In the inset 1D PXRD pattern (below 2θ = 10°), TIIG-Bz nanomaterials showed five diffraction peaks at 2θ = 5.563°, 5.933°, 7.121°, 7.734°, and 9.023°. In comparison, new diffraction peaks (indicated by the arrows) were obvious in the 1D PXRD pattern of TIIG-Bz/2-BBA nanowires, where six diffraction peaks appeared at 2θ = 5.563°, 5.716°, 5.933°, 7.146°, 7.261°, and 9.023° (Figure S2). The diffraction peaks of both TIIG-Bz and TIIG-Bz/2-BBA assemblies were assigned to a triclinic lattice structure (Figure S2 S1) increased with the addition of 2-BBA into the TIIG-Bz assembly. The PXRD results indicated that 2-BBA molecules in TIIG-Bz/2-BBA nanowires limit the growth of TIIG-Bz-only crystals and make the crystal structure of the original TIIG-Bz assembly a bit larger. As shown in the SEM image in Fig. 3(b) and (c), it seems that the addition of 2-BBA is advantageous for 1D nanowire growth of TIIG-Bz assembly.</p><p>The existence of both XB and CB in TIIG-Bz/2-BBA nanowires was confirmed by analyzing XPS spectra, as shown in Fig. 5. The changes of the binding energies in the XPS spectra for each atom and compound are summarized in Table 1. XB and CB are intermolecular interactions between an electron donor and an electron accepter that partially give and take electron pairs. Bromine or chalcogen atoms that have the electron-deficient σ hole can act as the electron accepter in this interaction; these elements can be bound to electron-rich oxygen in carbonyl groups and partially receive electrons via XB and CB. As a result, the electrons of these elements are increased and the binding energies of each electron become weaker. The decrease in the binding energy of the bromine atom in 2-BBA was observed upon assembly of 2-BBA with TIIG-Bz, as shown in Fig. 5(a). The binding energy of bromine 3d 5/2 in 2-BBA was 70.86 eV and that of the TIIG-Bz/2-BBA nanowires decreased by 1.60 eV to 69.26 eV, which is a characteristic of XB 62,63 . In addition, the binding energies of the chalcogen atom, sulfur in TIIG-Bz, 2p 3/2 , and 2p 1/2 decreased from 164.02 and 165.20 to 163.94 and 165.12 eV by 0.08 eV, respectively, as shown in Fig. 5b. The larger change in binding energies of bromine compared to sulfur was due to the stronger binding of XB than CB as the σ hole of the bromine atom is more positive than that of sulfur; this is consistent with a recent report related to the XB mechanism 62 .</p><p>On the other hand, the binding energies of N 1 s in both TIIG-Bz and TIIG-Bz/2-BBA nanowires were the same (399 eV), as shown in Fig. 5(c). This result is explained as follows. It was shown previously that the electron donor of XB and CB is usually a non-covalent electron pair of nitrogen 30,64,65 . However, in TIIG-Bz, the non-covalent electrons of nitrogen are delocalized in the thienoisoindigo moieties and are oriented perpendicular to the conjugated plane. In addition, ethyl hexyl chains sterically hinder the close association of the nitrogen atom with 2-BBA. Hence, the nitrogen in TIIG-Bz is unable to bind with electron acceptors via XB or CB. Instead of nitrogen, electron-rich oxygen in the aromatic carbonyl of TIIG-Bz can contribute to XB or CB as electron donors. However, it was difficult to confirm the effect of the addition of 2-BBA in the TIIG-Bz composite using the O 1 s binding energy spectrum due to the presence of multiple external peaks, including methanol or water (~534 eV), carbon dioxide (~532 eV), and the Si substrate (~532 eV). Figure 5(d) shows peaks at 530.76 eV that were attributed to contributions from O 1 s electrons in the aromatic carbonyls of TIIG-Bz. The strongest peak for both TIIG-Bz and TIIG-Bz/2-BBA nanowires was at 532.14 eV and was assigned to the Si-O peak from the SiO2 wafer substrates. Noticeably, the binding energies of O 1 s electrons in TIIG-Bz increased by 0.08 eV (from 530.76 eV to 530.84 eV) where their intensities significantly decreased, indicating electron donation from O 1 s electrons to XB or CB. Meanwhile, O 1 s electrons in the aliphatic carbonyl group of 2-BBA should have a binding energy at 532.5 eV 66,67 . However, their binding energy increased by 0.46 eV to 532.96 eV when 2-BBA was assembled with TIIG-Bz (Fig. 5(d)). One of the most plausible explanations for these peak changes in the O 1 s spectra is that oxygen in the aromatic carbonyl of TIIG-Bz interacts with the σ hole in bromine of 2-BBA via XB, while the other oxygen in the aliphatic carbonyl of 2-BBA reacts with the σ hole in sulfur of TIIG-Bz via CB. Thus, XPS characterization suggested that one TIIG-Bz and two 2-BBA molecules were associated with each other via both XB and CB in supramolecular assemblies of nanowires, forming slipped one-dimensional stacks 68 , as schematically illustrated in Fig. 1.</p><p>The configuration of one TIIG-Bz and two 2-BBA molecules in supramolecular assemblies (as shown in Fig. 1) was indirectly verified by measuring charge transfer efficiencies of OFETs based on thin films of TIIG-Bz and 2-BBA mixtures with various mixing ratios from 1:1 to 1:5. The devices had a BG/TC structure. Figure 5(a) and (b) show the output and transfer curves of an OFET based on a pristine TIIG-Bz thin film with a low hole mobility of 2.34 × 10 −4 cm 2 /Vs. In comparison, the output and transfer curves of a thin film OFET fabricated with a 1:2 molar mixing ratio of TIIG-Bz to 2-BBA (Fig. 6(c) and (d)) exhibited the highest hole mobility of 6.39 × 10 −4 cm 2 / Vs (about three times higher when we measured the hole mobilities of the OFETs with the different mixing ratios (Fig. 6(e) and Table 2; average values from seven devices); representative transfer and output curves are shown Fig. S3. This optimal molar mixing ratio was used to fabricate thin films of TIIG-Bz and 2-BBA, which clearly indicated that the TIIG-Bz/2-BBA film was the most well-ordered and the structure with one TIIG-Bz and two 2-BBA molecules was optimum for charge carrier transfer as TIIG-Bz has two positions available for interaction with 2-BBA via XB and CB. It should be noted that the assembly structure of the pristine TIIG-Bz film was not significantly changed by mixing it with 2-BBA for spin coating the film. GIXD profiles of the pristine TIIG-Bz film and TIIG-Bz/2-BBA with a 1:2 mixing ratio (Fig. S4) showed identical diffraction peak positions, indicating no significant differences in the lattice structures. However, the profile of the TIIG-Bz/2-BBA film showed stronger ordering in the assembly structure than that of the pristine TIIG-Bz film. These results suggested that the spin-coating time was not sufficient to produce a new lattice structure by the assembly of 2-BBA with TIIG-Bz, while the addition of 2-BBA enhanced structural ordering of TIIG-Bz assembly. The enhanced ordering at the optimal ratio of 1:2 strongly supports the hypothesis that two 2-BBA molecules interacted with one TIIG-Bz molecule. We propose that simultaneous XB and CB bonding between TIIG-Bz and 2-BBA increased the overall area of the conjugated plane, resulting in enhancement of the intermolecular transfer of charge carriers via the strongly ordered molecular structure. In the case of higher 2-BBA contents (i.e., the 1:3, 1:4, and 1:5 ratios), it also seemed that unbounded 2-BBA acted as an impurity on the TIIG-Bz/2-BBA complex and interfered with the formation of well-ordered complexes, resulting in reduced charge transfer mobility. The applicability of supramolecular nanowire assemblies based on TIIG-Bz and 2-BBA was demonstrated by fabricating organic nanowire FETs. We fabricated OFETs using both nascent TIIG-Bz nanowires and TIIG-Bz/2-BBA nanowires, and investigated the intrinsic charge transport properties. We used the BG/TC device structure for facile fabrication, as schematically illustrated in Fig. 7(a) and shown by the SEM image in Fig. 7(b). The basic transistor parameters for the devices are summarized in Table 2. The OFET based on the TIIG-Bz nanowires showed a hole mobility of 3.50 × 10 −3 cm 2 /Vs, about one order of magnitude higher than OFETs based on the pristine TIIG-Bz thin films (2.34 × 10 −4 cm 2 /Vs), as shown in the representative transfer curve of the TIIG-Bz nanowire OFET device (Fig. 7(c)). The nanowires in the OFET contributed to increasing device performance by improving hole transport mobility via the nanowires compared to the thin-film-based OFET. The improved mobility was due to one-directional assembly causing the holes to move directly to the cathode, as is widely accepted 3,4,9 . The OFET based on TIIG-Bz/2-BBA nanowires showed an even higher hole mobility (9.34 × 10 −3 cm 2 /Vs), three times higher than that of the OFET based on TIIG-Bz nanowires, as shown in Fig. 7(d) and Table 2. The highest hole transfer mobility was 0.01146 cm 2 It should be noted that there was excess 2-BBA in the chloroform solution, ten times more than the molar content of TIIG-Bz in the preparation of TIIG-Bz/2-BBA nanowires. However, the excess 2-BBA in the nanowire preparation process did not deteriorate the device characteristics, but rather, significantly improved the mobility. This suggests that the excess 2-BBA available for dissolution in the good solvents (chloroform and methanol) during bisolvent phase transfer process was excluded from the formation of TIIG-Bz/2-BBA composite nanowires without interfering with the formation of the well-ordered supra-molecular assembly. Overall, it was confirmed that the OFET based on TIIG-Bz/2-BBA nanowires assembled via XB and CB had significantly enhanced hole mobility compared to OFETs based on thin films and nanowires of TIIG-Bz; this was due to the supramolecular structure with preferred 1D orientation resulting in efficient charge carriers transfer. It should be mentioned that the charge transfer mobilities reported here were lower than or similar to those of TIIG-Bz-based OTFTs employing a different device structure, such as top gate/bottom contact and different dielectric materials 51 . However, these preliminary results offer</p><!><p>We demonstrated a supramolecular nanowire assembly of a TIIG-based organic semiconductor with 2-BBA and their FET device characteristics. The analysis of XPS data strongly supported the insertion of two 2-BBA molecules into the conjugated plane of one TIIG-Bz molecule via simultaneous XB and CB between TIIG-Bz and 2-BBA. This supramolecular arrangement was further supported by XRD analysis and the fact that the highest hole mobility of OFETs based on thin films was observed for a molar ratio of TIIG-Bz to 2-BBA of 1:2. When nanowires of TIIG-Bz were assembled via a bisolvent phase transfer method, an increased conjugated plane area and the insertion of 2-BBA enhanced intermolecular π − π interactions. The enhanced π − π interactions is more dominant than lateral packing by hydrophobic interactions of alkyl chains, promoting 1D crystal growth and narrow nanowire width. It was clear that the increased conjugated area of the TIIG-Bz/2-BBA supramolecular assembly allowed efficient hole transport in the crystallization direction, although the lattice structure of the original TIIG-Bz assembly become larger with incorporation of 2-BBA. The formation of TIIG-2BBA supramolecular structure suggests a new method simultaneously using XB and CB to control the self-assembled morphology and crystalline feature of organic semiconductors with increasing intermolecular interaction, improving crystallization, and achieving high charge transport mobility in OFETs.</p><!><p>Materials. TIIG-Bz (MW = 650.94 g/mol) was synthesized using a Pd-catalyzed Suzuki coupling reaction between (E)-2,2′-dibromo-4,4′-bis(2-ethylhexyl)-[6,6′-bithieno [3,2-b]pyrrolylidene]-5,5′(4 H,4 H′)-dione and phenyl boronic acid. Full details of the synthesis and characterization were published in our previous work 52 .</p><p>2-BBA (MW = 185 g/mol) were purchased from Alfa Aesar (United Kingdom) and used as received. Chloroform and methanol were obtained from the Dae-Jung Reagent Chemical Company (South Korea). Preparation of TIIG-Bz and 2-BBA nanowires. TIIG-Bz and 2-BBA nanowires were prepared via a bisolvent phase transfer method, as schematically illustrated in Fig. 1, following a published procedure 28,32,53 . TIIG-Bz (1.95 mg; 3 μmol) and 2-BBA (5.55 mg; 30 μmol, 10 times the molar content of TIIG-Bz) were individually dissolved in chloroform (5 mL) in two separate vials. We placed 0.5 mL of each solution in a 70 mL vial and sonicated it for 10 min to ensure complete mixing. Then, 30 mL of a methanolic 2-BBA solution with the same 2-BBA concentration as the mixed chloroform solution (0.555 mg/mL CHCl 3 ) was slowly and carefully added to the chloroform mixture containing TIIG-Bz and 2-BBA to maintain separated methanol (top) and chloroform (bottom) phases. The bi-solvent mixture was set aside for 16 h and nanowires of TIIG-Bz and 2-BBA self-assembled at the interface during diffusive mixing of the two solvents due to low solubility of TIIG-Bz. The prepared nanorods were filtered through a 0.2 μm membrane and re-dispersed in methanol to remove any residual 2-BBA.</p><p>Device fabrication. The organic nanowire FETs were fabricated by spreading 0.5 mL of the nanowire methanol solution over an n-doped Si/SiO 2 substrate. Shadow masks yielding a channel width of 1500 μm and channel length 100 μm were overlaid on the nanowires in the vertical direction of the channel and gold electrodes with a thickness of 140 nm were deposited to complete the bottom-gate top-contact (BG/TC) devices. For comparison, BG/TC OFETs based on thin films of only TIIG-Bz and TIIG-Bz/2-BBA mixtures were fabricated by spin casting (1500 rpm, 30 s, ramp-up speed: 0.1 sec) a 2.5 mg/mL TIIG-Bz chlorobenzene solution and TIIG-Bz/2-BBA chlorobenzene solutions with different mixing ratios onto Si/SiO 2 substrates. For a fixed amount of TIIG-Bz (2.5 mg/mL), five different 2-BBA contents were mixed at molar ratios from 1:1, 1:2, 1:3, 1:4 and 1:5 (0.71, 1.42, 2.13, 2.84, and 3.55 mg/mL of 2-BBA, respectively). The TIIG-Bz films were then annealed at 80 °C for 20 min, followed by deposition of 70-nm-thick gold electrodes via thermal evaporation through a metal shadow mask, yielding a channel length and width of 150 μm and 1500 μm, respectively. The field-effect mobility was determined in the saturation regime using the following relationship.</p><p>where I DS is the saturation drain current, L is the channel length, W is the nanowire width, C is the capacitance (~35 nF/cm 2 ) of the SiO 2 dielectric (100 nm), V G is the gate bias, and V th is the threshold voltage. The device performance was evaluated in air using an HP4156A Precision Semiconductor Parameter Analyzer (Agilent, U.S.A) [69][70][71] .</p><p>Characterization. The optical properties of TIIG-Bz and its nanowire assemblies with 2-BBA were examined with an ultraviolet-visible (UV-Vis) spectrometer (V-670, JASCO, USA). The UV-vis-NIR absorption spectra were also obtained using a JASCO V-670 UV-vis-NIR spectrophotometer equipped with a 60 mm BaSO 4 -coated integrating sphere, to prevent absorption spectra distortion due to light scattering by the nanowires. The measurements of UV-vis-NIR absorption spectra were carried out in a 1-cm path-length quartz optical cell. The polymer nanowire solution was dispersed in methanol (spectroscopy-grade), and ultrasonication was applied for 10-20 secs before the absorption measurements. A field-emission scanning electron microscope (FE-SEM; sigma, Carl Zeiss, USA) and an atomic force microscope (AFM; XE-100, PSIA, South Korea) were used to observe the morphologies of the nanostructures. Structures of the TIIG-Bz assemblies with 2-BBA were analyzed in detail using PXRD and GIXD experiments at a synchrotron facility (PLS-II 6D UNIST-PAL beamlines at Pohang Accelerator Laboratory, South Korea). For PXRD measurements, dry samples of TIIG-Bz and TIIG-Bz/2-BBA assemblies were placed into polyimide capillary tubes and continuously rotated during the measurements. For GIXD measurements, methanol dispersions of the assemblies were dropped onto slices of silicon wafers and dried. The molecular spacing and packing orientation relative to the substrates were characterized using GIXD profiles. XPS was carried out using an AXIS Ultra DLD instrument (Kratos, U.K.) in an advanced in-situ surface analysis system (AISAS; KBSI, Korea) operating at a base pressure of 1.6 × 10 −10 mbar at 300 K with a monochromatic Al Kα line at 1486.69 eV. The samples were prepared on a Si wafer substrate, or a Si wafer substrate coated with a 140-nm-thick gold layer, which were electrically grounded to the sample stage. The binding energy scales were calibrated by the C 1 s core level position at 284.8 eV as an internal reference and the Fermi edge of a gold standard. Survey and narrow spectrum scans were obtained with analyzer pass energies of 160 and 40 eV, respectively at 150 W. In order to separate the chemical bonding states in the spectra, the spectral line shape was simulated using Casa XPS software using a Shirley background and a GL (30) line shape (70% Gaussian, 30% Lorentzian).</p>

Scientific Reports - Nature

Following Particle-Particle Mixing in Atmospheric Secondary Organic Aerosols by Using Isotopically Labeled Terpenes

Atmospheric fine particles contain thousands of organic compounds. Natural compounds from trees are often terpenes, consisting of multiple isoprene units, which when oxidized produce hundreds of poorly understood product compounds, many of which have extremely low vapor pressures and partition to particles. The interactions of these compounds control many particle properties, but it is difficult to distinguish them from each other. By synthesizing isotopically labeled terpenes, we were able to follow the interactions of individual particles with precision.C. Mixing SVOCs gas particle exchange through evaporation and condensation!

following_particle-particle_mixing_in_atmospheric_secondary_organic_aerosols_by_using_isotopically_l

INTRODUCTION<!>The Bigger Picture<!>RESULTS AND DISCUSSION<!>Mixing of SOA from Limonene and Toluene Oxidation<!>Mixing of SOA from Monoterpenes<!>Conclusion<!>Synthesis of Deuterated a-Pinene<!>Two-Chamber Setup<!>Single-Particle Mass Spectrometry<!>SUPPLEMENTAL INFORMATION

<p>Probing changes in the chemical composition of suspended nanoparticles upon chemical exchange that may arise from mixing different types of aerosols is a major challenge. This difficulty is caused by the need to somehow mark, ideally without the use of external labels, the starting aerosol populations such that their constituents, and exchanges among them, can be uniquely identified in individual particles.</p><p>Although deuterium-labeling offers, in principle, the opportunity to probe chemical exchange among aerosol particles formed from organic compounds with a reasonably low level of invasiveness, doing so has been hampered by a lack of isotopologs other than those that are commercially available. Here, we have overcome this hurdle by using unlabeled and deuterium-labeled precursors to generate and characterize secondary organic aerosol (SOA), a class of aerosols made from the chemical oxidation and reaction of vapors. SOA is a class of atmospheric constituents that are among the least understood components in the climate system. 1,2 The particles, which have diameters of a few hundred nanometers or less, are suspended in air and act as individual nano-containers, among which we monitored exchange by using single-particle mass spectrometry with D:H readout. This approach is advantageous because SOA mass spectra from different terpenes are quite similar. However, by synthesizing isotopically labeled precursor molecules, we were able to generate SOA populations whose mass spectra are clearly distinguishable.</p><!><p>The exchange of constituents between distinct types of aerosols is relevant to many processes important to atmospheric chemistry, combustion, bio-threat detection, and consumer-product formulations. However, because of the high similarity of aerosol mass spectrometer signals, it is difficult to distinguish between different aerosol populations and to track constituent exchange. We have overcome this hurdle by synthesizing deuterium-labeled terpenes as precursors for secondary organic aerosols and studying mixing driven by semivolatile vapor exchange with particles formed from other unlabeled terpenes as well as toluene. We found that particles from isoprene and a-pinene ozonolysis absorbed vapors rapidly. Particles from limonene ozonolysis showed slower exchange, and particles from b-caryophyllene ozonolysis showed limited exchange. Our results show that molecular exchange among particles from terpene oxidation becomes slower and less extensive as the precursor carbon number increases.</p><p>As shown below, our work provides detailed compositional information needed to address the critical issue of how different particles and products from different precursors interact with one another. Interaction and potentially chemical exchange may be limited by volatility, 3,4 non-ideality, 5 or diffusion, depending on the particle viscosity, [6][7][8][9] which in turn may vary with relative humidity (RH) and temperature. 9,10 We therefore probed how particle-particle interactions, and chemical exchange among particles via semi-volatile partitioning, are modulated by conditions of varying RH and temperature. We used isotopic labeling of SOA by synthesizing selectively deuterated a-pinene, the most common biogenically emitted terpene over the boreal forest ecosystem, 11 to explore the interactions of aerosols derived from the oxidation of various terpene precursors emitted in the natural environment. Yet, although biogenic volatile organic compound (VOC) emissions dominate global budgets, biogenic SOA formation is also strongly correlated with the presence of anthropogenic pollutants. 12,13 This strong correlation indicates that an as yet undetermined chemical or physical mechanism couples biogenic precursors and anthropogenic perturbations to atmospheric chemistry. Therefore, we also prepared SOA from commercially available deuterated toluene, a well-known marker for combustion because of its anthropogenic activity, to study interactions among and the evolution of SOA populations derived from anthropogenic and biogenic emissions. This model, although idealized, is to explore possible consequences of mixing aerosol-containing outflow from urban or industrial centers with pristine background air. 14 Although many studies rely on rheology to infer diffusivity within SOA particles, 15,16 we directly investigated mixing driven by semi-volatile vapor exchange between different SOA populations. The timescales in our mixing experiments were on the order of several hours, comparable with atmospheric timescales of diurnal cycling and meteorological changes. In the experiments, we formed separate populations from two precursors and characterized, by using H:D readout, the chemical composition of individual particles with highly sensitive single-particle mass spectrometers before and after intermixing those two populations. We observed the populations for hours in a large reaction chamber and varied RH and temperature to explore potential physical and chemical changes to the particles under conditions typical of those in the atmosphere.</p><p>The key science questions that this work addresses are (1) whether SOA from different sources is readily miscible, (2) whether particles formed from the oxidation of various terpenes show significant limitations to diffusion and thus equilibration, and</p><p>what fraction of the SOA formed from various terpenes is effectively semi-volatile by showing mobility between particles on a timescale of less than several hours. As mentioned above, the SOA precursors we used were deuterated a-pinene prepared in house as well as commercially available deuterated toluene, along with several non-labeled commercially available terpenes. The SOA systems in our study are very common model systems used to mimic SOA formation in chamber experiments. Parameterizations of those experiments inform organic-aerosol representations in chemical transport models. Furthermore, they are also the model systems that have received the most attention in recent research into glassy SOA.</p><!><p>We provide full experimental details in the Experimental Procedures. In brief, we formed two aerosol particle populations in separate smog chambers and then brought them into contact to measure the composition of the two populations by using two single-particle aerosol mass spectrometers. We left one population (the chamber population) where it was formed, and we transferred the second population (the probe population) into that chamber, as shown in Figure 1. Because the chamber population was largely unperturbed, these experiments most directly targeted the behavior of the probe particles.</p><p>We conducted a series of mixing experiments involving SOA formed from at least one terpene to explore volatility and diffusion limitations for a sequence of terpene-derived SOA varieties. To explore interactions among different terpenes, we synthesized isotopologs of a-pinene, as described below. In Table 1, we list the precursors, SOA mass concentration (in mg m À3 ), and oxygen-to-carbon ratio (O:C) of the chamber population and the probe population immediately before we brought the two populations into contact for all mixing experiments. To track evolution of composition of the particles, we used two single-particle mass spectrometers along with other particle and gas monitors. One of the mass spectrometers permits a new method, event-triggering aerosol mass spectrometry (ET-AMS), which enables particle detection at a much higher rate (up to 200 events per second) than our previous light-scattering single-particle method (LSSP-AMS); this greatly improved the time resolution of the mixing experiments. We started with an experiment in which we investigated mixing between SOA from limonene and SOA from deuterated toluene (99.5% purity; Cambridge Isotope Laboratories) as an example to demonstrate the process of a typical mixing experiment. Then, we systematically explored SOA from terpene precursors with increasing carbon number. Two SOA populations are generated separately as shown in (A) by either ozonolysis (terpenes) or photo-oxidation (toluene). The ''chamber'' population is formed by condensing SOA onto preexisting ammonium sulfate seed particles. The ''probe'' population is formed by nucleation followed by condensation. After both populations stabilize, the probe population is transferred into the main chamber (B). Any semi-volatile organic compounds (SVOCs) produced from SOA formation will partition between the gas and particle phases on the basis of their effective activity coefficients, whereas low-volatile organic compounds (LVOCs) will remain in the particle phase (C). Single-particle mass spectrometers and other instruments collect real-time data of particle and gas concentration and composition.</p><p>In Table 2, we summarize the mixing experiments and their essential outcomes. Subsequently we discuss each experiment in detail.</p><!><p>We began with an experiment on SOA produced from ozonolysis of limonene (limonene SOA) as the probe population and SOA produced from photo-oxidation of D 8 -toluene (D 8 -toluene SOA) as the chamber population shown in Figure 2. This is a natural extension of previous experiments addressing interactions of a-pinene SOA and D 8 -toluene SOA 7,8 but with a terpene precursor that forms more highly oxidized SOA. The high time resolution of ET-AMS reveals the dynamics of vapor uptake over shorter timescales than experiments using LSSP-AMS (which we present in Figure S4). This experiment serves as a model for all experiments presented in this paper, and all other experiments follow a similar sequence of events.</p><p>In Figure 2, we show time sequences of chamber RH and temperature (upper panel), time sequences of single-particle data (middle panel), and normalized ion signal intensity from both populations for different periods of the experiment (lower panel). For the middle panel, we plot the single-particle data on the y axis according to a simple cosine similarity score with respect to the mass spectrum for pure limonene SOA. We also set the symbol color according to the sum of two mass fragments, f(C 2 H 3 O + ) + f(CHO + ), which are major fragments of the limonene SOA and almost absent in D 8 -toluene SOA. The black and brown curves in the middle panel of Figure 2 show the organic mass fraction of limonene SOA in each population. We show the time series of the bulk mass measurement from AMS in Figure S3.</p><p>At t = 0, we transferred some limonene probe SOA to the main chamber, which we had pre-filled with the D 8 -toluene SOA chamber population. Two hours after contact, we added water vapor to the chamber, increasing the RH to 30%. At t = 3.5 hr, we raised the chamber temperature from 22 C to 35 C. We know from prior experiments as well as calculations that particle coagulation is negligible on the fewhour timescale of these experiments for the number concentrations we used; thus, the only way the two particle populations can gain material from the opposite SOA types is via vapor condensation.</p><p>The high time resolution of the ET-AMS data allows us to observe that the mass fraction of D 8 -toluene SOA in the limonene SOA probe particles jumped to 0.15 almost immediately after contact, but also that the mass fraction continued to rise by roughly 0.05 hr À1 for the next 3 hr. This gradual change in composition proceeded unabated when we humidified the chamber but settled to a steady-state value before we increased the chamber temperature.</p><p>We plot the normalized ion signal intensity of each population from different periods of the experiment in the lower panel of Figure 2, showing signal associated with limonene-SOA in gold and signal associated with D 8 -toluene SOA in blue. The representative periods are (1) before mixing, (2) after mixing under dry conditions, and (3) after humidification to 30% RH. We neglected the ion signal at m/z 44 in the ET-AMS spectra (shown in gray) because of a large interference from gas-phase CO 2 . During period 1, the two populations were cleanly (and physically) separated, but during periods 2 and 3, the blue (chamber) material invaded the probe particles, causing a large increase in CHO + and C 2 H 3 O + in the probe population. The yellow (probe) material also invaded the chamber population, although to a much lesser extent.</p><p>The condensation of D 8 -toluene SOA onto the limonene-SOA probe particles confirms that the chamber aerosol contained semi-volatile organics, consistent with our expectations for the D 8 -toluene SOA. 7,8 However, the particles themselves resisted uptake of semi-volatile compounds (even between particles formed from deuterated and non-deuterated toluene oxidation) until RH a 30%. 8 In this study, on the few-hour timescale of these experiments, ''semi-volatile'' in practice means constituents with saturation concentrations C* >1 mg m À3 . To the extent that the D 8 -toluene SOA forms an ideal solution with the limonene SOA, the 30% mass fraction of semi-volatile material that eventually transfers to the limonene SOA probe particles is consistent with the mass fraction of semi-volatile D 8 -toluene SOA being 0.3. The multiple time constants in the data suggest that several phenomena could contribute to the overall behavior; the slower uptake could be rate limited by diffusion into the limonene SOA probe particles.</p><p>The temperature dependence of these mixing results is potentially complex. Assuming that these mixtures are close to ideal, heating will enhance mixing by encouraging vapor exchange, resulting in an equilibrium state consisting of a single homogeneous population; this happens when populations consisting of docosane isotopologs are heated above the docosane melting point. 7 However, in practice there are several competing effects. Increasing the chamber temperature by 15 C will increase the saturation concentration of any given species by roughly one order of magnitude, 4,17 driving down the gas-phase saturation ratio of ''semi-volatile'' species and causing them to evaporate from all particles while at the same time causing less volatile constituents in the particles to evaporate and exchange between the populations. Heat will also soften the particles and drive activity coefficients toward 1, which tends to anneal the aerosol toward a uniform composition. However, heating will also decrease the RH and thus the water content of the particles. For this first system, overall we observed that heating caused a slight decrease in the exchanged mass fractions of each population, suggesting that evaporation of semi-volatile species has the most influence. The RH and temperature for this experiment are plotted in the upper panel. In the middle panel, each particle is plotted according to their similarity score to the aggregated mass spectrum of limonene SOA. The symbol-connected lines show the mass fraction of the limonene SOA in both populations, also denoted as the extent of mixing, for every 3-min duration. Each particle is color coded by f(C 2 H 3 O + ) + f(CHO + ), which are two major peaks in the limonene SOA mass spectrum (lower panel). The brackets in the middle panel indicate the time periods corresponding to the aggregated mass spectra from both populations plotted in the lower panels. Before t = 0, the two populations had distinct mass spectra. After limonene SOA was transferred into the chamber at t = 0, it lost C 2 H 3 O + and CHO + as a result of evaporation. At the same time, it progressively took up vapors from D 8 -toluene SOA, indicated by the C 2 D 3 O + and CDO + mass spectra signals that are negligible before mixing. Two hours later, we raised the chamber RH to 30% from 10%, and limonene SOA became roughly 30% D 8 -toluene SOA. At the end of the experiment, the chamber temperature increased to 34 C, evaporating more volatile vapors from the condensed phase, and reduced the extent of mixing. The ion signal at m/z 44 was neglected (shown in gray) because of large interference from gas-phase CO 2 + .</p><p>We generally focused on uptake of chamber SOA into the probe population. Quantitative assessment of probe aerosol vapor uptake into the chamber particles was complicated by the added effects of dilution as well as uncertain vapor losses during probe aerosol transfer, but this interaction is nonetheless informative. In this case, the D 8 -toluene SOA chamber population also showed some uptake of vapors from the limonene SOA but to a lower extent, with little sign of an RH or temperature effect. We recently showed that toluene SOA particles have diffusion limitations that will delay equilibration only for RH < 20%, 8 and so the relatively small uptake into these D 8 -toluene-SOA chamber particles at higher RH suggests that the limonene-SOA probe aerosol has a small semi-volatile content.</p><p>Overall, our results indicate that constituents from limonene SOA and toluene SOA form a relatively ideal solution, but they also suggest that the mixing timescale of limonene SOA is on the order of 3-4 hr. There is relatively little evidence that increasing the RH from 10% to 30% changes the behavior of the limonene-SOA particles. However, the limonene SOA particles behave differently from a-pinene SOA particles, showing multiple timescales where the a-pinene SOA particles take up semi-volatile D 8 -toluene products in a rapid step. 7,8 Mixing of SOA from Isoprene Having shown that we can use these mixing experiments to explore semi-volatile vapor uptake by SOA from terpenes, we now systematically explore the mixing behaviors of SOA from C 5 to C 15 terpenes. We started with SOA from ozonolysis of isoprene (C 5 H 8 ), which contributes up to 30% of global particulate organic matter. 18 Isoprene is the lightest terpene and thus should produce the most volatile and diffusive SOA with relatively low viscosity. It is thought that the majority of SOA derived from isoprene consists of oligomers, for example, from the reactive uptake of monomers such as isoprene epoxydiol (IEPOX) and isoprene hydroperoxide (ISOPOOH). 19,20 So it is unclear whether this will cause any different behavior in the isoprene SOA.</p><p>As shown in Figure 3, we transferred SOA probe particles formed via isoprene ozonolysis into a chamber containing SOA derived from D-toluene photo-oxidation. We held the chamber at a constant 22 C and 10% RH for the entire experiment. As the data show, the SOA probe particles from isoprene ozonolysis absorbed semivolatile vapors from the chamber SOA derived from D 8 -toluene almost instantly upon entering the chamber and reached a mass fraction of 25%-30%. For the remainder of the experiment, the semi-volatile species from D 8 -toluene oxidation that had diffused into the isoprene SOA probe particles gradually re-evaporated, presumably as a result of vapor loss to the chamber walls, slightly reducing their mass fraction in the isoprene SOA. We observed a similar behavior for carefully prepared two-component mixtures of oleic acid (semi-volatile with C = 6 mg m À3 ) and squalane (nearly non-volatile with 0.01 ( C ( 0.1 mg m À3 ). 21 Figure S5 shows histograms of f 30 (CDO + ) + f 46 (C 2 D 3 O + ) in the isoprene SOA probe particles for t < 0 (before contact) and for t = 0.5, 1.5, and 2.5 hr after contact. As with the bulk composition, f 30 (CDO + ) + f 46 (C 2 D 3 O + ), originally from D-toluene SOA, slightly decreased with time after contact in isoprene SOA, indicating again that the semi-volatile organics from the D-toluene SOA gradually reevaporate after the initial mixing. The diffusion of semi-volatile organics into the isoprene SOA particles appeared faster than diffusion into SOA particles from limonene, possibly because of the lower molecular weight of the constituents in the condensed phase. Previous studies have also shown evidence that even when the RH is low, mass-transfer limitations are unlikely in SOA produced from isoprene and OH radicals. 22,23 This indicates that SOA from isoprene is probably liquid-like with low viscosity.</p><!><p>Our central objective was to explore cross-mixing among different types of terpene SOA. Without isotopic labeling, this would be difficult for different terpene precursors because of the similarity of the mass spectra and completely impossible for the crucial experiments involving mixing of SOA from chemically identical precursors, where entropic mixing is the only potential driving force. By synthesizing D 6 -a-pinene and D 3 -a-pinene as described in the Experimental Procedures, we generated two terpene SOA populations with distinguishable mass spectra. In this way, we could test whether these different SOA systems interacted with each other and explore the potential for diffusion and other limitations to mass transfer.</p><p>In Figure 4A, we show LSSP-AMS data from a cross-mixing experiment between H-a-pinene SOA (H-pinene SOA, probe population) and D 6 -a-pinene SOA (D-pinene SOA, chamber population). Given that the D-H substitutions were the only chemical difference in the two precursors, we had every reason to expect the two populations to form an ideal solution. As the brown aggregated curve shows, the probe particles took up vapors from the chamber population relatively rapidly over the first hour. After that, the mixing remained more or less constant even when we raised the RH to 20% and then to 40%. The slightly higher chamber fraction at 40% RH could possibly have been caused by water-induced chemistry or waterinduced partitioning of semi-volatile organic compounds (SVOCs). 24 However, the data do not suggest that there were any substantial diffusion limitations within the particles but rather that approximately 25% of the a-pinene SOA was functionally For the remaining 2.75 hr, the SVOCs that were absorbed by isoprene gradually re-evaporated and were lost to the chamber wall, leading to a slightly increased fraction of isoprene SOA in the probe population. D 8 -toluene SOA remained essentially pure.</p><p>semi-volatile under these conditions. The chamber particles likewise showed a corresponding uptake of probe SOA, indicating that they absorbed a combination of vapors from the probe chamber and vapors from probe-particle evaporation. The relatively rapid partial equilibration in both directions is consistent with previous studies where a-pinene SOA particles did not show significant diffusion limitations even at low RH. 7,8,22 At the end of the experiment, we increased the chamber temperature to 34 C, which forced some semi-volatile species to partition to the gas-phase, decreasing the fraction of materials originally from chamber population in the probe particles.</p><p>In Figure 4B, we show a mixing experiment for a-pinene SOA and limonene SOA using LSSP-AMS measurements. Here, we produced D-pinene SOA by using a mixture of D 6 -a-pinene and D 3 -a-pinene with a volume ratio of 8:5. Similar to the experiment shown in Figure 2, the vapor uptake into the limonene SOA particles took place progressively, although it accelerated when we increased the RH to 30% at t = 3 hr. The mixing of a-pinene SOA into the limonene SOA particles was slower than the mixing of a-pinene SOA into a-pinene SOA particles. A consistent explanation is that the relatively more oxidized and thus more polar products from limonene ozonolysis are somewhat more viscous and therefore show some diffusion limitations. In addition, the uptake of limonene SOA into the deuterated a-pinene SOA chamber particles was significantly less than the corresponding uptake of unlabeled a-pinene SOA vapors, which is consistent with the higher oxidation state of limonene SOA constituents resulting in lower volatility than a-pinene SOA.</p><p>A B Mixing of SOA from a Sesquiterpene Lastly, we used SOA derived from b-caryophyllene as the probe population and mixed it with SOA from a terpene (D 6 -a-pinene) and SOA from toluene. b-Caryophyllene, a sesquiterpene with a molecular formula of C 15 H 24 , also has two double bonds and a higher molecular weight than monoterpenes. It has very high SOA mass yields, although with a somewhat lower overall carbon oxidation state than limonene SOA. 25,26 The SOA from b-caryophyllene is also significantly less hygroscopic. The hygroscopicity parameter k for b-caryophyllene SOA is about 100 times smaller than k for a-pinene SOA at 1% supersaturation (k is a linear single parameter of aerosol hygroscopicity and is proportional to the inverse of water activity, a w ). 27 In Figure 5A, we show LSSP-AMS data from an experiment in which we exposed b-caryophyllene SOA probe particles to D-a-pinene chamber SOA at 7% RH, followed by RH steps to 20% and then to 75%. The temperature stayed at 22 C throughout the experiment. When the chamber was at 7% RH, b-caryophyllene SOA took up a very small amount (z5%) of D 8 -toluene SOA vapor. When we increased the RH to 20%, the vapor uptake increased slightly. We subsequently increased the RH to 75% and waited for 2 hr. The mass exchange remained no more than 10%.</p><p>Similarly in Figure 5B, the extent of mixing between SOA from b-caryophyllene and SOA from D 8 -toluene was much less than the mixing between isoprene or monoterpene SOA and D 8 -toluene SOA populations. After 2 hr at 50% RH, the extent of mixing was still less than 10%. In addition, the chamber population showed a small but non-zero uptake of vapors from the b-caryophyllene SOA.</p><p>We know from previous experiments that the chamber SOA in both cases shown in Figure 5 contains semi-volatile organics with activities between 0.2 and 0.3, so we would expect significant uptake into an ideal solution with no diffusion limitations. It is possible that the reduced uptake of semi-volatile constituents into the b-caryophyllene probe particles is because they are highly viscous. However, a significant diffusion limitation at 75% RH seems unlikely, although the hygroscopic growth for longifolene SOA (another sesquiterpene with a single exo double bond) is very low at 75% RH. 28 Another possible explanation is that constituents in b-caryophyllene SOA do not form an ideal solution with constituents in toluene SOA and a-pinene SOA.</p><!><p>By preparing SAO from isotopically labeled (deuterated) and unlabeled a-pinene, we explored the transfer of molecules among nanoparticles suspended in air. This would have been impossible without the isotopically labeled precursor because of the similarities among the aerosol mass spectrometer signals from terpene SOA samples. In addition, the efficiency of our single-particle method allows us to resolve composition changes in multiple populations with high time resolution after they are brought into contact during mixing experiments.</p><p>Four different phenomena can drive or limit uptake to or exchange between particles, and the isotopologs are critical to deconvolving the effects. First, exchange can be limited simply because the constituents are barely volatile, but non-volatile vapors will condense under almost all circumstances. Second, immiscibility (non-ideality) can limit exchange even of semi-volatile constituents. Third, exchange and uptake can be driven in part by condensed-phase reactions, especially involving at least one relatively volatile gas-phase species. Fourth and finally, in-particle diffusion limitations (viscous particles) can inhibit both semi-volatile and reactive uptake by sustaining a strong activity gradient between the particle surface and its interior. Aerosols generated from isotopologs under identical conditions should form ideal mixtures with each other and reach reactive equilibrium as well. Thus, exchange is limited only by low volatility or high viscosity. Because water plasticizes viscous particles, 29 RH ramps resolve this final ambiguity.</p><p>Oxidation of the precursors investigated here generated particles composed principally of low-volatility constituents (C % 1 mg m À3 ; p % 10 À5 Pa). However, up to one-third of the particle composition consisted of more volatile constituents that readily diffused into the SOA particles under most conditions. Unlike SOA from toluene, which resists vapor exchange when dry, 7,8 there are no significant diffusive limitations in particles derived from isoprene and monoterpene oxidation, although SOA derived from limonene shows signs of some diffusion limitations when dry. Under the conditions used here, when semi-volatile uptake was uninhibited and nearly ideal, probe SOA particles took up roughly 20% by mass vapors from a-pinene oxidation and roughly 30% by mass vapors from toluene oxidation, consistent with earlier experiments. 7,8 Only particles formed from oxidation of b-caryophyllene showed less uptake, even at 75% RH. The high time resolution of our single-particle measurements shows that the particles derived from limonene and b-caryophyllene take up vapors from deuterated toluene slowly, i.e., over the course of 2-3 hr, until they reach near equilibrium. Yet, we also found that the limiting mass fraction is roughly 0.3 in limonene SOA and less than 0.1 in b-caryophyllene SOA.</p><p>Our results suggest that under most circumstances in the planetary boundary layer, semivolatile vapors are sufficiently diffusive in SOA particles for equilibration to occur on relevant atmospheric timescales and that particles of biogenic and anthropogenic origin will mix with each other quickly. However, the fresh SOA we studied here had a lower O:C than aged ambient SOA. 30 Future experiments should explore particles with a wider O:C range, as well as the mixing of SOA formed from higher-molecular-weight intermediate VOCs, which are also important precursor molecules for atmospheric SOA. 31,32 EXPERIMENTAL PROCEDURES</p><!><p>The two isotopically labeled a-pinene compounds used in this study were synthesized with previously published methods. 33 Both (À)-a-pinene-10,10,10-d 3 and (À)-a-pinene-9,9,9,10,10,10-d 6 were accessed from the common intermediate, nopinone, which was synthesized through the ozonolysis of b-pinene (Sigma-Aldrich). (À)-a-Pinene-10,10,10-d 3 and (À)-a-pinene-9,9,9,10,10,10-d 6 can be accessed in two and eight synthetic steps, respectively, from the nopinone intermediate. Both routes enabled access to 100 mg quantities of each isotopically labeled a-pinene compound. Compound purity and percentage deuteration based on nuclear magnetic resonance (NMR) integration was determined to be 98% and R99%, respectively, for both compounds. That is more than sufficient to ensure that the resulting SOA particles are chemically defined by the desired compound and that the resulting particle mass spectra are easily separated. In the Supplemental Information, we provide 1 H NMR spectra for the isotopically labeled synthesized compounds and unlabeled a-pinene.</p><!><p>We exposed particles derived from terpene oxidation to semi-volatile vapors containing oxidation products from other SOA sources (either terpenes or toluene) and investigated the vapor uptake over several hours at different RH. We formed SOA from terpenes by dark ozonolysis and from toluene by photo-oxidation under high NO x conditions typical of a polluted urban plume. In the ozonolysis chamber, the ozone concentration in the experiments ranged from 200 to 500 ppb. In the photo-oxidation chamber, the NO x concentration was about 500 ppb. We focused on the mixing behavior of SOA formed via oxidation of terpenes.</p><p>Figure 1 shows the setup of the mixing experiments. 7,8 In each mixing experiment, we generated two SOA populations separately in two containers, a 10 m 3 chamber and a nearby 7 m 3 chamber (Figure 1A). The population in the 10 m 3 chamber (the ''chamber'' population) formed via condensation on pre-existing ammonium sulfate seeds. The seed particles were generated by atomizing 1 g/L ammonium sulfate solution, and the aerosol flow was subsequently dried in a diffusion dryer to well below the efflorescence RH. The dry seed particles in the chamber had a modal diameter of 90-100 nm. The population in the 7 m 3 chamber (the ''probe'' population) formed through nucleation. The mass signals from the non-volatile ammonium sulfate seeds in the particles assisted us in separating the two populations when analyzing the data. A single sampling line connected both chambers to an instrument suite via a three-way valve. We used one or two aerosol mass spectrometers (Aerodyne Research) to measure the chemical composition both of the ensemble and of single particles. We also measured particles and conditions in the chamber by using a scanning mobility particle sizer (SMPS, TSI 3081), an ozone monitor, a NO x monitor, and a temperature and RH sensor. After both aerosol populations stabilized, meaning that changes in mass concentration were dominated by particle wall loss instead of new mass formation, we transferred a portion of the probe population into the 10 m 3 chamber by using two Dekati dilutors with output flow rates of about 40 lpm (Figure 1B). The mixing timescale of the main chamber was roughly 5 min. We observed the changing composition of individual particles within each aerosol population by using the single-particle mass spectrometers (Figure 1C). This allowed us to probe SOA mixing driven by SVOC exchange. SOA particles in our experiments had mobility diameters between 200 nm and 700 nm, and we used atmospherically relevant mass concentrations as shown in Table 1.</p><p>In all experiments, we formed one population by oxidizing an isotopically labeled precursor, generating particles with a unique mass spectrum. This included experiments with essentially identical SOA populations formed from the same precursor (i.e., a-pinene and isotopically labeled a-pinene). Analysis of these SOA populations is critical for establishing baseline aerosol properties under conditions where the SOA is completely miscible and where there is no reason to expect ongoing chemical reactions between reactive products, which may occur when chemically distinct populations are mixed. Our principal isotopically labeled precursors were synthesized D 6 -a-pinene and D 3 -a-pinene. 33 We also used commercially prepared D 8 -toluene (Cambridge Isotope Laboratories, D > 99.5%). We provide structures of deuterated a-pinene in Figure S1 and the NMR spectra of non-labeled a-pinene, D 3 -a-pinene and D 6 -a-pinene in Figure S2. Because of isotopic labeling, major fragments in the unlabeled SOA mass spectrum shifted from m/z 29 (CHO + ) and m/z 43 (C 2 H 3 O + ) to m/z 30 (CDO + ) and m/z 46 (C 2 D 3 O + ) in the labeled SOA mass spectrum.</p><!><p>We used two single-particle mass spectrometers in this study. Both are aerosol mass spectrometers built by Aerodyne Research. One is a light-scattering single-particle mass spectrometer, whose function has been described in detail elsewhere. 34,35 The other is an event-triggering aerosol mass spectrometer with a high-speed data acquisition card capable of triggering data acquisition on the basis of a signal pulse from an individual particle. In both instruments, particles enter a particle time-offlight region via an aerodynamic lens that focuses a beam of sub-micrometer particles onto a mechanical chopper with two 1% opening slits operating at 145 Hz. Under typical conditions, at most one particle is in the particle time-of-flight chamber during any given chopper cycle. At the end of the particle time-of-flight region, particles strike a 600 C tungsten vaporizer, and the resulting vapors are ionized by 70 eV electron ionization. Ions are then extracted and analyzed by a time-of-flight mass spectrometer (Tofwerk) with a mass resolving power (m/Dm) of 2,100 at 200 amu and high ion transmission efficiency. 36 In LSSP-AMS, a 405 nm laser intersects the particle beam at a right angle 16 cm downstream of the chopper, and the flight time between the chopper and the laser determines the particle aerodynamic size. Particles larger than 250 nm generate a sufficiently large scattering signal to trigger data acquisition for single-particle analysis. The final data product after processing by the Aerodyne software (Sparrow) contains the unit mass resolution mass spectrum of each particle. Mass spectra in all chopper cycles with a light pulse are downloaded and saved. Unwanted mass spectra are discarded in a post-processing step. These include chopper cycles containing no mass spectrum because the particle bounced off the vaporizer as well as chopper cycles containing mass spectra from more than one particle; truly coincident pulses are exceedingly rare at the particle concentrations we used.</p><p>In ET-AMS, the mass spectrometer runs continuously, and firmware on a fast data acquisition card evaluates an ion signal threshold within user-defined regions of interests (ROIs). Each ROI corresponds to a mass-to-charge range and its ion signal thresholds. Users also specify logical ''AND'' or ''OR'' filters to combine the ROI into a single Boolean trigger. If an event passes the threshold setting, its spectrum is downloaded. In addition to the mass spectrum at the triggering time, two pretrigger spectra and five post-trigger spectra are also downloaded, ensuring that we capture the entire particle and help us establish baseline values. Particle aerodynamic size is calculated from the flight time between the chopper and the ionizer. The data are processed by Tofware developed by Tofwerk.</p><p>For ET-AMS measurements, because of the fast data acquisition card and the ROI setting, ''filtering'' to select signals is conducted online. The advantages of ET-AMS are that (1) there are less data per particle because fewer spectra per particle are recorded and (2) no time is wasted downloading unwanted data. Data-acquisition efficiency is thus much higher in ET-AMS than in LSSP (as can be seen in the figures). Furthermore, because ET-AMS does not rely on scattered light to trigger data acquisition, it is not limited to particles larger than 250 nm. Particles smaller than 100 nm can be detected as long as they generate a sufficient ion signal. However, ET-AMS has an intrinsic chemical bias (or selectivity) in particle collection because it downloads only data satisfying the specified ROI thresholds, whereas LSSP obtains data on the basis of a physical light-scattering signal. In our study, we set three different ROIs (specified for each experiment described below) in order to minimize ET-AMS sampling bias. Quantitative Single-Particle Analysis We used the experiment presented in Figure 2 as a model for the rest of the experiments. In the middle panel of Figure 2, we plotted the single-particle data on the y axis according to the cosine similarity score with respect to the mass spectrum for pure limonene SOA. Electron ionization in the mass spectrometers produced only a few ions (at least six) per particle, resulting in single-particle spectra that are not statistically meaningful. However, our objective for this first stage of the data analysis was to separate the particles into two groups: the chamber and probe populations. This is similar to a clustering analysis except that it involves forced external mixture generating two clusters with known general properties. This task is simplified when one population also has a unique non-volatile seed, as is the case with ammonium sulfate in the D 8 -toluene SOA here.</p><p>Once we separated the particles into two populations, we then aggregated the spectra by adding the individual-particle spectra within every 3-min interval and only afterward dividing by the total ion signal to obtain a normalized spectrum for each population. We quantitatively analyzed the aggregated mass spectra for their composition (specifically the relative content of the two kinds of pure SOA) via a simple linear regression to reference mass spectra from the pure populations. We assumed that the (normalized) aggregated mass spectrum of mixed particles at any given time, f p , can be described as a linear combination of the (normalized) mass spectra for the two pure particle populations, f H and f D (in each case this applies to the organic fraction of the mass spectrum and not any inorganic seeds), with contributions a H and (1 À a H ):</p><p>An identical equation applies for the chamber population, f c (p for probe, c for chamber). The black and brown curves in the middle panel of Figure 2 show the results of this composition analysis by plotting the organic mass fraction of limonene SOA in each population.</p><!><p>Supplemental Information includes five figures and can be found with this article online at https://doi.org/10.1016/j.chempr.2017.12.008.</p>

Chem Cell

Overall water splitting by Pt/g-C₃N₄photocatalysts without using sacrificial agents

We report the direct splitting of pure water by light-excited graphitic carbon nitride (g-C 3 N 4 ) modified with Pt, PtO x , and CoO x as redox cocatalysts, while pure g-C 3 N 4 is virtually inactive for overall water splitting by photocatalysis. The novelty is in the selective creation of both H 2 and O 2 cocatalysts on surface active sites of g-C 3 N 4 via photodeposition triggering the splitting of water for the simultaneous evolution of H 2 and O 2 gases in a stoichiometric ratio of 2 : 1, irradiated with light, without using any sacrificial reagents. The photocatalyst was stable for 510 hours of reaction.

overall_water_splitting_by_pt/g-c₃n₄photocatalysts_without_using_sacrificial_a

<!>Conclusions

<p>Using photocatalysts to produce hydrogen sustainably by water splitting is the "holy grail" in modern science. Over the past 40 years, inorganic semiconductors, such as metal oxides and metal (oxy)nitrides, have been utilized as photocatalysts for hydrogen production. [1][2][3][4][5][6][7][8] However, direct water splitting in a wireless powder photocatalytic system to produce gaseous hydrogen and oxygen has not yet been achieved using conjugated polymers (CPs). These materials have already shown great promise for use in organic electronics and photovoltaic devices, such as solar cells, light-emitting diodes, and eld-effect transistors, due to their good processability and tuneable electronic structures. [9][10][11][12][13] The key challenge to using pristine CPs for direct water splitting is the insufficient hopping charge transport of the chains (usually below 10 À4 cm 2 V À1 s À1 ) and a poor stability in water and under light irradiation. 12 Increasing the structural dimensions of the CPs (e.g., from 1D chains to 2D architectures) is desirable because the hole mobility is greatly increased (up to 0.1 cm 2 V À1 s À1 ) by the remarkably reduced binding energies of the Frenkel-type excitons and the robust stability of the 2D extended p-conjugated units. 14 However, further progress in direct water splitting by CPs will rely on breakthroughs in combining stable CP light transducers with suitable redox cocatalysts (usually noble metals) to promote charge separation and to reduce charge build-up on the polymer surface to prevent photocorrosion. Indeed, the promise of this type of system has been demonstrated by the successful development of 2D graphitic carbon nitride (g-C 3 N 4 ) polymer and metal-based redox cocatalyst systems for CO 2 reduction, organic synthesis and water half-splitting reactions using sacricial reagents. [15][16][17][18][19][20][21][22] In contrast, it is difficult to achieve overall water splitting without using sacricial reagents because it depends not only on a rational chemical synthesis to tune the textural properties of the polymer but also on a rational design of the composite to control the reaction kinetics on the polymer surface. [23][24][25][26][27] Photocatalytic water splitting by a prototypical g-C 3 N 4 polymer was shown to be thermodynamically possible because the C 2p and N 2p orbital bands straddle the water splitting redox potentials, [15][16][17][18][19][20][21][22][28][29][30][31][32][33][34] but pure g-C 3 N 4 is typically limited by sluggish kinetics in photocatalyzing overall water splitting due to a lack of surface redox active sites. By optimizing the g-C 3 N 4 bulk and morphological properties and employing suitable redox cocatalysts (e.g., Pt for H 2 evolution and Co(OH) 2 for oxygen evolution), activities for the water half-splitting reactions (water reduction and oxidation) can be dramatically increased. [28][29][30][31][32][33][34] Therefore, if the appropriate water redox cocatalysts are simultaneously deposited on g-C 3 N 4 , pure water splitting to produce gaseous hydrogen and oxygen could be achieved. However, the rough deposition of cocatalysts by traditional chemical reduction (e.g., H 2 and NaBH 4 ) cannot fully amplify the activity. Besides, the densely stacked graphitic layer also causes trouble for charge separation and migration due to a long bulk diffusion distance, resulting in a low photocatalytic quantum efficiency. 15 It is advisable to reduce the diffusion distance by rational synthesis of a g-C 3 N 4 nanosheet together with suitable cocatalyst modication to achieve water splitting. Up to now, direct water splitting photocatayzed by g-C 3 N 4 CPs in the absence of sacricial reagents has never been realized and still remains a signicant basic science challenge. Here, we demonstrate that light-excited g-C 3 N 4 CPs can induce a one-step water splitting reaction via a four-electron pathway to generate gaseous H 2 and O 2 in a stoichiometric molar ratio of 2 : 1 when their morphology is modied and the reaction kinetics are improved by modication with Pt, PtO x , and CoO x via photodeposition. The optimal g-C 3 N 4 -based nanocomposite had a turnover number of 3.1 moles of H 2 and O 2 per mole of g-C 3 N 4 photocatalyst for the overall water splitting reaction. The nanocomposite was stable in water and under light irradiation.</p><p>The g-C 3 N 4 polymers used for photocatalytic water splitting were typically prepared by thermally polymerizing urea into heptazine units at 550 C which pack together like graphitic crystals. This structure was conrmed by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and Raman spectroscopy (Fig. S1 †). 15,[35][36][37] The g-C 3 N 4 optical properties measured by UV-vis diffuse reection spectroscopy (DRS) were characteristic of a semiconductor; g-C 3 N 4 had an optical absorption edge at 442 nm due to the excitation of electrons from its valence band to its conduction band (Fig. 1a). The conduction band minimum (CBM) and valence band maximum (VBM) of the g-C 3 N 4 semiconductor were determined to be À1.31 V and 1.49 V (vs. NHE, pH ¼ 7), respectively, from electrochemical Mott-Schottky plots (Fig. 1b and c), where an estimated at potential was directly used as the conduction band potential. Density functional theory (DFT) calculations revealed that the band gap was 2.56 eV with the CBM and VBM located at À1.0137 and 1.5505 V (vs. NHE, pH ¼ 7), respectively, which enables g-C 3 N 4 to act as a redox shuttle for the water splitting reaction (Fig. S2 †). This calculated band gap is consistent with the experimental data and further demonstrates that in theory, g-C 3 N 4 could be used to split water.</p><p>First, the effects of g-C 3 N 4 morphology on the photocatalytic activity were investigated. We prepared three types of g-C 3 N 4 using dicyandiamide (DCDA), ammonium thiocyanate (ATC) and urea as precursors. The results showed that aer in situ photo-deposition with Pt, the urea-derived g-C 3 N 4 exhibited signicant photocatalytic activity for the overall water splitting reaction, while the other samples were inactive for overall water splitting (Table S1 †). It should be noted that all pure g-C 3 N 4 polymers showed no activity for overall water splitting in the absence of cocatalysts, implying that surface kinetic control using Pt species was indispensable to achieve overall water splitting by g-C 3 N 4 based photocatalysts. N 2 sorption measurements revealed that the DCDA-and ATC-derived g-C 3 N 4 samples had smaller surface areas than the urea-derived samples (ca. 10 m 2 g À1 vs. 61 m 2 g À1 ). However, mpg-C 3 N 4 with a surface area of ca. 67 m 2 g À1 also exhibited no water splitting activity. This indicated that surface area was not the major factor in controlling the water splitting activity and the splitting of water on densely stacked g-C 3 N 4 polymers was indeed very difficult to achieve. To better understand the real mechanism of water splitting on the so surface of the CPs, we characterized the morphology of the above different polymers. TEM images of DCDA-and ATC-derived g-C 3 N 4 and mpg-C 3 N 4 samples revealed densely stacked polymer layers, which were very different from the silk-like thin nanosheets of the urea-derived one (Fig. S3 †). The fast evolution of O in the form of CO 2 or CO could accelerate the deamination rate. Thus, the texture, morphology and electronic properties of the CNU samples were optimized, and contributed to creating the active Pt/g-C 3 N 4 photocatalysts for overall water splitting. Evidently, accelerated charge separation and migration on the nanosheets can be obtained in comparison with densely stacked graphitic layers, which is elucidated well by the corresponding large decrease of PL emission intensity (Fig. S4 †). The nanosheet structure can also be certi-ed by an AFM experiment. As shown in Fig. 2a, the thickness of the nanosheet is determined as $2 nm. One can now easily conclude that the ultrathin 2D geometry of urea-derived g-C 3 N 4 is crucial for achieving overall water splitting as demonstrated by the fact that g-C 3 N 4 samples prepared from urea at different temperatures all have remarkable water splitting activities (Fig. S5 †) due to their similar thin nanosheet structures (Fig. S6 †). The CNU samples prepared at 550 C showed optimum activities. This is because when the temperature is lower than 550 C, the heptazine cycle doesn't completely form, while partial decomposition occurs when the temperature is higher than 550 C. Both of these two aspects may generate inactive CNU samples. DCDA-and ATC-derived g-C 3 N 4 and mpg-C 3 N 4 samples revealed densely stacked polymer layers, and the deposition rate of Pt nanoparticles on the surface of the polymer was very slow, in the absence of organic sacricial agents to react with the holes. Optimization of the deposition technique of Pt is needed to enhance the overall water splitting activities of this bulky g-C 3 N 4 .</p><p>We then investigated the effect of cocatalyst loading techniques on the photocatalytic water splitting activity. Three different cocatalyst loading techniques, in situ photodeposition, and H 2 and NaBH 4 reduction, were developed to decorate the g-C 3 N 4 nanosheets. As shown in Fig. S7, † evident water splitting activity was generated for photodeposition of Pt on the surface of the g-C 3 N 4 nanosheets, while only very slow H 2 and no O 2 evolution were found for both H 2 and NaBH 4 reduction modi-ed ones. In the rst case, when g-C 3 N 4 was irradiated with light, photoexcited charge carriers were generated and then immediately migrated to the surface of the g-C 3 N 4 nanosheets without recombination. The surface adsorbed Pt 4+ was then reduced in situ by the excited electrons and deposited on the active sites, which can efficiently promote the water splitting. For the other investigated techniques, Pt 4+ was reduced by H 2 or NaBH 4 and then randomly deposited on the surface, resulting in poor activities. The selective photodeposition of Pt on thin g-C 3 N 4 nanosheets resulted in a uniform dispersion of ultrane Pt nanoparticles ($1-2 nm) with a (111) crystal lattice spacing of $0.23 nm (Fig. 2b and c). The homogeneous deposition of Pt can be further proved by STEM imaging (Fig. 2d). However, serious particle accumulation occurred when the Pt cocatalysts were deposited by H 2 and NaBH 4 reduction (Fig. S8 †), which was the major hindrance which led to decreased water splitting activity.</p><p>We also investigated the chemical composition and valence state of the Pt species. As shown in Fig. 3a and b, electron energy loss spectroscopy (EELS) and XRD analysis conrmed the existence of a Pt (111) plane. 38 Besides, no evident structure variation occurred aer modication with the Pt cocatalysts, implying a robust stability of the g-C 3 N 4 CPs. [39][40][41] Three pairs of XPS peaks corresponding to Pt 0 , Pt 2+ , and Pt 4+ with binding energy at 72.13, 74.26 and 78.17 eV, respectively, were measured (Fig. 3c). Pt 0 was effective for H 2 evolution while PtO x were able to promote O 2 evolution. 42 However, two pairs of XPS peaks were deconvoluted for a NaBH 4 reduction modied one (Fig. S9 †), indicating the complete reduction of Pt 4+ into Pt 2+ and Pt 0 . To conrm that PtO x were active for the promotion of a water oxidation reaction, we evaluated the photocatalytic water oxidation activities of the as-prepared PtO x /g-C 3 N 4 . As shown in Fig. S10, † this material showed enhanced activity for water oxidation in comparison with the pure one, emphasizing the positive role of PtO x in improving the water oxidation rate. In addition, the water splitting rates and evolved H 2 /O 2 gas ratio (Fig. S11 †) could be nely tuned by simply adjusting the total loading from 0.2 to 5 wt% due to the change of the ratio of Pt and PtO x intensities (Fig. S12 and Table S2 †) and the alteration of particle size (Fig. S13 †). The creation of metal/polymer surface junctions promotes the interfacial redox reaction which can be conrmed by a rapidly decreased PL intensity (Fig. 3d). The optimum activity was achieved at a Pt loading content of 3 wt%. When Pt or PtO x were singly deposited on the g-C 3 N 4 nanosheets, the sample exhibited very poor activity in both cases, which once again highlighted that the simultaneous creation of both H 2 and O 2 evolution cocatalysts on the active sites was indeed essential for triggering the overall splitting of water.</p><p>The g-C 3 N 4 nanosheets modied by other noble metals (e.g., Rh, Ru, or Au) via in situ photodeposition all just showed trace H 2 and no O 2 evolution (Fig. S14 †), implying the importance of Pt for water splitting. The pH value and amount of polymer powders used for water splitting were also optimized (Fig. S15 and S16 †). The optimum water splitting rate was obtained for samples prepared by photodepositing 3 wt% Pt on 0.2 g of g-C 3 N 4 nanosheets under neutral conditions. We then evaluated their stability for long term reaction.</p><p>As shown in Fig. S17, † the optimized Pt/g-C 3 N 4 showed good water splitting stabilities under both UV and visible light irradiation for 580 hours of continuous reaction. It should be noted that N 2 gas was evolved along with H 2 and O 2 at the initial stage of the reaction. This arises from the self-oxidation of the surface un-condensed amino groups (-NH) by excited holes. [43][44][45] As the reaction proceeded, aer 80 hours almost no N 2 evolution was observed, suggesting a complete consumption of the -NH groups. When the Xe lamp was turned off, the amounts of the evolved gases quickly diminished in just four hours (Fig. S18 †), indicating a fast occurrence of the backward reaction of water splitting on the Pt species (H 2 and O 2 recombination for water formation). Thus, to further enhance the overall water splitting activity of the system, an efficient restraint of the backward reaction via rational structural design of the cocatalysts (e.g., core/shell nanostructure) should be considered.</p><p>The addition of cobalt species for in situ formation of cobaltbased cocatalysts can also sufficiently promote the water oxidation selectivity and efficiency of metal-free semiconductors such as g-C 3 N 4 and h-BCN. [43][44][45][46][47] As expected, the simultaneous evolution of H 2 and O 2 gases in a stoichiometric ratio of 2 : 1 by Pt-Co/g-C 3 N 4 under UV (l > 300 nm) (12.2 and 6.3 mmol h À1 ) (Fig. 4a) and visible light irradiation (l > 420 nm) (1.2 and 0.6 mmol h À1 ) (Fig. 4b) was signicantly enhanced aer 1 wt% CoO x were further modied for use as O 2 evolution cocatalysts, which can be determined by XPS analysis (Fig. S19 †). The slightly decreased activity in each run of reaction may be attributed to the stacked samples on the inner side of the reactor (Fig. S20 †). Furthermore, no obvious deactivation was observed aer 510 hours of reaction (Fig. S21 †), demonstrating the robust resistance of the composites to water and light corrosion at the so interface. The total amount of gaseous H 2 and O 2 collected reached $6.2 mmol, which corresponded to turnover numbers (TON) of 3.1 and 111.3 based on g-C 3 N 4 and Pt, respectively. The apparent quantum yield (AQY) for the overall water splitting reaction was calculated to be 0.3% at 405 nm (Fig. S22 †) and was monitored by an on-line gas chromatograph (Fig. S23 †). This is lower than the value of 2.5% of (Ga 1Àx Zn x ) (N 1Àx O x ) inorganic photocatalysts. However, it is a remarkable rst observation that photocatalytic overall water splitting can occur on the surface of an organic/polymer semiconductor via a 4-electron pathway. Optimization of the system to further improve the efficiency is ongoing in our lab.</p><!><p>The discovery of Pt/g-C 3 N 4 CPs that can split pure water without the use of sacricial reagents establishes a new chemical paradigm for exploiting clean, renewable solar energy using organic semiconductor light-energy transducers. Ongoing efforts are focused on modifying the electronic and textural structures of g-C 3 N 4 CPs and coupling them to low-cost kinetic promoters to facilitate photocatalytic cascade processes for water splitting and CO 2 xation that are relevant to sustainable energy production via articial photosynthesis. [48][49][50]</p>

Royal Society of Chemistry (RSC)

Engineering Functional Inorganic-Organic Hybrid System Advances in siRNA Therapeutics

Cancer treatment still faces a lot of obstacles such as tumor heterogeneity, drug resistance and systemic toxicities. Beyond the traditional treatment modalities, exploitation of RNA interference (RNAi) as an emerging approach has immense potential for treatment various gene-caused diseases including cancer. The last decade has witnessed enormous research and achievements focused on RNAi biotechnology. However, delivery of small interference RNA (siRNA) remains a key challenge in the development of clinical RNAi therapeutics. Indeed, the functional nanomaterials play an important role in the siRNA delivery, which could overcome a wide range of sequential physiological and biological obstacles. Nanomaterials-formulated siRNA systems have potential applications for protection of siRNA from degradation, improving the accumulation in the target tissues, enhancing the siRNA therapy and reducing the side effects. In this review, we explore and summarize the role of functional inorganic-organic hybrid systems involved in the siRNA therapeutic advancements. Additionally, we gather up the surface engineering strategies of hybrid systems to optimize for siRNA delivery. Major progress in the field of inorganic-organic hybrid platforms including metallic/non-metallic core modified with organic shell or further fabrication as the vectors for siRNA delivery is discussed to give credit to the interdisciplinary cooperation between chemistry, pharmacy, biology and medicine.

engineering_functional_inorganic-organic_hybrid_system_advances_in_sirna_therapeutics

1. Introduction<!>2. Biochemical properties of siRNA and RNAi<!>3. Bio-barriers of siRNA therapeutics<!>4. Surface Engineering Strategies of Inorganic-Organic Hybrid Nanoparticles<!>4.1. Covalent ligand conjugation<!>4.2. Amphiphilic polymer assembly<!>4.3. Electrostatic layer-by-layer assembly<!>4.4. In-situ ligand coating during synthesis<!>4.5. Host-guest supramolecular ligand self-assembly<!>4.6. Lipid shell coating<!>5. Metallic Core Hybrid with Organic Shell Delivery System<!>5.1. Gold Nanoparticles<!>5.2. Quantum Dots<!>5.3. Iron Oxide Nanoparticles<!>5.4. Upconversion Nanoparticles<!>6. Non-metallic Core Hybrid with Organic Shell Delivery System<!>6.1. Silicon Nano/Microparticles<!>6.2. Mesoporous Silica Nanoparticles<!>6.3. Carbon Vectors<!>7. Conclusion and Perspective

<p>In spite of the huge advancement achieved in research, drug and technology development for cancer treatment during the past decades, cancer still is a major public health problem and is a main leading cause of death worldwide. Human cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Additionally, human cancers consist of a mixed population of malignant cells, which carry multiple genetic mutations caused by external and internal factors, such as tobacco smoke, infectious organisms, unhealthy diet, inherited genetic burden, hormones, and immune conditions.1 These factors may act together or in sequence to cause new cancer cases and deaths. As shown in Figure 1A, around 1.6 million new cases of cancer were diagnosed and about 35% cancer deaths in US per year during last five years. Additionally, more than 50% cancer patients die annually according to statistics from 2000 to 2011 in China (Fig. 1B).2 Especially, there are 4.292 million new cases and 2.814 million cancer deaths in last year (Fig. 1C).3, 4 Moreover, the number of new cases and deaths are 14.1 and 8.2 million in the world in 2012 (Fig. 1D).5 Thus, cancer is a major issue to affect the healthcare. Currently, treatments include surgery, radiation, chemotherapy, hormone therapy, immune therapy, gene therapy and targeted therapy with their own limitations.6 However, many of cancers are driver mutations causing tumor initiation, progression, metastasis and drug resistance that results in urgent need for new technologies and strategies to fight cancer. Among the emerging technologies and anti-cancer therapeutics, small interfering RNA (siRNA) and RNA interference (RNAi) have been widely recognized to be capable of silencing many or perhaps all genes that results in modulation or targeted blockage of the biological processes, which are the defining hallmarks of cancer. Therefore, RNAi would pave the way for the cancer treatment as a promising biotechnology.7, 8</p><p>Since its discovery by Andrew Fire and Craig Mello, it was demonstrated in 1998 that long double-stranded RNA (dsRNA) mixtures were 10-100-folds more efficient at causing gene silencing than the single strand RNAs in Caenorhabditis elegans (Fig. 2A).9 Four years later, Science named RNAi "technology of the year". Fire and Mello were awarded jointly the Nobel Prize in Physiology or Medicine for their discovery of RNAi-gene silencing by siRNA in 2006 (Fig. 2A). 10 Then, the therapeutics based on siRNA as a potent drug candidate for gene regulation came when Elbashir and co-workers proved that the synthetic siRNA enabled sequence-specific gene down-regulation in a mammalian cell line. 11 Meanwhile, nanotechnology plays an important role in drug delivery including siRNA therapeutics, e.g. the first targeted nanoparticle-based siRNA therapeutics delivery in human cancer patients in 2010 (Fig. 2A).12–14 Indeed, a tremendous amount of effort has been put into development of siRNA cancer therapeutics over the last 20 years, and great progress has been achieved in research and development, both in academics and pharmaceutical industry. Although, in one period the big pharmaceutical factories closed the siRNA-related projects due to the disappointing results.15 Over the last 5 years, siRNA therapeutics has achieved a rapid development in fighting various diseases including cancer, which is indicated by over 30% cases that have already achieved Phase 2 or Phase 3 clinical trials (Table 1). Moreover, the leading regional markets of RNAi not only set up in developed countries, but also in developing countries (Fig. 2B). However, most of the leading companies of RNAi are in USA or EU5 ranging from 2015 to 2025 (Fig. 2C) (https://www.researchandmarkets.com/research/td7m2r/rnai). This trend indicates that the RNAi would be a strong promise therapy for treatment of various human diseases in the future. Such big progresses have brought new avenue for the siRNA therapeutics in clinical trials. Additionally, these achievements laid the foundations to employ RNAi as a key tool for next generation cancer drugs.</p><!><p>siRNA is a typical short/small double-stranded RNA molecules, approximately 21 nucleotides in length, and about 19 bp of the core region of siRNA (Fig. 3A) with the ability to mobilize the RNAi pathway.35 The interference effect has revealed immense potential for regulate the diseases caused by the overexpression or mutation of gene including cancer, genetic disorder and other diseases because of sequence-specific gene silencing. The structure is also well defined, siRNAs have phosphorylated 5' ends and hydroxylated 3' ends with two overhanging nucleotides. Additionally, naked siRNA has a large molecular weight (14 kD) and negative charge (polyanionic nature of the phosphate charges). Once the long siRNA or siRNA introduced inside the cytoplasm, the enzyme endoribonuclease Dicer would cleave the long siRNA into fragments siRNA (21 bp), then siRNA is incorporated into RNA inducing silencing complex (RISC), which consists of the Argonaute (Ago) protein as one of its main components, that results in splitting of each double-stranded siRNA into the passenger (sense) strand and the guide (antisense) strand. The sense strand is degraded, whereas the antisense strand is incorporated into RISC as an activated RISC complexes form that further direct the specificity of the target mRNA recognition via complementary base pairing. Later, Ago-2 catalyzes cleavage of the target mRNA thereby triggering the protein down-regulation and gene silencing (Fig. 3B).35 Comparing to the anticancer chemotherapeutic drugs, siRNA possesses a number of advantages due to its specific mechanism. The benefits of siRNA as a potential anticancer drug is the strongest supported by its effective inhibition of the any interest target mRNA or mutated gene in any type of cancer cells. Moreover, the synthesis of siRNA is relatively easy and its production costs less.</p><!><p>In spite of advancement in preclinical and clinical studies, no product has been launched into the market, because there are still many challenges to be overcome for siRNA clinical therapeutics. As well known, siRNA molecules have unfavorable physicochemical properties, such as negative charges, large molecule weight and size, and instability that impede the access through the cell membrane. Serum endonuclease easily degrades naked siRNA then easily eliminated through glomerular filtration. This implies that the double-stranded siRNA molecules have a very short half-life (10 min) and very low stability. This issue can be overcome by chemical modifications of the RNA backbone and by embedding the siRNA into nano-carriers. 35In order to have effect, siRNA needs to arrive in the cytoplasm of the cancer cell. From the region of injection (if systemic delivery is used) to the site of action, the siRNA molecule faces many biological obstacles. Challenges that need to be overcome are safety, stability and effective delivery.</p><p>From the blood circulation to the tumor cell, the RNAi and/or its transport mechanism needs to additionally overcome biobarriers. Biobarriers are biological surfaces (epithelia, endothelia, cellular, nuclear, endosomal membranes, etc.), which can be multi-cellular and/or multi-compartmental. These are elements of separation within defined compartments (e.g. vascular, cytoplasmic, stromal, etc.) and between different compartments. In the delivery of nanoparticles to tumor sites, biobarriers comprise all "barriers" that a nanoparticle needs to cross in order to reach the target cell. Using systemic administration (e.g. intravenous injection of nanoparticles), the biobarriers that need to be addressed are: 1) Enzymatic degradation; 2) Reticulo-Endothelial (RE) organs sequestration and phagocytes; 3) Tumor interstitium; 4) Crossing the cellular membrane; 5) Endosomal escape in order to prevent degradation in lysosomes and/or excretion from the cell (Fig. 4).36 An adequate negotiation of these barriers is therefore necessary in order to successfully deliver the siRNA into the cell obtaining a sufficient therapeutic index.</p><p>So far, the major barriers that prevent siRNA therapeutics from reaching the market are poor resistance to enzymes, such as RNase, short biological half-life, lack of cell/tissue targeting, ineffective cellular internalization, complicated escape from endosomes/lysosomes, toxicity and other related side effects and difficulties (Fig. 4).37 Therefore, the successful delivery of siRNA to the targeted cell/organs is the most important issue for treatment of cancer in the clinical application.</p><p>Therefore, to address these issues, a special system for siRNA delivery is needed. Delivery systems used nowadays can be categorized into physical methods, conjugation methods, methods with natural carriers, such as viruses and bacteria, and non-viral carrier methods.38 The use of viral vectors for the delivery of siRNA tends to be avoided because of insertional mutagenesis and immunogenicity problems that may arise due to the nature of the carrier. A new, non-viral way to overcome biobarriers, the added capability with respect to traditional cancer treatment methods of multi-functionality and specific cell targeting, has been proposed by nanomedicine research – nanoparticles.</p><p>Notably, the rational design of siRNA carriers is an important consideration because the use of high quantities of carriers can result in toxicity as a consequence of poor metabolism and elimination of the carriers. A great number of systems were reported for siRNA delivery based on biomaterial platforms, such as lipid, polymer, and nanoparticle.39–41 Here we put the concept of the inorganic-organic hybrid nanoparticles-based siRNA therapeutics. Additionally, we review the engineering strategies of inorganic-organic hybrid systems and examine the details of metallic and non-metallic core hybrid with organic moieties for siRNA delivery. Finally, we summarize the advantages of such hybrid systems for paving the way for the next generation siRNA therapy.</p><!><p>The application of nanomaterials in biomedical areas shows great promise and inorganic nanomaterials attract a particular interest for bioimaging, diagnosis, drug delivery and therapy applications. As regards to the above-mentioned siRNA delivery, inorganic nanomaterials also exhibit great potential to reveal the mechanism of siRNA transportation process and subsequently to construct well-designed vectors to circumvent the biological barriers of siRNA delivery. To endow inorganic nanostructures with siRNA transportation capacity, surface engineering with organic ligands should firstly be carried out. Many strategies have been developed for the surface engineering of inorganic nanomaterials, and they can be divided into the following categories: covalent ligand conjugation, amphiphilic polymer assembly, electrostatic layer-by-layer assembly, in-situ ligand coating during synthesis, host-guest supramolecular ligand self-assembly, and lipid shell coating (Fig. 5).</p><!><p>The surface chemistry of different kinds of inorganic nanoparticles has been extensively explored for surface engineering via covalent ligand conjugation. Several kinds of anchoring groups have been developed for ligand conjugation, such as thiol, carboxyl, phosphate terminal ligands (Table 2). Thiols have been widely used as an anchoring unit to fabricate inorganic nanoparticles, mainly metallic nanoparticles. Thiols have very high affinity with many metal ions, such as Au, Ag, Cu, Zn, etc. via coordination interaction. Thereby, thiol-containing cationic ligands can be used for binding to nanoparticle surface to generate hybrid nanoparticles for siRNA delivery. Alternatively, nanoparitlces can be bond with heterobifunctional ligand containing thiol and another functional group, then further modified with other positively charged ligands. Rotello et al. synthesized three thiol-containing dendron ligands with triethylenetetramine (TETA) terminal groups of different generations.42 These ligands were used to modify gold nanoparticles (AuNPs) through Au-SH coordinate binding. The as-prepared hybrid nanoparticles are cationic and resist aggregation, which were shown to be effective nanocarriers for siRNA delivery. Similar to thiol ligands, disulfide terminal group containing ligands also can bind with nanoparticle through S-metal bond. In addition to AuNPs, a lot of other inorganic nanoparticles can be modified with thiol or disulfide ligands, such as silver nanoparticles (AgNPs), quantum dots (QDs), CuS nanoparticles, and so on. Hydrophobic iron oxide nanoparticles (IONPs) also could be functionalized through covalent ligand exchange strategy. Xu et al. reported that bidentate ligand, dopamine, can coordinate to the surface of IONPs owing to improved orbital overlap of the five-membered ring and a reduced steric effect on the iron complex.43 The resulting IONPs showed excellent stability in physiological conditions. For non-metal inorganic nanomaterials, such as silica nanoparticles, carbon nanomaterials, they usually have affluent functional groups on the surface, which can be easily conjugated with other ligands.</p><!><p>Inorganic nanoparticles with high quality are usually prepared in organic phase with hydrophobic surface protection ligands because it allows higher reaction temperature. In addition to the above-mentioned ligand exchange strategy, amphiphilic polymer coating is another widely used method for hydrophobic nanoparticle surface engineering. The polymers encapsulate nanoparticles via hydrophobic interactions between hydrophobic portion of the polymer chains and initial hydrophobic surface coating of nanoparticles. Hydrophilic portion of the polymer chains faces the aqueous media, and renders the nanoparticles soluble in aqueous media. This approach keeps the initial surface coating of nanoparticles and can be more effective in preserving the physicochemical properties of as-synthesized nanoparticles. The representative amphiphilic polymers used for hydrophobic inorganic nanoparticle surface engineering are summarized in Table 3. Nie et al. used a high-molecular-weight (100 kDa) copolymer with an elaborate ABC triblock structure and a grafted 8-carbon (C-8) alkyl side chain to encapsulate hydrophobic ligand, tri-n-octylphosphine oxide (TOPO) protected CdSe/ZnS QDs.74 A key finding is that this polymer can disperse and encapsulate single TOPO-capped QDs via a spontaneous self-assembly process. As a result, after linking to PEG molecules, the polymer-coated QDs are perfectly protected, so their optical properties did not change in a broad range of pH (1 to 14) and salt conditions (0.01 to 1 M). Dai et al. reported the synthesis of several poly(ethylene glycol) grafted branched polymers based on poly(γ-glutamic acid) and poly(maleic anhydride-alt-1-octadecene) for functionalization of various nanomaterials including single-walled carbon nanotubes (SWNTs), gold nanoparticles, and gold nanorods, affording high aqueous solubility and stability for these materials.75 Moreover, the polymer-coated SWNTs exhibited remarkably long blood circulation upon intravenous injection into mice.</p><!><p>Electrostatic layer-by-layer assembly is another easy-handling strategy for inorganic nanoparticle surface engineering because of the strong electrostatic attraction between oppositely charged species. Li et al. have developed a layer-by-layer (LBL) assembly strategy that uses oppositely-charged linear polyions to generate water-soluble UCNPs.81 Upon sequential adsorption of positively charged poly(allylamine hydrochloride) (PAH) and negatively charged poly(sodium 4-styrenesulfonate) (PSS) onto the surface of nanoparticles, they successfully modified NaYF4:Yb/Er nanoparticles with stable amino-rich shells. The LBL assembly technique offers many advantages, albeit requiring repeated wash steps after each adsorption step, and these include simplicity, universality, and thickness control in nanoscale. More importantly, the high stability and biocompatibility of these polyions make them attractive as coating materials for a wide range of fundamental and technological applications.</p><!><p>Taking advantage of the easy-preparation nature of some kinds of inorganic nanoparticles, such as AuNPs, AgNPs, IONPs, the organic-inorganic hybrid nanoparticles could also be prepared in one-pot reaction containing the inorganic precursors and organic ligands. He et al. reported the synthesis of hyperbranched polyethyleneimine-protected silver nanoclusters (hPEI-AgNCs) using a facile, one-pot reaction under mild conditions.82 The hPEI-AgNCs were very stable against extreme pH, ionic strength, temperature, and photoillumination. IONPs also have been reported to be prepared in the presence of a variety of surface coating ligands, such as dextran, starch, glycosaminoglycan, and polyvinyl alcohol (PVA), to obtain biocompatible and thermodynamically stable dispersions of IONPs in aqueous media. Pierre et al. reported the detailed magnetic and structural properties of IONPs formed in the presence of dextran. The results suggested that the presence of dextran limits the particle size compared to particles prepared without the polymer.83</p><!><p>Host–guest interaction mediated self-assembly of hydrophilic ligands on the surface of hydrophobic nanoparticles can also be used as an efficient method to make the hydrophobic nanoparticles soluble in water. Li et al. has successfully developed a simple and efficient approach to draw Adamantane (Ad)-coated NaYF4:Yb, Er nanoparticles into water by the interaction between the host molecule β-cyclodextrin (β-CD) and the guest Ad molecules.84 This method provided a simple post-treatment through only stirring or shaking, rapid response time (<20 s), high conversion yield (>95%) and exhibited good capability for bioimaging. High quality inorganic nanoparticles are usually synthesized in organic phase with hydrophobic surface coating ligands, such as oleylamine, oleic acid, etc. To transfer the hydrophobic ligands coated nanoparticles into water-soluble forms, a host-guest strategy based on the interaction of α-cyclodextrin (α-CD) and hydrophobic carbon chain was developed. Hydrophobic IONPs, AgNPs and UCNPs were all reported to be transferred to aqueous solution through hydrophilic α-CD self-assembly on the nanoparticle surface to afford aqueous suspensions of nanoparticles with very good stability.85, 86 This approach is considered to be a generic method for the stabilization of hydrophobic ligands capped inorganic nanoparticles in aqueous solutions, which is an important aspect for the exploration of biological applications of inorganic nanoparticles.</p><!><p>Lipids are widely used as delivery vectors for small molecular drugs and nucleic acid gene drugs of various molecular weights. Lipids are considered to be highly biocompatible and non-immunogenic. Lipid shell coating is a very well-established method for inorganic nanoparticle surface engineering to produce hybrid nanosystem with high biocompatibility. Huang et al. reported a lipid-coated calcium phosphate (LCP) nanoparticle formulation for efficient delivery of siRNA to a xenograft tumor model by intravenous administration.87 After entering the cells, LCP would decompose at low pH in the endosome, which would cause endosome swelling and bursting to release the entrapped siRNA. Inorganic nanoparticles with hydrophobic surface ligands (such as QDs, IONPs) also can be hydrophilized by coating with lipid shell for biomedical applications.88, 89 Anderson et al. developed a simple and efficient method to coat IONPs with lipid molecules with low polydispersity.89 Cationic lipids-coated hybrid IONPs showed potent capability to deliver DNA and siRNA into cells. Moreover, the application of an external magnetic field further enhanced the efficiency of nucleic acid delivery.</p><!><p>There has been a long history for metals to be used in disease treatments since ancient times. Copper and iron were recorded for reducing inflammation and treating anemia, respectively, in ancient compendiums.90 More modern written accounts published at the beginning of the 20th century reported the use of sodium vanadate to lower blood sugar levels in diabetic patients and simple gold complex to treat rheumatoid arthritis.91 The greatly successful development of platinum-based anticancer drugs attracted tremendous attention for medicinal inorganic chemistry.90 Along with the advent of nanotechnology era, many kinds of metal-based nanoparticles have been prepared and great efforts have been paid to explore their biomedical applications. These metal-based nanoparticles, including Gold Nanoparticles (AuNPs), semiconductor quantum dots (QDs), iron oxide nanoparticles (IONPs), up-conversion nanoparticles (UCNPs), and so on, have been extensively developed for detection, imaging, delivery and therapy applications. Owing to their unique optical, magnetic, electronic and thermal properties, versatile functions can be endowed to the engineered delivery systems. For example, the real-time delivery process can be tracked by optical imaging or magnetic resonance imaging (MRI), smart cargo release can be realized through pH or thermal responsive design, and much more potent therapy efficiency can be achieved by combining the intrinsic therapeutic effects of the metal nanoparticles. Moreover, the composition, size, shape and surface properties of these nanoparticles can be flexibly and precisely tailored, which afford great superiority for different biomedical applications.92 In this section, we will introduce representative classes of metal-based nanoparticles engineered as inorganic-organic hybrid delivery systems of siRNA therapeutics.</p><!><p>Gold is known to be one of the least reactive metals, exhibiting incredible chemical resistance against both oxidation and corrosion. The biocompatibility of Au nanomaterials has been widely demonstrated by a number of in vitro and in vivo studies.93–95 Meanwhile, Au nanostructures can be readily synthesized with great diversity in size and shape, which is considered to be a crucial factor to determine the fate of nanocarriers, such as the blood circulation time, organ specific accumulation and cellular uptake mechanism. The size of synthetic AuNPs usually ranges from several nanometers to several hundred nanometers. Most commonly investigated morphologies include gold nanosphere, gold nanorod, gold nanoshell, gold nanocage, gold nanocluster, and so on (Fig. 6A). Moreover, gold nanostructures possess some other unique properties for biomedical applications, such as strong localized surface plasmon resonance (LSPR), X-radiation absorption ability, photo-thermal conversion properties, and so on.96 Therefore, when nanocarrier strategy emerged as a promising method to address the barriers of naked siRNA administration, gold nanoparticles immediately caught the attention of researchers as an excellent candidate to achieve this goal.97, 98 In the past decade, various kinds of AuNPs-based delivery systems have been developed through well-defined organic surface engineering with biocompatible polymers or biomolecules for siRNA delivery.</p><p>As introduced above, a well-characterized approach for surface engineering of gold nanostructures is utilizing their ability to form very strong and stable gold-thiolate bonds (Au-S) with molecules containing thiol (-SH) or disulfide groups (S-S) (Fig. 6B).93 Park et al.99 synthesized AuNPs in the presence of cysteamine hydrochloride to afford amine-functionalized AuNPs with positively charged surface. Then siRNA conjugated with PEG5000 with a disulfide linker was added to adhere to the surface of AuNPs through electrostatic interaction. The nanosized polyelectrolyte complexes could be efficiently internalized into human prostate carcinoma cells and release siRNA in reductive cytosol environment. Thus, enhanced intracellular uptake of siRNA and significant inhibition of the target gene expression were achieved. Anderson et al.100 employed a different strategy for siRNA-PEG co-loading with AuNPs by first decorating the 15 nm AuNPs with SH-PEG1000-NH2 and then conjugating siRNA by means of a disulfide crosslinker to the terminal of the PEG. A comparable loading of ∼30 strands of siRNA per nanoparticle has been reported. To enhance the cellular uptake and facilitate endosomal escape, these particles were further coated with different kinds of poly(β-amino ester)s (PBAEs), a new class of cationic biodegradable polymer. These nanoparticulate formulations finally realized high levels of in vitro siRNA delivery, achieving gene silencing as good or better than the commercially available lipid reagent, Lipofectamine 2000. Other authors have taken another way to address the endosomal escape issue and realize laser-triggered controlled release of siRNA by introducing gold nanoshells as the core structure of the delivery system. Gold nanoshells with peak plasmon resonance in the near-infrared (NIR) range are susceptible to local heating under irradiation of NIR light (Fig. 6C).101, 102 A poly-L-lysine peptide (cysteine-tyrosine-serine-lysine50) was used to modify gold nanoshell surface, affording a positive charge of 14.27 mV to capture siRNA electrostatically. Controlled release of siRNA and enhanced endosome rupture have been achieved by irradiating with lower power NIR irradiation without causing obvious cytotoxicity.102</p><p>Gold nanoparticles have also been developed for co-delivery of siRNA and small molecular chemotherapeutic drugs through well-defined surface organic ligand engineering.103, 104 Gong et al. reported the surface engineering of AuNRs for co-delivery of achaete-scute complex-like 1 (ASCL1) siRNA and doxorubicin (Dox).103 AuNRs were covalently modified with different functional components: a pH-labile hydrazone linkage tethering Dox to enable pH-controlled drug release, a polyarginine cationic polymer for complexing siRNA and a tumor-targeting ligand octreotide (OCT), to specifically target neuroendocrine cancer cells. Finally, the delivery system Au-DOX-OCT-siRNA resulted in significantly higher cellular uptake and gene silencing effect. The OCT targeting nanocarrier-mediated combination chemotherapy and RNA silencing exhibited the strongest anti-proliferative effect.</p><p>In addition to covalent modification of gold nanostructures with thiol-containing ligands, layer-by-layer assembly is an alternative way to engineer multifunctional surface for siRNA delivery.105–111 The commonly used AuNPs are synthesized in aqueous solution by a citrate reduction method, resulting in a negatively charged surface. Poly(ethylene imine) (PEI), a widely used positively charged polymer in gene delivery study, has been employed to conduct the layer-by-layer assembling process.105 In this work, two kinds of final delivery systems with different terminal surfaces were fabricated, one with a terminal siRNA layer while another one with a terminal PEI layer. The final size of these particles is reported to be within 20-25 nm range, with a loading density of about 780 siRNA per particle. The cellular particle uptake of siRNA/PEI-AuNPs was significantly more than PEI/siRNA/PEI-AuNPs; however, transmission electron microscopy (TEM) images showed siRNA/PEI-AuNPs particles primarily trapped within the endosome. Finally, GFP silencing studies showed that PEI/siRNA/PEI-AuNPs achieved 70% GFP knockdown 48 h post transfection while no knockdown for particles with siRNA/PEI-AuNPs, suggesting a need of endosome escape component of siRNA delivery vectors. Liang et al. reported the incorporation of an anionic charge-reversal polyelectrolyte (PAH-Cit, cis-aconitic anhydride-functionalized poly(allylamine)) to assemble with PEI and siRNA on AuNPs surface through layer-by-layer method (Fig. 6D).106, 107 The PAH-Cit can undergo a charge reversal from negative to positive inside the lysosome during a pH change from 7.4 to 5.0, which will disassemble the PEI/siRNA/PAH-Cit/PEI-AuNPs layer-by-layer complex to accelerate siRNA release. The silencing efficiency of targeting gene was reported to be 80%, compared to only 20% for complexes formed with noncharge-reversal polymer. Confocal images revealed that enhanced gene silencing effect benefited from the increased endosomal escape ability of this charge-reversal complex. This layer-by-layer strategy was also used to fabricate PEI modified gold nanorods (AuNRs) for siRNA delivery with combination of photothermal therapy.109–111 Cancer is a complicated disease that usually requires combination therapy of several treatment modalities. Photothermal therapy is an effective and noninvasive cancer treatment procedure, which generally utilizes a NIR light source to activate tumor localized photothermal agents to trigger local hyperthermia. AuNRs exhibit strong photo-thermal conversion effect under NIR light irradiation because of the surface plasmon field enhancement of the absorption. We demonstrated that PEI-Au NRs not only protect siRNA from degradation, but also facilitate endosomal escape, both of which are prerequisites for successful gene silencing.109 As a result, the combined anticancer activity of PEI-Au NR/siRNA, PKM2 gene inhibition and photothermal effect showed very potent anticancer efficiency against breast cancer cells.</p><p>Moreover, taking advantage of their ease of synthesis property, gold nanoparticles surface engineering with positive organic ligands for siRNA delivery could be readily realized in-situ during the process of particle synthesis.112–114 Wang et al. reported the manufacturing of PEI-capped AuNPs by directly using PEI25k as the reductant and stabilizer, in place of citrate.112 This resulted in AuNPs with a positive surface charge which loaded siRNA molecules via electrostatic interactions. It was reported that PEI-AuNPs induce more significant and enhanced reduction in targeted green fluorescent protein expression in MDA-MB-435s cells, because of more siRNA internalization, as evidenced by confocal laser scanning microscopy observation and fluorescence-activated cell sorting analyses. Furthermore, lower cytotoxicity of PEI-AuNPs than pure PEI was observed at siRNA concentration of 120 nM. Another study took a similar in-situ strategy and synthesized AuNPs in the presence of a block polymer p(HPMA70-b-DMAPMA24).113 This resulted in AuNPs with diameters about 7 nm surrounded by a HPMA block, which serves as a hydrophilic, sterically stabilizing shell (similar to the role of PEG). The DMAPMA block functionalized as the cationic part for binding siRNA. The gene silencing efficiency on luciferase-expressing KB cells were evaluated to up to 50% under the condition of 100 nM siRNA, 6 h transfection and 24 h incubation. In recent years, fluorescent gold nanoclusters have attracted a lot of attention due to their unique features, such as ultrasmall sub-nanometer size, great biocompatibility and excellent photostability, making them ideal fluorescent labels for biological applications.115, 116 In a recent study, positively charged gold nanoclusters (GNC) were synthesized through on-step reduction of Au3+ in the presence of thiolate-containing GSH and oligoarginine CRRRRRRRRR.114 The prepared GNCs had a well-defined core structure with diameters around 2.6 nm. It was reported that the GNCs had a loading capacity of 226 μmol siRNA per gram GNCs. Nerve growth factor (NGF) siRNA was loaded for pancreatic cancer treatment. The GNC-siRNA complex exhibited several attractive properties, increasing the serum stability of siRNAs, prolonging the blood circulation lifetime of siRNA and enhancing the cellular uptake and tumor accumulation of siRNA. Subsequently, GNC-siRNA complexes effectively down-regulated the NGF expression in both Panc-1 cells and pancreatic tumor model. Finally, effective inhibition of the tumor progression was achieved in three pancreatic tumor models (subcutaneous model, orthotopic model and patient-derived xenograft model) without adverse effects.</p><!><p>In the past two decades, quantum dots (QDs), also known as semiconductor nanoparticles, have become one of the dominant classes of fluorescent dyes in various biomedical areas since the pioneered works at 1998.117–121 QDs are generally made from hundreds to thousands of atoms of group II and VI elements (e.g. CdSe and CdTe) or group III and V elements (e.g. InP and InAs) with diameters on the order of 2-10 nm.122 QDs exhibit unique optical and electronic properties, such as size-tunable emission, resistance to photobleaching, superior signal brightness, and broad absorption spectra of excitation (Fig. 7A).121 QDs also have a versatile surface chemistry, thereby allowing further surface engineering of functional groups to facilitate drug loading and cellular uptake. Therefore, QDs have been considered to be promising nanoscale scaffold for designing multifunctional nanosystems with both imaging and therapeutic functions. Though the direct use of QDs as drug delivery vectors remains questionable due to their potential long-term toxicity, QDs offer great potential as a proof-of-concept model to investigate the fate of nanoparticle-based drug delivery systems (NDDS) in biological systems, especially in vivo.121, 123 Owing to these unique features of QDs, they also have been considered to be a promising candidate for siRNA delivery system to evaluate cellular uptake mechanism,124 siRNA release process tracking,125–128 cancer cell targeting,129, 130 endosome escape,131 in vivo distribution,132, 133 and so on.</p><p>QDs were for the first time introduced into siRNA delivery system by Bhatia et al. for monitoring the delivery process and improving gene silencing effect.134 In this study, QDs were first modified with mercaptoacetic acid and thiol-PEG5000, and then incorporated into cationic liposomes along with siRNA. Take advantage of the superior photostability and tunable optical properties of QDs, they used flow cytometer to track delivery of nucleic acids, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Moreover, a multiplexed gene knockdown study was achieved using two kinds of QDs different colors for different targeting siRNA labeling. For cellular siRNA delivery, endosome escape is a critical issue, because delivery complexes are usually internalized by cells though endocytosis pathway, easily resulting in enzymatic degradation of the payload siRNA.124, 131 Gao et al. reported the engineering of QDs surface by coating with proton-absorbing polymer, with a balanced composition of tertiary amine and carboxylic acid groups (Fig. 7B).124, 131 The intracellular behavior of QD-siRNA complexes, including uptake, transport, and localization in live cells were investigated by fluorescence tracking. It was demonstrated that the proton-sponge coating on the QD surface leaded to efficient siRNA release from intracellular vesicles, which achieved 10-20-fold improvement in gene silencing efficiency.</p><p>Cancer cell targeted siRNA vector further improves the cancer specificity and therapeutic efficiency, and active tumor targeting moieties are usually conjugated to the carriers for molecular recognition of unique cancer-specific markers. With excellent fluorescent properties, siRNA nanocarriers engineered from QDs offer great superiority for targeted siRNA delivery tracking, mechanism study and therapeutic efficiency evaluation.129, 130, 135, 136 Lee et al. developed a multifunctional delivery platform based on QDs for targeted EGFRvIII gene down-regulation (Fig. 7C).129 RGD and HIV-TAT peptides were conjugated to QDs surface to target the integrin receptor protein αvβ3 overexpressed on U87 cells and to enhance the cellular transfection efficiency of QDs-siRNA complex. Target-specific delivery of siRNA was demonstrated by tracking the QDs fluorescence in a novel co-culture system containing the U87-EGFP cell line with other less-tumorigenic cell lines, such as PC-12 cells. In another study, chimeras composed of aptamer targeting prostate-specific membrane antigen (PSMA) and siRNA were delivered by positively charged QDs which were engineered from coating hydrophobic QDs with amphiphilic copolymers poly(maleic anhydride-alt-1-tetradecene) and then conjugating with PEI.130 The central concern of this work is to evaluate the cellular uptake efficiency of chimeras-QDs complex established through two different approaches: one-step direct adsorption chimaeras on QDs and two-step method by sequential adsorption siRNA and conjugation aptamer to siRNA. Finally, fluorescence tracking of QDs demonstrated that the two-step approach could greatly retain the conformation and high accessibility of the targeting aptamer moieties to achieve significantly higher transfection efficiency and targeting gene down-regulation ability.</p><p>With well-defined surface organic ligand engineering, QDs could also be used as siRNA and small molecule co-delivery vectors. A series of biocompatible QDs modified with amino acid grafted β-cyclodextrin (β-CD) derivatives have been reported.137–139 The arginine-β-CD modified QDs (Arg-CD-QDs) not only have positively charged surface to absorb siRNA, but also a hydrophobic cavity of β-CD, which serves as molecular capsule to encapsulate doxorubicin (Dox) (Fig. 7D).139 The siRNA was designed to target and silence the multidrug resistance gene (MDR-1), which is responsible for multidrug resistance in cancer cells. By simultaneous transportation of siRNA and Dox across the cell membrane to down-regulate the expression of P-Glycoprotein (P-gp), the multidrug resistance could be reversed and as a result the efficiency of Dox can be improved in multidrug resistant cancer cells.</p><!><p>Magnetic iron oxide nanoparticles (mostly maghemite, γ-Fe2O3 or magnetite, Fe3O4) are well-established nanomaterials that possess unique magnetic properties. IONPs are being actively investigated as new generation of magnetic resonance imaging (MRI) contrast agents (Fig. 8A & B).56, 140–143 Owing to their unique characteristics, including efficient contrast effects, excellent biocompatibility and versatile surface functionalization capability, IONPs are extensively explored for various biomedical applications, such as medical diagnosis, detoxification of biological fluids, protein purification, cell separation, chronic iron deficiency anemia treatment, hyperthermia, drug/gene delivery, and so on.144–146 The excellent biocompatibility of IONPs has been widely demonstrated in a number of studies in vitro and in vivo, because iron is one of the most abundant endogenous metallic elements in living organisms and is essential for various biological processes. IONPs can be degraded in the body and subsequently incorporated into iron pools or used in metabolic processes.147 Several IONPs-based nanoformulations have been approved or in the clinical trial as MRI contrast agents or therapeutic agents.148–150</p><p>MRI has a number of unique advantages including deep tissue penetration, high spatial resolution, and excellent soft tissue contrast.140 Therefore, IONPs-based delivery systems possess great promise for simultaneously in vivo transportation tracking, biodistribution imaging, and drug accumulation evaluation. Moore et al. developed a dual-purpose probe by surface engineering of IONPs for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution MRI and near-infrared in vivo optical imaging (Fig. 8C).38 The multifunctional delivery vector is synthesized through a step-by-step method IONPs was first coated with dextran and then conjugated with Cy5.5 dyes and covalently linked to siRNA molecules specific for model (GFP) or therapeutic (survivin) targets, and this nanocarrier is also modified with myristoylated polyarginine peptides (MPAP) serving as a membrane translocation module. The in vivo transportation of siRNA mediated by the IONPs could be monitored by MRI and optical imaging. In addition, the imaging results of gene silencing process could correlate with histological data. In another study, a similar all-in-one strategy is used for siRNA delivery and simultaneously in vivo transportation monitoring. Bovine serum albumin (BSA)-coated manganese-doped magnetism-engineered iron oxide (MnMEIO) nanoparticles were used as the core material.151 The MnMEIO NPs were further modified with Cy5 dyes for subcellular imaging and cyclic RGD peptides for cancer cell targeted delivery. The multifunctional theranostic nanovector was designed to enable highly accurate imaging to be carried out simultaneously with the delivery of therapeutic siRNA, which can minimize invasiveness and deleterious side effects.</p><p>To engineer IONPs for specific biomedical applications, surface functionalization with proper organic ligands should be first carried out to make them water-soluble, biocompatitable, or to introduce functional groups for further conjugation. The strategies for IONPs surface engineering for siRNA delivery could be divided into several categories: amphiphilic polymer coating,152–154 ligand exchange155–157 and in-situ coating during the synthesis process.158 Chen et al. used amphiphilic Alkyl-PEI2k as surface engineering ligand to assemble with hydrophobic IONPs to form cationic clusters for binding siRNA (Fig. 8D).159 The cluster nanocarrier could protect siRNA from enzymatic degradation in serum, and release complexed siRNA efficiently in the presence of polyanionic heparin. The excellent gene silencing efficiency of the siRNA-loaded system is assessed with 4T1 cells stably expressing luciferase (fluc-4T1) and with a fluc-4T1 xenograft model. Moreover, unlike high-molecular-weight analogues, the Alkyl-PEI2k-coated IONPs show good biocompatibility. Pierre et al. synthesized two types of polymers, i. e. homopolymers poly(oligoethylene glycol) methyl ether acrylate (p(OEG-A)), poly(dimethylaminoethyl acrylate) (p(DMAEA)) and block poly(DMAEA-b-OEG-A) polymers, as the surface exchange ligands.160 IONPs with a diameter of about 8 nm were obtained. These polymers were grafted separately to IONPs using the strong affinity of phosphonic acid terminal group to IONPs surface yielding IONPs@p(OEG-A), IONPs@p(DMAEA) and IONPs@p[(DMAEA)-b-(OEG-A)]. These polymers confer a good stability to IONPs in aqueous solution and 50% fetal bovine serum (FBS)medium because P(OEG-A) chains effectively mask the positive charge originating from p(DMAEA), thereby limiting protein adsorption on these particles. Hybrid nanoparticles were exploited for siRNA complexation, thereby generating IONPs/siRNA nano-carriers with anti-fouling p(OEG-A) shells. The excellent gene silencing efficiency was achieved on human neuroblastoma SHEP cells both in the presence and in the absence of a magnetic field in FBS containing medium. Take advantage of the well-established silica coating chemistry, a layer of silica shell could be first deposited on the IONPs surface as a platform for further modifications to generate multifunctional delivery system. Magnetic mesoporous silica nanoparticles (M-MSNs) were constructed to load siRNAs into the mesopores of M-MSNs, followed by polyethylenimine (PEI) capping, PEGylation and fusogenic peptide KALA modification.161 The resultant delivery system exhibited prolonged half-life in bloodstream, enhanced cell membrane translocation and endosomal escapablity, and favorable tissue biocompatibility and biosafety. The therapeutic effect of this delivery system carrying vascular endothelial growth factor (VEGF) siRNA was proved on both subdermal and orthotopic lung cancer models with remarkable tumor suppression, while tumor metastasis was also significantly reduced, overall leading to improved survival.</p><!><p>Lanthanide-doped upconversion nanoparticles (UCNPs) exhibit unique luminescent property to utilize sequential absorption of multiple photons through the use of long lifetime and real ladder-like energy levels of trivalent lanthanide ions to produce higher energy anti-Stokes luminescence. It thereby converts two or more long-wavelength excitation photons, generally NIR light, into shorter wavelength emissions (e.g., NIR, visible, and UV) through a process known as photon upconversion (Fig. 9A).162 UCNPs possess a lot of advantages for bioimaging, such as high tissue penetration depth, low autofluorescence background, sharp emission bandwidths, large anti-Stokes shifts, high resistance to photobleaching, and high temporal resolution.163–166 In recent years, UCNPs have been extensively developed as a new class of luminescent agents that have become promising candidates for use in various biomedical applications comprised of imaging, drug delivery, and therapy. 167–169</p><p>Owing to the unique and amazing luminescence property of UCNPs, they have been considered to be promising core structure for light-triggered delivery systems. Light-triggered drug delivery platforms have emerged as an elegant and non-invasive system for drug payload release in a remote spatiotemporal controllable manner at the desired site and time. This control is considered crucial to boost controlled local effective drug accumulation while minimizing side effects, therefore resulting in improved therapeutic efficacy.170–173 UCNPs offer an excellent choice for this task due to their utilization of NIR-excitation wavelength exhibiting ability to penetrate deeply into living tissues without causing phototoxic effects, which shows great superiority over high energy UV/visible light and expensive high intensity pulsed laser to activate the photosensitive component. In recent years, great breakthroughs have been successfully achieved by utilizing UCNPs system to realize NIR light-trigged drug release for chemotherapy. This concept has also been successfully applied for siRNA delivery and NIR light-triggered activation.174–178 Zhang et al. reported the exploitation of the potential of NIR-to-UV UCNPs to achive photoactivation of caged siRNA for photo-controlled gene regulation (Fig. 9B).175 In this study, siRNAs were caged with light-sensitive 4,5-dimethoxy-2-nitroacetophenone (DMNPE) and loaded into the pores of mesoporous silica coated UCNPs by physical adsorption. DMNPE-caged siRNA molecules were shown to be uncaged and turned to functional gene as designed to silence the targeting gene after activated with NIR light irradiation. This concept was fully proved by the significant decrease in GFP fluorescence for caged GFP siRNA delivery in GFP expressing B16 cells and further more in vivo in animal models with deep tissue penetration. In another study, Xing et al. developed UCNPs with surface functionalization by cationic photocaged linkers through covalent bonding, which could effectively adsorb anionic siRNA through electrostatic attractions (Fig. 9C).176 The UCNPs-siRNA complexes could be easily internalized by living cells. Upon NIR light irradiation, the photocaged linker on the UCNPs surface could be cleaved by the upconverted UV light and thus initiated the intracellular release of the siRNA. The in vitro agarose gel electrophoresis and intracellular imaging results indicated that the UCNPs-based gene carrier system allowed effective siRNA delivery. The applications of NIR light instead of direct high energy UV irradiation may greatly guarantee less cell damage.</p><p>UCNPs are also shown to be an alternatively new choice for photodynamic therapy (PDT) with NIR light excitation.179, 180 This promising approach was introduced for efficient siRNA delivery and therapy by combination with photochemical internalization, PDT or photothermal therapy (PTT) to achieve synergistic tumor therapy effect.181–183 Positively charged UCNPs were engineered via a layer-by-layer strategy and further loaded simultaneously with Chlorin e6 (Ce6), a photosensitizing molecule, and siRNA, which targets the Plk1 oncogene (Fig. 9D).182 Under excitation by a NIR light at 980 nm, cytotoxic singlet oxygen can be generated via resonance energy transfer from UCNPs to photosensitizer Ce6, while the residual upconversion luminescence is utilized for imaging. The silencing of Plk1 induced by siRNA delivered with UCNPs could induce significant cancer cell apoptosis. As the result of such combined photodynamic with gene therapy, a remarkably enhanced cancer cell killing effect is realized. Gd-doped UCNPs were synthesized and modified with cationic polymers, which were further conjugated with cypate and loaded with HSP70-siRNA against heat shock protein 70 (HSP70).183 Cypate, a type of organic carbocyanine fluorophore, displays high molar extinction coefficient for NIR light, thereby exhibiting high photothermal conversion efficiency for cancer photothermal therapy. HSP70 are usually upregulated under heat stress, thus protecting cells from hyperthermic damage. Therefore, this UCNPs-cypate-siRNA system could simultaneously generate photothermal effect to destroy cancer cells and inhibit the expression of HSP70 to achieve synergistically enhanced antitumor therapy effect. Moreover, this multifunctional platform provided a multimodal imaging-guided manner for precisely controllable antitumor theranostics.</p><!><p>The intrinsic physical and chemical properties of non-metallic materials also play a critical important role in the development progress in medicine and other industries. As well known that the arsenic trioxide, also named pi-shuang in traditional Chinese medicine.184 It has long been of biomedical interest and now used to treat cancer like acute promyelocytic leukemia (APL). Another case, selenium (Se) is an essentials dietary component for humans, recent increasing reports and evidences to show a promising cancer chemopreventive element.185 With the rapid development of nanotechnology, it has brought great opportunities to use those non-metallic materials as nanocarriers for gene therapies, chemotherapies and other small molecule drugs. Se-based nanoparticles were successfully used to deliver siRNA to inhibit epidermal growth factor receptor signaling, or tumor microenvironment responsive delivery system to enhance the nanotherapeutics.186, 187 Specifically, both the silicon and carbon elements in group IV, they are most abundant and significant non-metallic substances in human tissues. These two kinds of typical materials have been designed, synthesized and applied in various research fields including nanomedicine. The advantages of these systems including large surface, stability, biocompatibility, facile synthesis, easy surface functionalization, high penetration capacity to biological barriers, ultra-high loading capacity and so on.188 Silicon based delivery system, such as mesoporous silica nanoparticles (MSNs), nano size silicon particles and nanoporous silicon microparticles (also named multistage vector, MSV). Additionally, carbon based platform including carbon dots, carbon tubes, graphene, fullerene, nanodiamond and so on.189–191 Both of them have emerged as a booming area for the development of delivery vehicles and for diagnostic agents and anticancer drugs. Following, we expand the siRNA therapeutics based on silica nanoparticles, silicon nano/microparticles and carbon vectors with further fabrication of organic shells for treatment human diseases.</p><!><p>Silicon nanoparticles have been investigated since 1990s. Silicon offers specific physical and chemical properties, size-dependent multicolor reflection of light, photobleaching stability and favorable non-toxicity.192 Silicon is an "indirect band-gap material" unlike other semiconductors and shows particular changes when its size approximates the bulk Bohr radius (4 nm). Porous silicon with the advantages of biocompatibility, biodegradability and unique physical properties has great potential as a drug delivery platform for various therapeutic agents including chemotherapeutics, gene, and small molecular diagnostics.193, 194 The in vivo biosafety and efficacy are highly dependent on the surface functionalized properties (size, fluorescent dye, peptides, targeting moieties and geometries), administration dosage and routes (Fig. 10A).195 The typical example of silicon carriers for siRNA delivery is the multistage vector systems (MSVs), this system encompasses three components and each optimized stage to address a different set of biological barriers. 1) Porous silicon microparticle as a first stage component that can be loaded with different nanoparticles. 2) The second stage includes micelles, liposomes, polymeric nanoparticles or metallic nanoparticles, etc. 3) The third stage is the payload drugs including siRNA, chemotherapeutics, miRNA, and antibodies (Fig. 10A).196</p><p>Up to now, the protocol for mass production of MSV microparticle is easily produced according to the United Stated Food and Drug Administration's (FDA's) good manufacturing particles (cGMP). Discoidal MSVs with different diameters (500-2600 nm), various heights (200-700 nm), pore sizes (5-150 nm), and high porosity (40-90%) can be adjusted by modulating the electrochemical etching and photolithography process (Fig. 10B).197 Consequently, such physical properties of MSVs enable generation of particles with various performance attributes, nanoparticle-loading capacity, drug release profile, biodistribution and degradation kinetics. Additionally, MSVs could successfully stain in the tumor blood vessels because of its geometries properties and the significant difference structure between the tumor and normal vessels. When the silicon microparticles gradually degrade, second stage nanoparticles are released into tumor tissues. These second nanoparticles could further protect the therapeutic drugs from degradation and promote intracellular uptake in cancer cells. Finally, the third stage anticancer drug is typically released from the second stage nanoparticles into targeting cells (Fig. 10C).195 The MSVs with the novelty of multistage design could sequentially overcome the biological barriers to enhance the accumulation in the disease tissues and improve the drug concentration in the targeting cells.</p><p>Based on MSVs system, Ferrari's groups encapsulated the liposome or polymer siRNA formulated second nanoparticles inside the nanopores successfully applied on treatment of triple negative breast cancer, bone marrow metastatic breast cancer, metastatic ovarian cancer and inflammatory disease.198–203 Shen et al. used the 1000 nm (diameter) and 400 nm (height) MSVs with further optimized through 3-aminopropyl triethoxysilane (APTES) to obtain a slight positive charge, which facilitated loading of slightly negative liposomes into the nanopores. Additionally, APTES fabricated MSVs reveal a more stable ability in the PBS/FBS (10% FBS) mixture solutions. By day 10, most of the porous silicon particles maintain a clear edges and well-defined nanoporous structure. This system could successfully deliver siRNA inside the SKOV3 (Fig. 10D).198 Meanwhile, the siRNA with a sustain release from the MSVs to keep the down-regulated protein expression from day 3 to day 9. The in vivo results also indicated that this platform could suppress the EphA2 protein expression trigged reduction of total number of microvessels in tumor samples with a dose-dependent behavior. Importantly, tumor growth was completely inhibited when mice treated with MSV/EphA2 siRNA in combination with paclitaxel.</p><p>Beside above concept, Shen et al further developed conceptually different from the multistage system. Therapeutic siRNA or microRNA are not pre-packaged, nor are they loaded in the form of nanoparticles in the polycation nanoporous silicon (PCPS) system.204, 205 Instead, they bind directly to the polycation inside the nanopores, thus providing a very convenient approach to load a large quantity of therapeutic agents. The therapeutic agents and polymer will be slowly released and self-assembled nanoparticles upon silicon particle degradation, thus avoiding a sudden burst of release, their study has clearly demonstrated effective cellular internalization and tumor enrichment (Fig. 10E).205 Release of therapeutics is achieved during porous silicon degradation, which is another difference from the multistage vector system. This platform with following advantages: 1) with high loading capacity, 2) with low or no toxicity, 3) with a friendly production protocol, and 4) stable during transportation and storage. Additionally, the self-assembled nanoparticles could successfully help siRNA polyplexes to escape from endosome and lysosome to cytoplasm with the benefits from the PEI moieties trigged the proton sponge effects. Furthermore, systemic delivery of PCPS/STAT3 siRNA in murine model of MDA-MB-231 breast cancer enriched particles in tumor tissues and reduced STAT3 expression in cancer cells, causing significant reduction of cancer stem cells in the residual tumor tissue.205 Totally, their new multistage concept has a high loading capacity and no detectable toxicity. These inorganic (silicon) and organic (liposome or polymer) hybrid systems not only address the biological barriers for siRNA delivery to the targeting tissue and cell, also could maintain a sustain gene silencing expression.</p><!><p>Recently, mesoporous silica nanoparticles (MSNs) have been drawn great attention for nucleic acid transportation due to its biocompatibility, definable morphology, large surface area for massive loading amount of cargos.206–208 For delivery application, MSNs exhibited unique features on chemical modifications of outer and inner surfaces of MSNs with various bioactive macromolecule including PEG, dendrimers, antibodies, aptamers, peptides and cationic polymer.208–213 With these various modifications on the surface of MSNs, the siRNA condensed tightly to MSNs by coupling with the cationic polymers and was able to quick uptake due to the target moiety conjugation and following rapid intracellular releasing (Fig. 11A). Therefore, MSNs have also been successfully administered as effective gene delivery devices in different cancers.214, 215 For instances, Xia et al. studied transfection efficiency of MSNs/siRNA delivery system on GFP-HEPA cells by noncovalent attachment of different molecule weight polyethyleneimine (PEI) polymers to the surface of MSNs as siRNA delivery platform(Fig. 11B).216 After comparation the cellular uptake, cytotoxicity and transfection efficiency of PEI-coated MSNPs, they demonstrated that 10kDa PEI-coated particles not only bind siRNA with high affinity, but also enable to achieve efficient nontoxic cellular delivery of these payload. Additionally, due to their unique mesoporous scaffold, MSNs can be easily loaded with both chemotherapy drugs and siRNAs and effectively co-deliver the payloads into tumor cells with synergistic effect.217–219 Huan et al reported that administration of MSNs loaded concurrently with Doxorubicin and siRNA targeting the P-glycoprotein (P-gp) protein to overcome multiple drug resistance (MDR). They showed that this co-delivery strategy of DOX and siRNA by MSNs was capable to increase the intracellular drug concentration to levels exceeding that of free DOX which resulted in an increased killing of DOX compared with the free form on KB-1V MDR cancerous cell, which demonstrate that it is possible to use the MSNs platform to effectively deliver a siRNA targeted the drug exporter gene that can be used to improve the chemotherapeutic agent sensitivity (Fig. 11 C).220</p><p>During blood circulating of nanoparticles before reaching their site of action, undesirably releasing and degradation of loaded active siRNA in MSNs may suffered due to the mesoporous structure, which is a major challenge that should be avoided as much as possible.221 To fulfill this purpose, the porous of MSNs could be capped by materials that only sensitive to the tumor microenvironment, in which that capping molecules respond, following by the opening of MSNP-locked valves to release the siRNA and other active reagent such as chemo drug. Lin et al reported a MSNs based siRNA delivery system with capping the porous by poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) cross-linked with reductive sensitive linker (Fig. 11D).222 The results showed that the ssCP-MSNs exhibited an excellent siRNA binding capacity. After cellular uptake of ssCP-MSNs, high concentration of intracellular GSH triggers the release of loading siRNA by decapping the PDMAEMA. Moreover, this capping siRNA system exhibited obvious tumor suppression of HeLa-Luc xenograft murine model after following systemic administration. Overall, considering the further evaluation of the anti-cancer activity of MSNs based drug and gene delivery platform, tumor microenvironment-responsive capped MSNs loaded with hydrophobic or hydrophilic chemotherapy drugs in core siRNAs in mesoporous are the most advanced and effective strategy so far.223, 224 Such responsive capping reagent is decapped because of the specific environment difference of tumor site such as acidic and high redox nature. Administration of such MSNs with the least premature drug and siRNA release behavior significantly will improve the outcome of gene therapy (Fig. 11 E). 225, 226</p><!><p>Up to now, carbon-based nanomaterials (CBNs) have been also actively investigated due to their advantageous chemical and physical properties. (i.e., thermal effect, electrical conductivity, and high mechanical strength).227–229 This perspective highlights different types of carbon-based nanomaterials such as carbon nanotubes (CNTs), graphene, fullerene, carbon quantum dots and carbon fiber currently used in biomedical applications.230–233 Among the different applications, CBNs are hosting enormous interests as siRNA delivery platform due to their extremely large surface area, with every atom exposed on its surface, which allows for ultra-high functionalization and loading capacities, high affinity with the biology molecule and facile surface functionalization (Fig. 12A).234–236</p><p>Carbon nanotubes are hollow 1-dimension CBNs with a typical diameter of 1-2 nm and length ranges from 50 nm to 1000 nm. Their tubular morphology endows them to easily and efficiently for cellular uptake by acting as nano-shape needles that make the CNTs become the most widely used CBNs in bio-application .237–239 Meanwhile, they are capable to encapsulate of cargos and maintain its function with controlled release of loaded molecules which is becoming increasingly important in gene delivery application. For siRNA delivery, both single-walled (SW) and multi-walled (MW) carbon nanotubes were modified or conjugated with siRNA for various disease treatments. Zhang et al. proposed an efficient vector based on SWNTs for siRNA to knockdown murine telomerase reverse transcriptase expression in murine tumor cells on both in vitro and in vivo levels for controlling of key signaling regulators in cancer cells.240 Guo et al, they established the capacity of cationic MWNT to deliver the apoptotic siRNA against PLK1 (siPLK1) in Calu6 tumor xenografts, which express high transfection efficiency comparing with cationic liposome. Moreover, the MWNTs/siRNA complexes significantly improve the xenografts animal survival rate. This study demonstrated the potential therapeutic efficacy of cationic MWNT as a promising safety siRNA delivery (Fig. 12C).241 Notably, a novel "smart" single-walled carbon nanotubes (SWNTs) to achieve nanotube-siRNA conjugates with reductive cleavable disulfide bond to enable intracellular controlled siRNA releasing from nanotube surfaces was developed by Liu et al as a nonviral molecular transporters for the delivery of siRNA into human T cells and primary cells. This smart siRNA platform exhibited efficient RNA inference of CXCR4 and CD4 receptors on both human T cells and peripheral blood mononuclear cells (PBMCs). The cellular penetrate ability and RNAi efficiency of nanotubes are far exceeding those of existing liposomes due to the underlying hydrophobic interactions on nanotube-mediated molecular delivery (Fig. 12B).242</p><p>Graphene, a newly discovered two-dimensional carbon nanosheet structure,243–245 was demonstrated with additional remarkable colloidal stability, easily tunable surface functionalization, and good biocompatibility as a promising nano-carrier for safe and efficient gene transfection.246–248 Recently, its oxidized form of graphene oxide (GO) were used in a pioneering study by Zhang et al. for PEI and PEG dual-modification step as complexing Ckip1-targeted siRNA, following by deposited onto the pre-prepared Titania nanotubes surface via cathodic electrodeposition to obtain the nGO-PEG-PEI/siRNA biofunctionalized implant for enhanced osteogenesis (Fig. 12D). 249 The results demonstrated that together with Ckip-1 siRNA of osteogenic potential, the both osteogenic differentiation on NT-GPP/siCkip-1 implant surface in vitro and the new bone formation around implant in vivo was significantly promoted which presents a promising implant biomodification siRNA delivery system based on functionalized GO.249 Ren et al, prepared a non-viral carrier (GO-PLL-SDGR) to deliver VEGF-siRNA for targeting cancer therapy. This GO-PLL-SDGR/siRNA complex could deliver VEGF-siRNA into tumor cells and down-regulate the expression of VEGF effectively to inhibit the tumor growth in a S180 xenograft tumor model.250 These results indicated that targeting cationic GO /siRNA delivery system has great application potential in cancer therapy. Moreover, a GO based siRNA/drug co-delivery system was developed by Zhang et al, which concluded a PEI-grafted GO nanocarrier for delivery of Bcl-2 siRNA and doxorubicin. The results reveal that the PEI-GO not only shows significantly lower cytotoxicity but also leads to significantly enhanced anticancer efficacy that may provide insight into designing and constructing GO based novel drug/gene co-delivery nanocarriers (Fig. 12E).251</p><p>Other CBNs such as carbon quantum dots (Cdots) are also widely applied on drug/gene delivery due to their photoluminescence natural.252, 253 For instance, Wu et al developed a novel targeting theragnostic nanocarrier based on fc-rPEI-Carbon quantum dots (rPEI-Cdots)/siRNA which can be used simultaneously in lung cancer diagnosis and gene therapeutics. The in vitro results showed that treating with fc-rPEI-Cdots/ pooled siRNA complex for 3 days is significantly reduced to nearly 30%.253 Wang et al also demonstrated a Cdots-based and PEI-adsorbed complexes both as imaging agents and Survivin siRNA nanocarriers, which indicate that Cdots-based nanocarriers is a promising platform of both siRNA delivery and imaging for cancer treatment.254</p><p>Overall, extensive researches have elevated the CBNs as one of the highlighted nanocarrier platform for siRNA delivery owing to their unique combinations of structure, energetic, and electrical properties. The major disadvantage of CBNs to siRNA is their non-biodegradability that may induce a range of adverse health effects. This point has been demonstrated in several studies and need for more future investigations.255, 256</p><!><p>It is encouraging that the recent announcement of Alnylam on the remarkably successful phase III results of Patisiran after two decades of the discovery of RNAi.257 Meanwhile, resurgence in clinical trials using RNAi occurred in 2012, there are more than 30 RNAi-based therapeutics currently in clinical trials, and several of these are Phase III trials. However, considering the complicated biosystem and time consuming preclinical study, there are still a number of issues to address due to instability of siRNA in body circulation and low bio-application. Luckily, it has been revealed that one of the major challenges indeed for RNAi-based therapy is the development of delivery system. To solve this problem, a large number of carriers have been reported for siRNA delivery including virus and non-virus based platforms during the past two decades.</p><p>Among all kinds of siRNA carriers, inorganic-organic hybrid nanoparticles emerge as a diverse set of versatile platforms for both fundamental studies and potential clinical translation applications. (Fig. 13) Here, we summarized the various choices for a broad range of covalent or non-covalent approaches for inorganic nanoparticle surface engineering to provide the ability to further optimize siRNA delivery in vitro and in vivo. Through the results, it could be concluded that the size and morphology of the inorganic cores showed great flexibility of tailoring to achieve better cellular uptake, blood circulation and specific bio-distribution. Meanwhile, the unique optical, magnetic and thermal properties of inorganic nanoparticles also have been extensively used to explore the crucial mechanisms of siRNA transportation in vitro and in vivo, such as endosome escape, reticuloendothelial system (RES) capture, kidney clearance, and enhanced penetration and retention (EPR) effect, etc. Decades of studies demonstrated that the therapeutic capability of inorganic-organic hybrid systems for siRNA delivery in a broad range of disease models, showing great promise for future therapeutic applications. However, the remaining obstacles such as undesired systemic toxic of hybrid systems hinder the translation of these delivery systems into clinic. Therefore, a lot of efforts are still needed to fill the space between basic research and clinical translation for these delivery systems (Fig. 13), for example, engineering and manufacturing siRNA-loaded inorganic nanoparticles with homogeneous size, composition, and surface charge in a simple, fast, and inexpensive manner; revealing the safety effect of siRNA-loaded nanoparticles in regards to administration, clearance route, and potential long-term immune response. Especially, with the development of immunology, more careful evaluation of the immune responses of biological system to the siRNA-nanoparticle complex after administration should be developed. For instance, the potential potently immune system response induced by siRNA molecules and delivery vectors, which is considered to be a great challenge - the contradiction among therapeutic effects from target-specific, RNAi-mediated gene silencing and those caused by nonspecific stimulation (i.e. inflammation/toxicity) of the innate immune system. Better understanding of the fundamental aspects of nanomaterial interactions with biological systems such as organismic, tissue, and cellular levels will further promote the development of this area.</p><p>Owing to the great properties of some inorganic nanoparticles, such as the excellent biostability of AuNPs and IONPs, the biodegradability and biocompatibility of silicon materials, great potential for clinical translation applications has enlightened the pathway to the future. Taken together, RNAi therapy based on siRNA has the potential to revolutionize treatment of various kinds of cancers and other diseases. Innovative delivery systems based on inorganic-organic hybrid nanoparticles have great promising to further enrich the fundamental theory of siRNA therapy, and develop safe and efficient delivery platforms for personalized cancer therapy in the future.</p>

PubMed Author Manuscript

Ultrafast charge separation dynamics in opaque, operational dye-sensitized solar cells revealed by femtosecond diffuse reflectance spectroscopy

Efficient dye-sensitized solar cells are based on highly diffusive mesoscopic layers that render these devices opaque and unsuitable for ultrafast transient absorption spectroscopy measurements in transmission mode. We developed a novel sub-200 femtosecond time-resolved diffuse reflectance spectroscopy scheme combined with potentiostatic control to study various solar cells in fully operational condition. We studied performance optimized devices based on liquid redox electrolytes and opaque TiO 2 films, as well as other morphologies, such as TiO 2 fibers and nanotubes. Charge injection from the Z907 dye in all TiO 2 morphologies was observed to take place in the sub-200 fs time scale. The kinetics of electron-hole back recombination has features in the picosecond to nanosecond time scale. This observation is significantly different from what was reported in the literature where the electron-hole back recombination for transparent films of small particles is generally accepted to occur on a longer time scale of microseconds. The kinetics of the ultrafast electron injection remained unchanged for voltages between +500 mV and -690 mV, where the injection yield eventually drops steeply. The primary charge separation in Y123 organic dye based devices was clearly slower occurring in two picoseconds and no kinetic component on the shorter femtosecond time scale was recorded.Dye-sensitized solar cells (DSCs) are promising candidates for solar energy conversion applications. These devices do not rely on rare or expensive materials, so they could be more cost-effective than cells based on silicon and thin-film technologies. Recently, DSCs device efficiency has reached a maximum power conversion efficiency of over 12% using donor-bridge -acceptor (D-π -A) zinc porphyrin dye in combination with a cobalt-based redox mediator 1 .The performance of DSCs is based on kinetics competition between the electron injection from the sensitizer to an electron collecting material, usually TiO 2 , regeneration of the oxidized dye with redox electrolyte and unwanted back reactions of injected electrons recombining with oxidized dye molecules or oxidized species of redox electrolyte 2 . A challenge in this field is that the kinetics of charge carriers may be altered in complete devices showing top performances. A deep understanding of many parameters controlling the overall performance is crucial for achieving improvements in performance. Despite numerous studies, there is still a debate on the electron injection time scale for the optimized solar cells and according to the proposed "kinetics redundancy", the optimized solar cells might not have ultrafast electron injection kinetics 3 . Existing studies are mainly based on the classical pump-probe transient absorption spectroscopy, which is widely used to measure the kinetics of electron injection processes. Since optical transparency is required, to perform transient absorption studies, only

ultrafast_charge_separation_dynamics_in_opaque,_operational_dye-sensitized_solar_cells_revealed_by_f

<!>Results and Discussions<!>Femtosecond diffuse reflectance spectroscopy on operational DSC device based on Z907<!>Methods<!>Solar cell fabrication.<!>Photovoltaic characterization.

<p>model systems based on a transparent TiO 2 thin film sensitized with various dyes and semiconductors in different environments (solid samples or in solution) were investigated so far [3][4][5][6][7] . It should, however, be noted that the most efficient liquid-based solar cell devices are not transparent. Indeed, these devices are based on a double layer of TiO 2 film, which contains a scattering layer made of 400 nm TiO 2 particles deposited on top of a mesoporous transparent layer 8 . The resulting light transmittance of the cell is less than 15% in the visible region and, hence, conventional transient absorption spectroscopy in transmission mode cannot be applied in this case. Despite the importance of the subject, the kinetics of electron injection in actual optimized, opaque dye-sensitized solar cell devices under working conditions has not so far been reported.</p><p>We aim here to investigate the dynamics of charge carriers directly in fully functional devices, using potential control and state-of-the-art pump-probe diffuse reflectance spectroscopy. Despite the great potential of the latter technique, only a few studies can be found in literature investigating its implementation and application. We aim to demonstrate that diffuse reflectance spectroscopy is of great value for time-resolved analysis of photophysical processes in opaque or highly absorbing materials. Time-resolved diffuse reflectance spectroscopy was first reported by Wilkinson et al. 9 in microsecond time regime in 1981 followed by Bowman et al. 10 and Asahi et al. 11,12 . The technique was utilized on scattering systems like powders of organic microcrystals, and by Furube et al. 13 on DSCs under open circuit condition. Here we have developed an ultrafast time-resolved pump-probe diffuse reflectance spectrometer with a sub-200 femtoseconds time-resolution. This required application of novel optical design for the collection of diffuse reflected light. In addition and for the first time, we combined the femtosecond time-resolved diffuse reflectance laser spectroscopy with potential control and photovoltage measurements.</p><p>Furthermore, the technique has enabled us to investigate the charge separation kinetics in DSCs based on photoanodes of other TiO 2 film morphologies. For example, we studied samples of anodized nanotubes on Ti foil 14,15 and nanostructured fibers 16,17 . These samples have exhibited promising behavior in cell performance but are not optically transparent and are not suitable for investigation with pump-probe transmission based transient absorption technique.</p><p>Our studies reveal that the charge separation dynamics in Ru-based dye in the complete device is ultrafast and is indeed affected by the morphology of the TiO 2 film. We observed an early charge recombination in scattering TiO 2 particles, TiO 2 fibers and anodized TiO 2 nanotubes. These recombinations had different amplitudes and were not previously reported for small particles and are rationalized in terms of different electron mobility and trapping states in different TiO 2 films. Under an applied voltage bias condition from + 500 mV up to -690 mV, the kinetics of the electron injection from the dye excited-state into the oxide remains ultrafast. However, the injection yield decreases at the bias point of -690 mV. In contrast to Ru-complex based dye, the organic D-π-A dye Y123 exhibited slower charge injection kinetics. The excited-state lifetime of Y123 dye is measured to be 50 ps. The time constant of the electron injection process is measured being 1.1 ps. While this classifies as ultrafast, it is about one order of magnitude slower than for Ru-based dyes, which was measured to have features in femtosecond time scale.</p><!><p>Figure 1a shows the schematics of the standard optimized high-performance liquid solar cell. In the conventional DSC scheme, the mesoporous layer is made of 20 nm-diameter interconnected TiO 2 particles. Although this structure offers a large surface area for dye adsorption, Rayleigh scattering with this size of TiO 2 particles is small, resulting in high transparency of the dye-sensitized film in a broad spectral region. A significant amount of light (70% in the near infrared region) is transmitted without interacting with dye molecules in the cell. The working electrode applied in highly efficient devices is based on a TiO 2 double layer film 18 , sensitized with dye molecules on top of a TiCl 4 -treated conductive glass. The structure of these samples is shown in Fig. 1a. The first layer is a transparent mesoporous anatase TiO 2 film, consisting of interconnected spherical nanoparticles (20 nm). Another layer made of 400 nm-diameter TiO 2 particles is deposited on top of the transparent layer. Figure 1b shows the total transmittance, total reflectance and total absorptance of the Z907 dye-sensitized TiO 2 double layer film based DSC photoanode. The 400 nm particles act as light scattering centers enhancing light absorption by increasing the light pathway within the film. Consequently, the total transmittance of the cell in the visible and near-infrared region is less than 15% as it can be seen in Fig. 1b. This suggests that the diffuse reflectance spectroscopy is the only versatile optical laser spectroscopy technique capable of studying such devices. The Kubelka-Munk function, F(R) spectra is derived from diffuse reflectance of the film according to equation (3), presented in the method section. The F(R) spectrum is compared with the absorptance spectrum of the opaque photoanode in Fig. 1b. As it is seen, the Kubelka-Munk spectrum follows the shape of the absorptance curve, and the similarity in both spectra is observed. The peak around 520 nm corresponds to the Z907 dye ground state absorption that serves as an absorbing medium. The shoulder at 380 nm corresponds to the absorption of TiO 2 substrate that serves as the scattering media in Kubelka-Munk theory.</p><p>Two types of liquid electrolyte-based devices were selected for the present study. The first type of DSC is prepared with a Ru-complex sensitizer (Z907) in combination with an iodide/ triiodide based redox electrolyte in 3-methoxypropionitrile solvent. This combination was reported to result in highly stable devices when subjected to light and thermal stress during long-term aging 19,20 . The second type of cell is based on the organic D-π -A, sensitizer Y123 and a cobalt complex-based redox electrolyte. This type of device yielded a power conversion efficiency of over 9% 21,22 and over 12% in combination with a porphyrin dye 1 . The thickness of both TiO 2 layers affects both photocurrent and photovoltage, which were optimized in earlier studies in terms of final power conversion efficiency 8 .</p><!><p>sensitizer. In order to unravel the electron injection dynamics in dye-sensitized opaque solar cells, we utilized pump-probe diffuse reflectance spectroscopy. We applied this technique to the study of Z907 dye-sensitized TiO 2 films, which are from the same family of N719 and N3 Ru-based dyes. According to earlier transient absorption studies on N719 (cis-bis (isothiocyanate)bis(4,4′ -dicarboxylic-2,2′ -bipyridyl) ruthenium(II)) and N3 (cis-di(thiocyanate)bis(2,2′ -bipyridyl-4,4′ -dicarboxylic acid)ruthenium(II)) dye-sensitized TiO 2 films, the transient absorption spectrum around 800 nm is assigned to the oxidized dye molecules 13,[23][24][25] and the absorption spectrum around 1200 nm is attributed to absorption by conduction band electrons 13,26,27 . The kinetics of electron injection in the complete device is studied by monitoring the evolution of oxidized dye molecules and photo-injected electrons in TiO 2 . The oxidized dye molecules are monitored at the characteristic absorption onset in the visible wavelength region at 670 nm or in the NIR region at 840 nm, and photo-injected electrons are monitored at 1200 nm.</p><p>Figure 2a,b compare the early and later time evolution of absorptance of oxidized dye molecules anchored on three different TiO 2 films in the presence of MPN solvent. The samples are excited at 600 nm. The time delayed diffuse reflected probe beam is measured at 840 nm. Transient absorptance change is extracted from the measured transient diffuse reflectance change given by equation (2), depicted in the method section. In Fig. 2a,b, it is seen that in the presence of MPN solvent, the kinetics of electron injection in double layer film resembles that of transparent film made of small TiO 2 particles. All samples have an instrument response-limited transient absorptance onset within 200 fs and a slow rise of the signal with a time constant of 1.1 ps.</p><p>The first ultrafast response limited rise of the signal corresponds to the ultrafast electron injection from dye to TiO 2 conduction band. Ultrafast fluorescence studies performed by Chergui and coworkers 28 on N719 sensitized TiO 2 small particles films revealed that the electron injection occurs with a time constant of 10 fs for non-thermalized levels of the dye and 120 fs from the thermalized level. The second slow rise component up to 5 ps is observed in all three TiO 2 films was also previously reported in measurements of transparent films by different research groups 29,30 . Studies by Wenger et al. 29 assigned this feature to the presence of dye aggregates in the films, which have a larger distance for electron injection and, therefore, less electronic coupling for electron transfer process. Sundström et al. proposed another description in terms of a two-state mechanism. They assigned the fast and slow components to the injection from the singlet and the triplet excited-states of the ruthenium complex, respectively 30 .</p><p>Figure 2c illustrates the long time kinetics of the evolution of oxidized dye molecules up to 500 ps after excitation in the working cell based on TiO 2 double layer film. Remarkably, the kinetics features a decay component in several hundred picoseconds after excitation. The trace is fitted by an exponential decay function with a time constant of 4 ns. It should be noted that all measurements shown in Fig. 2 are performed at very low excitation intensities. For measurements shown in Fig. 2, the excitation energy of each pulse at the sample is 200 nJ. The repetition rate is 0.5 kHz. The beam diameter is close to 500 μm. Under these conditions, the excitation irradiance is 102 μJ cm −2 . With having the repetition rate of 0.5 kHz, the excitation power is 51 mW cm −2 , which is equivalent to 50% of the sun irradiance power at AM 1.5 condition. In addition, the number of photons normalized to the volume of TiO 2 within the irradiation area is about 2.15 × 10 11 . This is significantly smaller than the number of adsorbed dye 3) depicted in the method section. The total transmittance of the cell in the visible and infrared region is less than 15%. The Kubelka-Munk function spectrum follows the shape of the absorptance spectrum. molecules on the microscopic surface of TiO 2 film, within the irradiated area which is 9.8 × 10 16 (assuming one monolayer of adsorbed dye molecules). Hence, our observations are not due to some non-linear effects.</p><p>The excitation intensity dependence of the observed kinetics is also presented in Supplementary information, Figure S4. All traces measured at low excitation intensities can be fitted by a single exponential function. This early decay kinetics is again observed when the evolution of oxidized dye molecules is monitored in the visible wavelength region at 670 nm (Supplementary Figure S5). Moreover, the same depleting kinetics can be observed (Fig. 3b) when photo-injected electrons are monitored at 1200 nm in the presence of the electrolyte. Therefore, we assign the early decay kinetics observed for the complete opaque DSC in the presence of MPN solvent, to the early back recombination of photo-injected electrons with oxidized dye molecules.</p><p>The kinetics of back recombination is even more strongly accelerated in the presence of the electrolyte. Traces in Fig. 3a compare the measurements in the presence (black markers) and absence of redox electrolyte (red markers) on double layer TiO 2 film. In the vicinity of the redox electrolyte, the formation of the signal is again ultrafast, and the sub-200 fs ultrafast component is still present. As it can be observed, 26% of the signal of the oxidized dye molecule decays with a time constant of τ 1 = 9 ps. The same fast decay kinetics are also present in the samples dipped in the redox-inactive ionic liquid, 3-methyl-1-ethylimidazolium bis (trifluoromethane) sulfonimide (EMITFSI), (see Fig. 4d). Therefore, the observed kinetics cannot be assigned to processes like reductive quenching of the excited-state of the dye molecules by redox electrolyte. The accelerated charge recombination in the presence of both redox active and redox inactive electrolyte in some picoseconds after excitation is rationalized by electric fields induced by the charges in the electrolyte and charge screening effects. The local electric field induced by ions present in the redox electrolyte or ionic liquid at the surface is accelerating the charge recombination between electron-hole geminate pair after initial charge separation.</p><p>Figure 3b provides more evidence, as photo-injected electrons in TiO 2 are directly monitored at 1200 nm. The trace is fitted with two exponential decay functions. The rate constant of the fast decay component of photoelectrons measured at 1200 nm is 1.1 × 10 11 s −1 and for the slower decay kinetic is 5.917 × 10 8 s −1 , giving a lifetime of 8.9 ps and 1.7 ns, respectively. Interestingly, these time constants are consistent with the kinetics fit values of measurements at 840 nm monitoring oxidized dye molecules. Taken together, the observed decay kinetics at 840 nm and the mirror kinetics at 1200 nm are due to an early back recombination of photo-injected electrons with the oxidized dye molecules anchored on the surface of TiO 2 particles. Therefore, a significant observation in our measurements of the DSC devices based on scattering particles and all other TiO 2 morphologies (as it is discussed below), is that the kinetics of electron back recombination with oxidized dye molecule has features in the picosecond to nanosecond time scale. This observation is remarkably different to what is normally stated in the literature for model systems of transparent films made of small particles. The small particles based films are the only morphology that has been studied to date. In earlier studies of small TiO 2 particles sensitized with Ru dyes based on transient absorption spectroscopy at visible or NIR region or by 2D-IR spectroscopy 23,[31][32][33] , such electron back recombination was not observed and the oxidized dye molecule was accepted to be stable until much longer time scale of microseconds. We observe that the kinetics is accelerated in full device relative to what is so far accepted for small particles. In a complementary study, presented in Supplementary Table S1, the photovoltaic, optical and structural characteristics of the transparent and double layer based devices are depicted. The value of photocurrent normalized to the light absorptance of the scattering layer is about 30% less than that for transparent layer based device. This indicates that in big particles some fraction of photoelectrons are lost and is consistent with the early back recombination of electrons and oxidized dye molecules observed in laser spectroscopy measurements.</p><p>In addition, the pump-probe diffuse reflectance technique has also enabled us to investigate the electron injection profiles in many other DSC devices. These studies include measurements of different opaque nanostructured TiO 2 films such as TiO 2 nanofibers 16 and TiO 2 nanotubes. TiO 2 nanotubes are prepared by anodization of Ti foil as the substrate 14 . Due to opacity, these samples could never be studied by transmission based transient absorption technique.</p><p>Figure 4 shows the dynamics of electron injection on Z907 dye-sensitized TiO 2 films of different morphologies monitored at 840 nm. Panel a shows the kinetics of charge separation in Z907 dye-sensitized standard double layer based DSC device in the presence and absence of redox electrolyte. Panel b depicts the pump-probe diffuse reflectance measurements on Z907 dye-sensitized anodized TiO 2 nanotube film on Ti foil. The same studies are performed on dye-sensitized TiO 2 nanostructured fibers. Measurements of fibers in the presence of cobalt-based electrolyte (CO II /CO III ) and also redox-inactive electrolyte (EMITFSI), are shown in panel c and d, respectively. It is interesting that in all measurements the electron injection is still in the ultrafast regime and recombination features with different amplitudes comparable to that of double layer film is present. In standard full DSC based on double layer film 30% of the signal decays in 20 ps after excitation, this compares to only 16% for DSCs based on anodized nanotubes.</p><p>The observed kinetics in the presence of redox-inactive ionic liquid suggests that the decay kinetics cannot be due to the reductive quenching of the dye excited-state by redox electrolyte. The observed difference in early back recombination of photoelectrons in the TiO 2 films of nanoparticles and anodized nanotubes is rationalized in terms of morphological parameters of the two films such as trap state distribution.</p><p>We hypothesize that photo-injected electrons may get trapped in the TiO 2 particle surface states where they form geminate pairs with holes (oxidized dye molecules) and result in the observed early fast recombination in different TiO 2 morphologies with different amplitudes. It should be noted that, our control studies (Figure S3) shows that at low excitation intensities of 0.25 sun irradiance at AM1.5, the excited dyes inject more than 3.8 × 10 16 cm −3 electrons into the TiO 2 , which is much smaller than the trap state density (10 18 cm −3 to 10 20 cm −3 ) The observed kinetics in the vicinity of electrolyte measured at 1200 nm monitoring the kinetics of photo-injected electrons in TiO 2 . The solid lines correspond the fit to the result by exponential function. The time constant of exponential fit to the measurements at 840 nm is τ 1 = 9.22 ps and for trace measured at 1200 nm is τ 1 = 8.9 ps. The pump wavelength is 530 nm and pulse intensity is 200 nJ. reported for TiO 2 films 14 . One should also consider the energetic distribution of these traps. For instance, the trap state distribution in the anodized nanotubes is measured using macroscopic techniques like charge extraction experiments by Hagfeldt et al. 14 . The nanotubular electrodes have a trap state distribution significantly different from nanoparticulate electrodes. Nanotubes possess relatively deeper traps with a characteristic energy of the exponential distribution more than twice than that of nanoparticulate electrodes. Throughout time-resolved terahertz studies, Schmuttenmaer et al. 34 have claimed that the low mobility in polycrystalline TiO 2 nanotubes is not only due to scattering from grain boundaries or disorders as is in other nanomaterials but instead results from a single sharp resonance from exciton-like trap states. These observations are in good agreement with our spectroscopy studies. Indeed, electrons can get more localized in energetically deeper traps in nanotubes films and, therefore, less early back recombination with oxidized dye molecule is observed in these films in comparison with nanoparticles based films.</p><p>Our observations show direct evidence of an ultrafast electron injection occurring in a complete Ru-dye based DSC device. This has not been confirmed so far for DSCs having all components like scattering layer, electrolyte, conductive glass, etc. Charge injection from the amphiphilic Ru II (bipyridyl) Z907 dye in all different TiO 2 morphologies was observed to take place in the sub-200 fs time scale. We observed that the kinetics of charge separation is indeed influenced by the morphological parameters of the TiO 2 substrate. The photo-injected electron in TiO 2 fibers, nanotubes, and 400 nm particles shows prompt back recombination kinetics with oxidized dye molecules in the picoseconds-nanoseconds time scale after excitation. Electronic parameters like density of trap states and energetic of trapped electrons, i.e. how deep electrons are trapped, and consequently the mobility of electrons, might play a vital role in the behavior of photo-injected electrons after initial interfacial charge separation. We have combined the diffuse reflectance spectroscopy with potentiometric techniques to monitor the electron injection process in DSC standard device under working condition. Figure 5a shows the photovoltaic characteristics of the Z907 sensitizer and iodide based redox electrolyte device. The photovoltaic parameters of the device at full sunlight are; short circuit current density (Jsc) of 15.5 mA/cm 2 , open circuit photovoltage (Voc) of 698 mV, fill factor (FF) of 0.71 and power conversion efficiency (PCE) of 7.6%. Figure 5b presents the typical transient absorptance of the cell in short circuit condition and under different bias voltages of + 500 mV, − 500 mV (close to max power point) and − 690 mV. Transient absorptance traces are not easily distinguishable. All traces in this figure can be fitted by exponential function with close fitting parameters; a component with a lifetime of 50-70 ps, and the flat behavior until hundred picoseconds, which is shown in inset. By raising the bias to − 690 mV close to open circuit condition, the amplitude of the observed signal is almost half of the others while the kinetics remain similar. In other words, as the amplitude of the pump-probe signal is proportional to the number of oxidized dye molecules, with increasing the applied bias voltage the quantum yield of electron injection is reduced. The energy level diagram, which contains the energy level of the conduction band of TiO 2 (CB), HOMO and LUMO level of Z907 dye and iodide-based redox electrolyte (Eredox) and trap state distribution are illustrated in Fig. 5c. Increasing the forward bias voltage would raise the quasi-Fermi level position by filling up the trap states to some extent in TiO 2 , which is highlighted in Fig. 5c. As it is observed by shifting the quasi-Fermi level toward the LUMO level of the dye, the energy difference gets noticeably smaller. In example the energy difference at the bias level of − 600 mV is 75% of the energy difference at the bias level of − 500 mV and further reduces to 50% at a higher bias level of − 700 mV.</p><p>It should be noted that at the bias level of − 690 mV, the amount of voltage in the films is less than this value due to the dark current. The voltage drop in the cell at a voltage bias of − 700 mV is estimated as 90 mV. By taking account of this voltage drop, the respective amount of the cell photocurrent measured at bias voltages up to − 500 mV and at − 690 mV is consistent with the respective amplitude of the pump-probe signals. In our control studies (Supplementary Figures S6 and S7), the photovoltage induced by each laser pulse is estimated to be only of μV order when the cell is biased in the conventional voltages of hundreds of mV. This indicates that our pump-probe measurements under potentiostatic condition can be considered as a perturbation technique. Electron injection in DSC based on D-π-A organic sensitizer and cobalt electrolyte. One of the most significant advances in design of light-harvesting materials is the so-called donor-conjugated linker-acceptor (D-π -A) organic dyes. In comparison with Ru-based dyes, organic dyes have higher molar extinction coefficient and can be readily designed for a desired absorption spectrum 1,[35][36][37] . These molecular structures look attractive in terms of electron donor-acceptor interactions 35 . In order to understand the electron injection dynamics in these systems, femtosecond diffuse reflectance spectroscopy is applied to DSC devices containing Y123 and a cobalt complex based redox electrolyte. Three different morphologies of the photoanode are used, and the results are compared in Fig. 6. In these measurements, the pump beam wavelength is 600 nm to excite the dye, and the probe beam is 840 nm. Upon laser excitation, an ultrafast formation of the signal happens in 200 fs, followed by a fast decay of the signal to 50% of its amplitude in 2 ps. After two picoseconds, the signal reaches a plateau in all the 3 morphologies of the TiO 2 layers. Unlike Ru-based dyes, no obvious difference is observed in the kinetics for measurements in the presence of MPN solvent and redox electrolyte and the medium has no influence on the observed kinetics (measurements in the presence of MPN solvent are provided in Supplementary information Figure S9). The slow growth component of the signal in hundred picoseconds time scale seen in the films made of scattering particles or the double layer film, is assigned to the electron injection from dye aggregates. This component is removed from the signal when the sample is immersed in acetonitrile solvent for several hours (Supplementary Figure S10). TiO 2 films prepared with scattering particles might have enough space to accommodate aggregated dye molecules within the pores, which are loosely in contact with the TiO 2 layer. This makes a larger distance for electron transfer between the dye and TiO 2 .</p><p>In order to study the mechanism of the very fast decay of the signal in 2 ps, we performed the transient broadband absorption measurements. We compared the broadband transient absorption of Y123-sensitized TiO 2 film with that of Y123 dye in solution as a reference sample where interfacial electron transfer process is deactivated. By comparison of the recorded spectra of the dye-sensitized films with the dye in solution we can clearly resolve the spectral absorption contribution of the excited-state and oxidized state of the Y123 dye. The transient absorption spectra of the samples measured at NIR region is also shown in Supplementary Figure S11. The ground state optical absorption spectrum of the dye is provided in Supplementary Figure S8. For the dye in solution (Fig. 7a), the negative peak at the characteristic ground state absorption of the dye around 520 nm, is attributed to the ground state bleaching of the dye formed upon photo-excitation. In Fig. 7a, a positive transient absorption is observed in the wavelength region from 630 nm up to 700 nm. This transition is assigned to excited-state absorption of dye molecule. The dye excited-state relaxation time constant is 52 ps and has a mirror-like kinetics to ground state relaxation. The transient absorbance spectrum of the dye-sensitized TiO 2 films is presented in Fig. 7b. In this sample, the ground state bleaching is observed at 580 nm, which is around 60 nm shifted with respect to the steady-state absorption onset of the dye.</p><p>This red-shift in the transient absorption spectrum is an evidence of a Stark-shift of the absorption spectrum of the dye molecule. The Stark-shift is explained by the shift of ground state absorption of the dye molecule due to the local electric field induced by the electric dipole of the neighbor dye molecules. This effect was also previously reported for the same family of D-π -A dyes 36,37 . For the dye-sensitized TiO 2 film the positive absorption feature is extended over the 630 nm wavelength regions. This positive feature is now assigned to a contribution of both of the excited-state absorption of the dye and the absorption by oxidized dye molecule formed upon injection of electrons into TiO 2 conduction band.</p><p>Figure 8 compares the kinetics of a Y123 sensitized TiO 2 film probed in two different wavelength regions of 690 nm and 740 nm. According to Fig. 7a, at 690 nm the excited-state of Y123 has absorption while at 740 nm the excited-state does not absorb. Therefore, the blue trace in Fig. 8 recorded at 740 nm, represents a pure monitoring of the kinetics of the electron injection process. The formation of the signal, which reflects the electron injection time, is occurring in picosecond time scale and is fitted with an exponential growth function with a time constant of 1.1 ps. As a result, we observe that in the Y123 D-π -A dye, the charge injection kinetics is not as fast as in Ru-based dyes, as reported by Chergui and co-workers 28 .</p><p>The difference in the electron injection time in the Y123 with Ru-based dyes should be due to the D-π -A structure of this dye and its coupling with the TiO 2 film. In the Y123 dye, the electron donor part is the triphenylamine unit, and the acceptor orbitals are located on the cyanoacrylate group. Cyclopentadithienophene (CPDT) is working as a π -bridge between the donor and acceptor parts for the conjugation of electrons. This bridge helps increasing the dipole moment and enhancement in the molar extinction coefficient of the dye molecule 38 . Our results suggest that the relaxation of the excited-state is fast with a time constant of 52 ps. This process competes with electron injection into the lower lying conduction band of TiO 2 . Moreover, non-adiabatic charge transfer from a molecular electronic excited state into a continuum of acceptor levels constituted by the conduction band of a semiconductor can be described by Fermi's golden rule 2 . Due to the very high density of acceptor levels, the nuclear factor in the equation tends to a constant value. As a consequence, the thermodynamics of the process, the temperature, and the reorganization energy are not expected to affect the injection dynamics. The electronic coupling between the donor and the acceptor (electronic coupling matrix element squared |H] 2 ) is then likely to control in a large extend the electron transfer rate. As |H| 2 depends exponentially upon the charge transfer distance, the adsorption geometry and the electronic coupling between the dye's HOMO and the empty d 4 orbital manifold of Ti IV sites on which the dye is anchored must be determining the electron injection time.</p><p>In summary, our experimental technique allowed us to reveal the charge separation dynamics in a complete opaque solar cell device under applied bias voltage. Also, the interfacial charge separation in different dye-sensitized opaque TiO 2 nanostructured interfaces was determined. We showed that this technique could be a powerful and sensitive tool for measurements of opaque and highly absorbing materials. Coupling diffuse reflectance spectroscopy with potentiometric characterization tools gives a unique possibility to study charge carriers in devices under real operational conditions. In Ru-complex based solar cells, the kinetics of electron injection is confirmed to be ultrafast and is not affected by the bias voltage. In standard opaque and other TiO 2 morphologies based devices, after ultrafast electron injection, an early recombination of photo-injected electrons with oxidized dye molecules is observed with features in the picosecond to nanosecond time scale. The respective amplitude of this recombination process is influenced by morphological parameter i.e. trap states in TiO 2 films. We found that the ultrafast electron injection kinetics is not influenced by trap state filling upon increasing forward bias up to − 500 mV. By applying a higher voltage close to open circuit conditions, and shifting the Fermi level of TiO 2 closer to the dye excited-state level, the electron injection is less efficient but the injection kinetics is still ultrafast. For a DSC with a Y123 organic dye, the dynamic of the excited-state of the dye and the kinetics of electron injection process significantly differ from Ru-base dyes. The excited-state relaxation in the Y123 dye molecule competes with electron injection into TiO 2 . In contrast to Ru-complex based dyes, which show ultrafast electron injection in femtoseconds time scale, the electron injection for the Y123 dye is precisely monitored to occur within 2 ps after excitation of the dye.</p><p>Finally, the technique proposed here will be an excellent tool to be implemented for studies on highly absorbing materials, such as the new emerging perovskite based devices and can open up a new avenue of characterization research.</p><!><p>Pump-probe femtosecond diffuse reflectance spectrometer. In principle, the configuration of the diffuse reflectance spectroscopy is similar to the traditional transient absorption in transmission mode. The differences are: firstly in the probe beam geometry to collect the diffuse reflected light, which carries the information of transient species, secondly the sample structure and thirdly the optical model in data treatment. Here, a new optical scheme for collection of diffuse reflected light is designed which gives a unique time-resolution of sub-200 fs (Supplementary Figure S1). In this configuration, diffuse scattered light from the sample is collected, collimated, and focused onto the detector with two coupled off-axis 90° parabolic mirrors. Having this configuration, the large solid angle of light collection results in improved signal to noise ratio. The other advantage of using parabolic mirrors over lenses is that no further dispersion is introduced to the pulses; therefore, a better time-resolution is expected. Indeed, in this configuration, the time-resolution is limited by the time broadening of the beam in the diffusive sample. This time broadening is typically measured as about 30 fs in our samples 39 . For the transient absorption measurements, the pump beam at a defined wavelength is produced using a twostage non-collinearly phase-matched optical parametric amplifier (NOPA). It is modulated using a synchronized chopper at a frequency of 0.5 kHz, which is half the repetition frequency of the laser. It is focused onto the sample at an angle of about 60° from normal. The pump beam has a diameter of 500 μm at the surface of the sample and typical energy of about 100-200 nJ/pulse. The probe beam is provided by a second NOPA having less energy than the pump on the sample to avoid multiple excitations and is focused having the spot size of around half of that of the pump. The polarization between the pump and probe beams is at the magic angle (54.7°). The transient response of the sample is measured by collecting the diffuse reflected pulses of the probe. The light scattered by the sample is focused onto the detector (photodiode: Nirvana detector, New Focus, model 2007). The signal of the detector is amplified by use of a lock-in amplifier. Lock-in parameters are set as integration time 1s, dwell time 4s, time constant 1s for measurements. A power supply (Weir) is used to apply a fixed bias voltage on the solar cell for diffuse reflectance measurements on the cell under voltage bias condition. The bias voltage between the two electrodes is changed from − 690 mV to + 500 mV and time-resolved diffuse reflectance of the device is measured at each applied bias. In these measurements, the pump and probe beam are irradiated to the cell from the backside (photoanode side), similar to the photovoltaic measurements. All the rest experimental details of experiments are similar to that previously explained. Time-resolution of diffuse reflectance setup and linearity tests. The time-resolution of the setup is defined by using optical Kerr gating technique. In this technique pump and probe beams are focused and spatially overlapped on a non-linear media (SF10 crystal or glass substrate). The cross-correlation of pump and probe beam on a Kerr-media is measured at an angle of 45° between the polarization of pump and probe. We performed the cross-correlation experiment in the diffuse reflectance mode. As the Kerr-media once the SF10 crystal and another time the scattering sample with cover glass is used. The reason is to compare the broadening of the cross-correlation peak when the diffuse reflectance is measured on these types of substrates. The time-resolution of the setup is sub-200 fs. It should be noted that the time-resolution of this technique is limited by the time broadening of the beam in the sample due to the scattering effect. This time broadening is measured to be 120 fs for our samples. This is in contrast with the transmission based transient absorption technique in which the time-resolution is solely determined by the pulse duration of pump and probe and their cross-correlation. The setup has an unprecedented sensitivity as it enables measurements of transparent, non-reflective samples (see green curve in Fig. 6) with a reasonable signal to noise ratio. In order to check the linearity of measurements, the intensity of the pump beam is changed over a broad range of energy from 0.047 μJ/pulse to a high intensity of 0.950 μJ/pulse. The diffuse reflectance of dye-sensitized sample at a fixed time delay (i.e. 50 ps) is measured. Both absorptance and Kubelka-Munk formalism show a very good linear fit to the measurements over the whole intensities of excitations (Supplementary Figures S2 and S3).</p><p>Data acquisition and treatment. In the configuration of reflectance measurements, the transient absorptance ( )  change can be measured and corrected by determining the absolute amount of diffuse reflected light with and without pump beam. This is practically achieved by chopping the pump pulse at half repetition frequency of the laser. The time-resolved diffuse reflectance of samples is measured by varying the delay time between the pump and probe pulses. Therefore, transient absorptance is displayed as:</p><p>In case of opaque samples that the optical transmittance of sample is negligible, absorptance change (  ∆ ) can be deduced only from reflectance change, as:</p><p>Where T is the intensity of the transmitted light and R and R 0 represent the intensity of the diffuse reflectance of probe pulse with and without excitation, respectively. The linearity of absorptance change upon excitation intensity is tested in our control studies, over a wide range of excitation intensities. Another theory describing the optical behavior for a tightly packed isotropic absorbing and scattering medium is Kubelka-Munk 40,41 , in which the Kubelka-Munk function relates the measurable so-called diffuse reflectance of the sample to the ratio of the absorption coefficient (K) and scattering coefficient (S). In the case of diluted medium, K is linearly dependent on concentration of absorbing species (c), in the same way, the Lambert-Beer Law is also valid in solutions; equation (3):</p><p>ln (10) (3)</p><p>For quantitative simulation, use of the Kubelka-Munk function is essential, however, treating the transient reflectance of the samples with both equations of (2) and (3), does not change the kinetics.</p><p>Broadband transient absorption setup. The pump− probe technique uses a compact CPA-2001, 1 kHz, Ti: Sapphire-amplified femtosecond laser (Clark-MXR), with a pulse width of about 120 fs at a wavelength of 775 nm. The pumping beam is generated using an NOPA tuned to 600 nm to generate pulses of approximately 8 μJ, that are then compressed in an SF10-glass prism pair down to duration of less than 60 fs (at FWHM). At the sample, the excitation pulse energy is decreased to a few hundred nJ. The probe beam is a white light continuum generated in a sapphire plate and splits before the sample into signals and reference beams in order to account for intensity fluctuations. Both beams were recorded shot by shot with a pair of 163 mm spectrographs (Andor Technology, SR163) equipped with 512 × 58 pixels back-thinned CCD cameras (Hamamatsu S07030-0906). The polarization of pump and probe pulses was set at a magic angle.</p><!><p>Dye-sensitized solar cells were fabricated using a double-layered photoanode made of mesoporous TiO 2 film. A transparent, 9 μm-thick layer of 20 nm particles was screen-printed onto an FTO glass plate (NSG-10, Nippon Sheet Glass). Subsequently, a 5 μm-thick layer of scattering particles (400 nm diameter) was deposited by screen-printing. The surface area of TiO 2 film was 1 cm 2 . The TiO 2 film was sintered up to 500 °C by a stepwise heating program. Prior and after TiO 2 deposition a TiCl 4 treatment was performed on the samples. The BET surface area of the mesoporous transparent film and scattering film were 85 m 2 g −1 and 27 m 2 g −1 .</p><p>The values of the two films porosity were 70% and 65% for transparent film and scattering film respectively. Prior to dye loading, photoanodes were sintered again at 480 °C for 30 minutes. Afterward, substrate was cooled down to 80 °C and immersed in the dye solutions for overnight. After rinsing with the acetonitrile, the stained substrates were sealed with pieces of thermally platinized electrode. The platinized electrode was made using a solution of H 2 PtCl 6 on FTO glass (TEC15, Pilkington), and served as a counter electrode. The working and counter electrodes were separated by 25 μm-thick hot melt ring (Surlyn, DuPont) and sealed by heating. The electrolytes were introduced to the cells via pre-drilled holes in the counter electrodes.</p><!><p>The setup used for standard photovoltaic characterization (J-V curve) consisted of a 450 W Xenon lamp (Oriel), whose spectral output was matched in the region of 350-750 nm with the aid of a Schott K113 Tempax sunlight filter (Präzisions Glas & Optik GmbH), and a source meter (Keithley 2400) to apply potential bias and measure the photocurrent. A set of metal mesh filters was used to adjust the light intensity to a desired level. A black metal mask defined the cell active area to be 0.158 cm 2 .</p>

Scientific Reports - Nature

Toward the Detection of Cellular Copper(II) by a Light-Activated Fluorescence Increase

A new type of Cu2+ fluorescent sensor, coucage, has been prepared with a photosensitive nitrophenyl group incorporated into the backbone of a coumarin-tagged tetradentate ligand. Coucage provides a selective fluorescence response for Cu2+ over other biologically relevant metal ions. Coordination of Cu2+ dims the fluorescence output until irradiation with UV light cleaves the ligand backbone, which relieves the copper-induced quenching to provide a turn-on response. Experiments in live MCF-7 cells show that coucage can be used for detecting changes in intracellular Cu2+ upon the addition of excess exogenous copper. If improvements can be made to increase its affinity for copper, this new type of turn-on sensor could be used as a tool for visualizing the cellular distribution of labile copper to gain insight into the mechanisms of copper trafficking.

toward_the_detection_of_cellular_copper(ii)_by_a_light-activated_fluorescence_increase

<p>Copper, the third most abundant transition metal in the human body, plays a critical role in many fundamental physiological processes; however, it also catalyzes the production of highly reactive oxygen species that damage biomolecules.1 Due to copper's dual nature, cells have developed strict regulatory processes to control its cellular distribution.1 Alterations in copper homeostasis are linked to neurodegenerative diseases such as Menkes and Wilson diseases, Alzheimer's, familial amyotrophic lateral sclerosis, and prion diseases.2 Being able to visualize the cellular distribution of copper in both its physiological oxidation states, Cu+ and Cu2+, would offer insight into how cells acquire, maintain, and utilize copper while suppressing its toxicity. Whereas reliable fluorescence sensors exist for Cu+, there are fewer options for detecting Cu2+ in living cells.3</p><p>A common strategy in designing fluorescent probes for metal ions is to link a ligand to a fluorophore such that metal binding causes an increase in fluorescence only in response to the target ion. Cell permeable fluorescent sensors have proven useful for investigating intracellular metal ion distribution, particularly for Ca2+,4 Zn2+,5 and Cu+.6 The development of this type of "turn-on" sensor for Cu2+, however, is hampered by the fluorescence quenching effect of this paramagnetic metal ion. As a consequence, many Cu2+ sensors have a "turn-off" mechanism,7 which is generally less sensitive, gives false-positive results, and offers limited spatial resolution. Several examples of turn-on sensors have appeared recently,3,8 but limitations include sensing mechanisms that operate only in organic solvent or at non-physiological pH,8a-d low quantum yields in aqueous solution,8e or potential off-target responses.8f-i Therefore, there is a need to develop new strategies that provide a fluorescent turn-on response in order to investigate intracellular Cu2+. We present here coucage, a new type of fluorescent sensor that uses UV light to uncage a Cu2+-dependent fluorescence response.</p><p>Coucage is based on our previously reported copper caging ligand H2cage,9 but adapted with coumarin as a fluorescence reporter that is quenched upon Cu2+ coordination. The nitrophenyl group incorporated into the backbone of the fluorescent tetradentate chelator is the caging element that blocks activity until activated with light.10 Exposure to UV light induces bond cleavage, as shown in Scheme 1, which triggers two-fold activity: release of copper by decreasing ligand denticity, and restoration of fluorescence by disengaging the copper-induced quenching.</p><p>Coucage displays an absorbance band at 432 nm that gives a corresponding fluorescence emission maximum at 479 nm with a quantum yield of 0.054. Fig. 1a shows that its fluorescence at pH 7.4 is quenched by 75% when saturated with Cu2+, giving a quantum yield of 0.016 and a conditional dissociation constant, Kd, of 7.3 ± 0.9 μM. The 1:1 coucage:Cu2+ ratio for complex formation was confirmed by the method of continuous variation (Supp. Info.).</p><p>The depressed fluorescence of solutions containing coucage and Cu2+ can be restored to nearly half the original intensity by irradiation at 350 nm, as shown by the thick spectral trace in Fig. 1a. The emission maximum of photolyzed samples shifts slightly to 475 nm, with a quantum yield of 0.023. The fluorescence of the photolyzed products does not return to initial levels for at least two reasons, the first being that the quantum yield of independently synthesized photoproduct 1 (0.030) is inherently lower than coucage. The second is that Cu2+ retains some quenching effect on the photoproducts, although to a much lesser extent than on intact coucage (see Fig S5).</p><p>Unlike the response observed with Cu2+, no significant fluorescence changes are observed for coucage in the presence of other metal cations, as shown in Fig. 1b for Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu+, and Zn2+. When Cu2+is added back to these solutions, the fluorescence decreases by 70% (Fig. 1b, purple bars), confirming coucage's high selectivity for Cu2+ over other biologically important metal ions. The fluorescence can again be partially restored upon irradiation, as shown by the black bars in Fig. 1b.</p><p>The increase in fluorescence upon irradiation of [Cu(coucage)] is apparent immediately, and cleavage of the ligand backbone is complete in approximately 3 min. The quantum yield of photolysis for coucage and coucage in the presence of Cu2+ is 0.51 and 0.68 respectively, indicating that coordination by Cu2+ does not decrease photolysis efficiency, as previously observed for [Cu(cage)].9 Analysis of the reaction mixture by liquid chromatography mass spectrometry (LC-MS) revealed 1 and 2 as photoproducts (Fig. S1).</p><p>In order for coucage to bind tightly to Cu2+, all three amide protons must be deprotonated. The fact that only 75% fluorescence quenching is achieved at pH 7.4 suggests that the amide proton closest to the coumarin is not fully deprotonated at this pH, setting up a H+/Cu2+ competition that precludes maximum fluorescence quenching. Indeed, increasing the pH of coucage/Cu2+ solutions above 8 dramatically decreases fluorescence, leaving only a residual 10% signal by pH 9 (Fig. S4). Although the greatest fluorescence quenching is observed at high pH, coucage remains biologically applicable since a Cu2+ turn-off response is observed at pH 7.4.</p><p>To test coucage in living cells, we treated human breast carcinoma MCF-7 cells with coucage and Cu2+ and observed the intracellular fluorescence of irradiated vs. non-irradiated cells using scanning confocal microscopy. MCF-7 cells incubated with coucage alone initially show a high fluorescence response, as shown in Fig. 2b (see also Supp Info). After addition of excess Cu2+ to the cell culture medium and incubation for 20 min, the intracellular fluorescence signal decreases by 70%, indicating that Cu2+ has coordinated to coucage inside the cells (Fig. 2c). Cu2+-treated cells exposed to UV light from a Rayonet photoreactor for 4 min exhibit bulk fluorescence restoration up to 67% of the original intensity, as seen in Fig. 2d. Control experiments in the absence of fluorophore show no background fluorescence, and photobleaching of coucage results in less than 2% intensity loss during the 3 s excitation times used to collect images. (Supp Info). Brightfield images after coucage, Cu2+, and UV exposure show that cells remain viable throughout the imaging experiment. In these experiments, cells receive only 0.28 kJ/m2 of UVA irradiation, which is significantly lower than the 50–300 kJ/m2 doses known to induce DNA damage and cell death.11 Cells were also irradiated directly on the microscope (See Fig. S14). Although this method provides a less distinct fluorescence increase, it demonstrates the possibility of observing the same cells before and after photolysis.</p><p>In conclusion, we have presented a new strategy for achieving a fluorescence turn-on response to detect Cu2+ in living cells. The sensor relies on a coumarin-tagged ligand that selectively binds Cu2+ over other biometals to induce fluorescence quenching, which is subsequently relieved upon UV irradiation to provide the turn-on response. In essence, the strategy reports on the memory of where Cu2+ had been available for chelation by the 7 μM binder. Because the probe is destroyed during the readout, this strategy inherently cannot provide real-time monitoring of cellular Cu2+ fluctuations. Experiments in live MCF-7 cells demonstrate that coucage is cell permeable and can detect an increase of intracellular Cu2+ under conditions of excess (between 25 and 125 μM) exogenous copper. Copper is imported in its reduced Cu+ oxidation state, and intracellularly is believed mostly to remain in its reduced form. However, subcellular microenvironments may support Cu2+, and the coucage strategy introduced here might find utility in providing snapshots of such Cu2+, provided that improvements can be made to the ligand to make it more sensitive. Future investigations are therefore aimed at improving the quenching efficiency of the copper complex at physiological pH and increasing the binding affinity in order to create a more sensitive probe, as well as applying photoactive fluorescent ligands to other biologically interesting metal ions.</p><p>a) Fluorescence decrease of 1 μM coucage with 0–100 equiv. Cu2+, along with the subsequent increase following UV exposure (thick black trace). Inset: Emission at 479 nm vs. added Cu2+. b) Blue bars: the unchanged fluorescence of 1 μM coucage in the presence of 1 mM Na+, K+, Mg2+, Ca2+ and Zn2+ or 50 μM for others; Purple bars: quenched emission upon addition of 50 μM Cu2+; black bars: restored fluorescence after 4 min of UV exposure. All samples prepared in 10 mM Hepes buffer at pH 7.4 with 10% DMSO and excited at 430 nm.</p><p>Confocal fluorescence images of coucage and Cu2+ in MCF-7 cells; each panel shows an independent view from the same well. a) Bright-field transmission image. b) Cells incubated with 5 μM coucage for 20 min. c) Image taken 20 min after addition of 25 equiv of Cu2+ to coumarin-incubated sample. d) Image taken after 4 min of UV light exposure to coumarin/Cu2+-treated sample. Bar graph represents the average, background-corrected intensity from 10 randomly selected fields of view collected for each condition.</p><p>Synthesis and Photolysis of [Cu(coucage)]</p>

PubMed Author Manuscript

Genes associated with Parkinson\xe2\x80\x99s disease: regulation of autophagy and beyond

Substantial progress has been made in the genetic basis of Parkinson\xe2\x80\x99s disease (PD). In particular, by identifying genes that segregate with inherited PD or show robust association with sporadic disease, and by showing the same genes are found on both lists, we have generated an outline of the cause of this condition. Here, we will discuss what those genes tell us about the underlying biology of PD. We specifically discuss the relationships between protein products of PD genes and show that common links include regulation of the autophagy\xe2\x80\x93lysosome system, an important way by which cells recycle proteins and organelles. We also discuss whether all PD genes should be considered to be in the same pathway and propose that in some cases the relationships are closer, whereas in other cases the interactions are more distant and might be considered separate.

genes_associated_with_parkinson\xe2\x80\x99s_disease:_regulation_of_autophagy_and_beyond

<!>Mitophagy: Parkin, PINK1, and Fbxo7<!>Autophagy: \xce\xb1-synuclein, Vps35, LRRK2<!>Lysosomal genes: GBA and ATP13A2<!>Sporadic PD<!>Linking the pathways: could we and should we?<!>Future directions<!>Conclusions

<p>Autophagy refers to a series of processes related to how cells can recycle specific components, the term being derived from the Greek for self-eating (Zhang and Baehrecke 2015). Autophagy in its various forms has been linked to many aspects of fundamental biology, but also to some specific disease states (Feng et al. 2015). In this review, we will discuss the various lines of evidence suggesting that dysregulated autophagy might play a causal role in the pathogenesis of Parkinson's disease (PD), a common neu-rodegenerative condition.</p><p>The interested reader is directed toward many high-quality reviews on the basic biology of autophagy, but before discussing PD we will highlight a few essential concepts. Most importantly, 'autophagy' covers at least three distinct phenomena. Macroautophagy functions to degrade unnecessary or damaged proteins and organelles using a double membranous organelle, the autophagosome (Feng et al. 2014). In a series of regulated events, the autophagosome matures and fuses with lysosomes. In contrast, microautophagy involves direct invagination of lysosomal membranes to engulf cellular components (Mijaljica et al. 2011). Chaperone-mediated autophagy (CMA) is a distinct pathway dependent on recognition of proteins by a complex including the 70 kDa heat-shock cognate protein (Hsc70) that then is transferred across the lysosomal membrane (Cuervo and Wong 2014). In each form of autophagy, the net result is that the material to be degraded will be subject to hydrolysis by lysosomal enzymes and the final products are then available to the cell for re-use. Autophagy is therefore important in homeostasis, maintenance of cellular pools of nutrients such as amino acids, as well as in triggering some forms of cell death. Autophagy also plays a role in infectious diseases, by regulating the removal of bacterial or viral agents. Therefore, in broad concept there is no single 'autophagy' pathway but a series of inter-related events using overlapping sets of machinery that have multiple biological functions.</p><p>In recent years, it has been recognized that disruption of autophagy has specific effects in the central nervous system. For example, genetic ablation of key autophagy genes results in neurodegenerative phenotypes in mice (Komatsu et al. 2006). In parallel, the identification of many genes associated with PD and related conditions led to substantive understanding of how neurodegeneration can occur in this disease (Bonifati 2014). Interestingly, some of these genes appear to have specific roles in how autophagy is regulated. Here, we will critically discuss the evidence for regulation of autophagy in PD, focusing on gene products that appear to have related functions.</p><!><p>Mutations in parkin were first reported in an early onset form of PD in 1998 and mutations in PTEN-induced novel kinase 1 (PINK1) were identified in several families with similar phenotypes a few years later (Kitada et al. 1998; Valente et al. 2004). Looking at the primary sequence of the encoded proteins, it was recognized that parkin is an E3 protein-ubiquitin ligase and PINK1 was a serine/threonine protein kinase with a mitochondrial targeting sequence. Mutations were associated with loss of protein function, in that several were large deletions or truncating mutations (Bonifati 2014). However, it was not immediately obvious what the function of these proteins was in cells and whether PINK1 and parkin had any relationship with each other.</p><p>Two sets of results in very different systems demonstrated that PINK1 and parkin were involved in related pathways. First, when PINK1 knockout flies were examined they were seen to have phenotypes that were related to the accumulation of damage to mitochondria in a variety of tissues (Clark et al. 2006; Park et al. 2006). This phenocopied a set of phenotypes previously reported in parkin knockout flies (Greene et al. 2003), and through a series of genetic experiments it was shown that PINK1 was genetically upstream of parkin function. Some of the key data include that double knockout PINK1/parkin flies had no worse phenotype than each knockout alone and that parkin could rescue PINK1 knockout but not vice-versa (Clark et al. 2006; Park et al. 2006).</p><p>Second, while parkin is present predominantly in the cytoplasm and nucleus (Cookson et al. 2003), it was shown first by the Youle group that parkin could be recruited to mitochondria under conditions that induce mitochondrial depolarization (Narendra et al. 2008). Subsequently, it was demonstrated that this was a PINK1-dependent phenomenon (Matsuda et al. 2010; Narendra et al. 2010; Vives-Bauza et al. 2010). PINK1 is normally subject to rapid processing by proteases in mitochondria that are themselves inactivated by loss of membrane polarization (Jin et al. 2010; Deas et al. 2011; Meissner et al. 2011; Sekine et al. 2012). Thus, when mitochondria are damaged, there is an accumulation of PINK1 on the surface of that subset of the organelle. More recently it was shown that the key enzymatic events were the phosphorylation of Ubiquitin by PINK1 (Kane et al. 2014; Koyano et al. 2014), which then acts as a receptor for parkin (Okatsu et al. 2015). Parkin is simultaneously activated, likely by one or more phosphorylation events (Ordureau et al. 2014), and attaches phosphorylated ubiquitin to a series of proteins on the outer mitochondrial membrane (Chan et al. 2011; Sarraf et al. 2013). The key steps of this pathway are shown in Fig. 1.</p><p>After the ubiquitylation of mitochondrial membrane proteins by activated parkin, the marked mitochondria are degraded by a specialized form of macroautophagy called mitophagy (Narendra et al. 2008). It has been proposed that the turnover of mitochondria, specifically those that have lost mitochondrial membrane potential, that is, that are depolarized as above, is a way in which damaged mitochondria can be removed from the normal mitochondrial pool. It is likely that mitophagy is an important homeostatic mechanism that prevents accumulation of organelles that are diminished in their ability to make ATP, a critical function of mitochondria (Youle and Narendra 2011).</p><p>The PINK1/parkin pathway may also involve other genes related to inherited parkinsonism. For example, it has been shown that Fbxo7, a rare cause of recessive disease (Bonifati 2014) interacts physically with parkin and is required for mitophagy (Burchell et al. 2013). Interestingly, in Drosophila, Fbxo7 over-expression can rescue phenotypes linked to parkin deficiency, which suggests that that physical interaction with parkin is not required for at least some protective effects including in vivo.</p><p>In this way, PINK1, parkin, and Fbxo7 have been shown to be critically linked to mitophagy. While there are many details that still need to be mechanistically resolved, what is perhaps most interesting about these data are that they show how two different forms of protein degradation pathways, namely the ubiquitin–proteasome system and macroautophagy, co-operate to control a fundamental aspect of cellular homeostasis. Where there is less clarity is how disruption in these processes leads to a neurodegenerative phenotype.</p><p>At this point, it is worth noting that the phenotype in people with disease related to parkin or PINK1 has some features that are distinct from 'typical' sporadic PD. Although the number of autopsied cases are rather few, neurodegeneration in parkin/PINK1 seems to be limited to loss of dopamine neurons in the substantia nigra pars compacta (Poulopoulos et al. 2012). While loss of nigral neurons is a major and important feature of sporadic PD, it has been known for some time that many other brain regions can also be involved (Langston 2006). In addition, sporadic PD tends to progress substantially over time, whereas PINK1 and parkin cases generally show slower progression and sustained response to low dose dopaminergic medication (Bonifati 2012). Thus, in some ways, these recessive diseases represent a very dopamine-focused form of PD. Mutations in Fbxo7 can be variable clinically and include features such as chorea (Gündüz et al. 2014).</p><p>The reason why the phenotype of PINK1/parkin/Fbox7 merits discussion is that there is no obvious link between the physiological functions of dopamine neurons and a specific need for mitophagy. Autophagy in general, and mitophagy specifically, are very general cell biological phenomenon conserved over a large evolutionary space from yeast through humans. So why, at least in humans, there might be any specific cells that are lost with aging after disruption of mitophagy is difficult to resolve.</p><p>Adding to this difficulty, there has been some controversy about the role of PINK1/parkin-dependent mitophagy in neurons. While some studies have reported that neurons are susceptible to depolarization-induced parkin recruitment (Cai et al. 2012; Joselin et al. 2012; Ashrafi et al. 2014; Ye et al. 2015), others have not (Van Laar et al. 2011, 2015). Some studies have suggested that excessive excitatory stimuli might be sufficient to trigger parkin recruitment in neurons (Van Laar et al. 2015), with a potential implication being that pathological rather than physiological situations might have a bigger impact on parkin function. One key idea is that the bioenergetics of neurons are distinct from other cell types. Our laboratory has shown that hexokinase, which converts glucose to glucose-6-phosphate and is the first committed step in glycolysis, is required for parkin recruitment at least in cultured cells (McCoy et al. 2014). While it is often stated that neurons use glycolysis only sparingly and instead predominantly rely on oxidative metabolism, recent measurements of ATP suggest that both major pathways can be used to support synaptic function in cultured hippocampal neurons (Rangaraju et al. 2014). To what extent dopamine neurons, which are reported to have unusual bioenergetic demands, might use different ways to generate ATP is not clear but worth pursuing in the future.</p><p>It is also important to note that mutations in PINK1 and parkin may impact mitochondrial regulation by mechanisms that are potentially separate from mitophagy. For example, loss of PINK1 is associated with lower mitochondrial complex I activity possibly because of direct phosphorylation of the inner mitochondrial membrane component of complex I ndufa10 (Morais et al. 2009, 2014). Work in Drosophila models has suggested that ndufa10 can rescue PINK1 deficiency independent of effects on mitophagy or parkin (Pogson et al. 2014). PINK1 and parkin can increase turnover of proteins of the respiratory chain in vivo (Vincow et al. 2013) and have been shown to influence the translation of mRNA species that are important for respiratory chain function, which may represent a mechanism of mitochondrial quality control again independent of mitophagy (Gehrke et al. 2015).</p><p>These results collectively suggest that while the regulation of mitophagy is likely to explain some aspects of the function of PINK1 and parkin, it is also possible that the overall pathway is more complex than a simple linear relationship, as others have discussed (Scarffe et al. 2014). Some of the additional functions assigned to either PINK1 or parkin include regulation of mitochondria along neuronal axons (Weihofen et al. 2009; Liu et al. 2012; Birsa et al. 2014) and control of mitochondrial biogenesis (Pacelli et al. 2011; Shin et al. 2011). There are also important data supporting the idea that parkin can play an important role in ER-mitochondrial cross talk (Van Laar et al. 2015), including control of the translocation of a subset of mitochondrial proteins to the ER during mitophagy (Saita et al. 2013), suggesting that parkin may impact multiple organelles in the neuron.</p><p>The considerations illustrate a central contention that we will expand upon later in this article; that although there are sometimes direct mechanistic links between proteins encoded by genes that cause PD, the pathways that link genes can be more complex than implied by a simple linear relationship. In a sense, this is an unsurprising thought as a given cell has many different processes to co-ordinate under variable physiological conditions. Thus, the genes involved in recessive parkinsonism may have functions outside of mitophagy that allow for the control of distinct cellular events that need to be tied together either spatially or temporally, as indicated by the boxes distinct from the mitophagy pathway in Fig. 1. Interestingly, Mendelian inheritance of mutations in some of the proposed effector genes for these functions (such as Miro1, ndufa10, or Pcg1α) is extremely rare, which might indicate that disruption of the central functions of mitochondria is poorly tolerated.</p><p>There is substantial evidence that mitochondrial turnover occurs by additional, PINK1/parkin-independent pathways. Mitophagy can be triggered by diverse stimuli including hypoxia (Liu et al. 2014), mitochondrial toxins such as rotenone (Dagda et al. 2013), or the mitochondrial lipid cardiolipin (Li et al. 2015), and it is not clear if PINK1/parkin is always required for these effects. For example, in cell culture, iron chelators (Allen et al. 2013) or the protein AMBRA1 (Strappazzon et al. 2015) can promote mitophagy in the absence of parkin. In vivo, parkin deficiency does not result in the accumulation of damaged mitochondria in a model of chronic mitochondrial complex I impairment in dopaminergic neurons, the mitopark mouse (Sterky et al. 2011). Similarly, there are circumstances where mitophagy can proceed in the absence of PINK1 (Dagda et al. 2009; East et al. 2014). In some tissues, the mitochondrial protein Drp1 acts in a parallel, parkin-independent pathway to control mitochondrial ubiquitylation and turnover (Kageyama et al. 2014), and Drp1 can rescue phenotypes resulting from the loss of function of PINK1 in vivo (Liu et al. 2011).</p><p>Collectively, these considerations indicate that PINK1 and parkin are regulatory proteins that are particularly important in mitophagy after exposure to some cellular signaling pathways but are unlikely to be central players in all forms of mitophagy. This point, that PD genes play modulatory rather than essential roles, will be returned to when discussing some of the dominant genes that appear to have roles in the regulation of other forms of autophagy.</p><!><p>α-Synuclein is one member of a group of small proteins predominantly found in the pre-synaptic terminals of neurons (Clayton and George 1999). In 1997, mutations in familial cases with Parkinson's disease were discovered in the gene encoding α-synuclein, SNCA (Polymeropoulos et al. 1997). Subsequently, α-synuclein was found to be a major constituent of Lewy bodies and Lewy neurites, the pathological hallmarks of PD (Spillantini et al. 1997). In 2003 Singleton and colleagues reported a triplication of the SNCA locus in a large family with PD and dementia (Singleton et al. 2003). Subsequently, duplications and triplications of the α-synuclein locus were found in several families (Farrer et al. 2004; Ibáñez et al. 2004; Ikeuchi et al. 2008). The mRNA and protein levels of α-synuclein in triplication cases are twice the amount present in controls (Miller et al. 2004), showing that increased α-synuclein levels can cause Parkinson's Disease. Remarkably, genome wide association studies (GWAS) identified the α-synuclein locus as contributing to lifetime risk of sporadic PD (International Parkinson Disease Genomics Consortium et al. 2011).</p><p>As increased α-synuclein levels are implicated in PD pathogenesis, a logical question is therefore to examine the pathway by which α-synuclein is degraded in cells. For example, Cuervo et al. (2004) found that α-synuclein is a substrate for the specialized form of autophagy, CMA. The amino acid sequence of α-synuclein contains a CMA-like recognition motif that is sufficient to allow translocation of α-synuclein through isolated intact lysosomes and further degradation in a CMA-dependent fashion. It was subsequently shown that CMA is a major pathway for degradation of α-synuclein in primary neuronal cultures and human neuronal cell lines (Vogiatzi et al. 2008). The siRNA-mediated knockdown of lysosome-associated membrane protein 2 (LAMP2a) resulted in slower turnover of wild-type α-synuclein, and α-synuclein mutant lacking the CMA motif exhibited slower degradation compared to wild type. In addition, inhibition of CMA can lead to the formation of insoluble, oligomeric α-synuclein species (Vogiatzi et al. 2008). Knockdown of LAMP2a in SH-SY5Y cells also results in increased half-life of α-synuclein (Alvarez-Erviti et al. 2010).</p><p>In addition to these effects of CMA on α-synuclein, mutant, or modified forms of the protein may inhibit CMA. Specifically, phosphorylated α-synuclein (Martinez-Vicente et al. 2008), A53T mutant (Cuervo et al. 2004; Xilouri et al. 2009), and dopamine-modified forms of the wild-type protein (Martinez-Vicente et al. 2008; Xilouri et al. 2009) lead to CMA dysfunction, and may induce compensatory macroautophagy (Xilouri et al. 2009). Indeed, pharmacological inhibition of macroautophagy resulted in α-synuclein accumulation in the lysosome (Webb et al. 2003; Vogiatzi et al. 2008) and activation of macroautophagy facilitated the degradation of both wild-type and mutant forms of α-synuclein (Webb et al. 2003; Spencer et al. 2009). A study in transgenic mice over-expressing α-synuclein under the regulatory control of the Thy-1 promoter provided evidence that CMA provides a major mechanism for α-synuclein degradation in vivo. Enhanced lysosomal content of α-synuclein, and up-regulation of CMA-related proteins (LAMP2a, Hsc70) were directly correlated with the level of α-synuclein over-expression throughout the brain (Mak et al. 2010). Impairment of the autophagy system in general, and CMA in particular, is therefore likely to increase the amount of α-synuclein in brain, thus contributing to PD development. There are additional genes that may have roles in macroautophagy and CMA that, therefore, may affect α-synuclein through overlapping mechanisms.</p><p>Mutations in leucine-rich repeat kinase 2 (LRRK2) (Paisán-Ruíz et al. 2004; Zimprich et al. 2004) are a relatively common genetic cause of late-onset PD, accounting for up to 40% of familial cases in some populations (Bras et al. 2005; Deng et al. 2006; Ozelius et al. 2006; International Parkinson Disease Genomics Consortium, Nalls M. A., Plagnol V., Hernandez D. G., Sharma M., Sheerin U.-M., Saad M., et al. 2011). Similar to α-synuclein, GWAS have also identified variants at the LRRK2 locus as having increased risk for sporadic PD (International Parkinson Disease Genomics Consortium, Nalls M. A., Plagnol V., Hernandez D. G., Sharma M., Sheerin U.-M., Saad M., et al. 2011). LRRK2 encodes a large (286 kDa) protein with multiple protein–protein interaction domains, namely ankyrin repeats, leucine-rich repeats, C-terminal WD40 domain, and two enzymatic regions, the ROC-COR bidomain that acts as a GTPase and kinase. Although many LRRK2 mutations have already been described, only a few have been proven to cause PD: G2019S, R1441C/G/H, Y1699C, I2020T (Cookson 2010). Interestingly, pathogenic mutations tend to cluster within the three domains that form the enzymatic core of LRRK2, namely the ROC-COR and kinase domain.</p><p>LRRK2 has been implicated in multiple functions, including cytoskeletal organization, endosomal vesicle trafficking, translational control, and miRNA processing (Cookson 2010), but here we will discuss the connections to autophagy. An important set of observations linking LRRK2 with macroautophagy came from cellular localization investigations. A study of HEK293 cells expressing low levels of LRRK2 localized the protein to several structures associated with autophagy, including microvilli/filipodia, neck of cave-olae, multivesicular bodies, and autophagosomes (Alegre-Abarrategui et al. 2009). In human brains, LRRK2 is present in cytoplasmic punctae corresponding to multivesicular bodies and autophagic vacuoles (Biskup et al. 2006). LRRK2 can be co-localized with the late endosomal markers Rab7, and less frequently with the lysosomal marker LAMP2 (Higashi et al. 2009). LRRK2 is also found in autophagic vesicles when expressed in cells or mouse models (Biskup et al. 2006; Plowey et al. 2008; Alegre-Abarrategui et al. 2009; Ramonet et al. 2011).</p><p>Protein interactions of LRRK2 may also provide clues as to its function. It has been shown to interact with early endosomal marker Rab5b (Shin et al. 2008; Heo et al. 2010). More recent data suggest that LRRK2 might phosphorylate Rab5b and activate its GTPase activity, negatively regulating its signaling (Yun et al. 2015). A Drosophila melanogaster homolog, lrrk, physically interacts with the late endosomal GTPase rab7 and to negatively regulate rab7-dependent perinuclear localization of lysosomes (Dodson et al. 2012). Gómez-Suaga and colleagues reported that pathogenic LRRK2 mutations reduce Rab7 activity in mammalian cells, causing a delay in trafficking of Rab7 out of late endosomes (Gómez-Suaga et al. 2014).</p><p>Physical interaction with another homolog of Rab7, Rab7L1 has been reported by our lab and by others (MacLeod et al. 2013; Beilina et al. 2014). In our hands, we found that LRRK2 forms a complex with Rab7L1, Hsc70, chaperone regulator BCL2-associated athanogene 5 (Bag5), and cyclin G-associated kinase (GAK) to promote the removal of Golgi derived vesicles by autophagy-dependent mechanisms (Beilina et al. 2014), as shown in Fig. 2. Interestingly, GAK and Rab7L1 genes have been nominated as risk factors for sporadic PD by GWAS (Simón-Sánchez et al. 2009; International Parkinson Disease Genomics Consortium, Nalls M. A., Plagnol V., Hernandez D. G., Sharma M., Sheerin U.-M., Saad M., et al. 2011; Sharma et al. 2012). Previous functional studies demonstrated that GAK is recruited to the trans-golgi network (TGN) (Zhao et al. 2001; Kametaka et al. 2007) and promotes the uncoating of endocytosed clathrin-coated vesicles (Greener et al. 2000; Lee et al. 2006). Experimental evidence demonstrated that depletion of GAK in cells impaired the sorting of cathepsin D to lysosomes (Kametaka et al. 2007).</p><p>Collectively, the observations of LRRK2 interaction with one or more small GTPases in the Rab family support that LRRK2 plays a critical role in sorting of a variety of vesicles in the cell. However, this does not indicate a direct role of LRRK2 in autophagy specifically. Strong evidence supporting the role of LRRK2 in autophagy comes from studies of LRRK2 knockout mice (Herzig et al. 2011; Tong et al. 2012). These mice appeared to have kidney and lung pathology with lipofuscin accumulation and biphasic alteration in the autophagy–lysosomal markers. Interestingly, striking accumulation and aggregation of α-synuclein was observed in kidney of LRRK2 −/− mice at 20 months of age (Tong et al. 2012).</p><p>Studies using iPSc-derived dopaminergic neurons bearing the G2019S mutation in LRRK2 showed a basal increase in autophagy LC3- and p62-positive puncta and increase in LC3II protein levels compared to control cells (Sánchez-Danés et al. 2012). In addition, inhibition of autophagy in these dopaminergic cells resulted in mutant-specific reduction in autophagic flux measured by LC3II immunoblotting, suggesting problems in clearance of autophagosomes (Sánchez-Danés et al. 2012). Increased levels of α-synuclein were observed in iPSc-derived G2019S mDA neurons and these increases are not associated with increased mRNA levels of α-synuclein, suggesting impaired degradation of α-synuclein (Sánchez-Danés et al. 2012; Reinhardt et al. 2013). As discussed above, because α-synuclein is degraded in part by CMA, a reasonable interpretation is that inhibition of general macroautophagy by LRRK2-G2019S would secondarily result in excessive stability of α-synuclein because of the disruption of the more specialized form of autophagy, CMA. It has also been reported that LRRK2 may inhibit CMA directly, perhaps by a similar mechanism to that reported for α-synuclein (Orenstein et al. 2013), suggesting that LRRK2 mutations may have effects on both bulk macroautophagy and CMA. Whether LRRK2 mutations also affect other specialized forms of autophagy, and whether the mechanisms by which they do are shared with a more general inhibition of macroautophagy, is not resolved at this time.</p><p>There is also some evidence that this observation with G2019S might generalize to other mutations in LRRK2. In response to starvation, fibroblasts from patients with different LRRK2 mutations across multiple functional domains showed lower levels of lipidation of LC3 compared to wild-type controls (Manzoni et al. 2013b). Conversely, inhibition of kinase activity of LRRK2 enhanced LC3 lipidation and the effects of kinase inhibitors required the presence of LRRK2 (Manzoni et al. 2013a). Consistent with the reports in LRRK2 knockout mice, these results suggest that LRRK2 normally acts to suppress autophagy, as measured by LC3-II formation. How these results relate to the observations of LRRK2 on various vesicular structures marked by Rab GTPases remains to be clarified. That LRRK2 can be found in several different compartments, including the TGN, endosomes, and autophagic vesicles, is consistent with earlier localization data (Alegre-Abarrategui et al. 2009).</p><p>Despite this substantial body of literature suggesting that LRRK2 plays a regulatory role in macroautophagy, there are some unresolved questions about the mechanisms and direction of effect. For example, expression of G2019S LRRK2 increased the number of LC3-positive punctae, presumably representing autophagic vacuoles, in SH-SY5Y cells (Plowey et al. 2008), whereas G2019S expressed at endogenous levels in human fibroblasts has been reported to decrease LC3-II levels under starvation conditions (Manzoni et al. 2013b). While some of the discrepancies could be because of technical differences between studies, such as the use of slightly different measures in different cell lines, it is also possible that some important biology could be discerned by investigating the mechanism(s) underlying how LRRK2 impacts macroautophagy. For example, the observation that LRRK2 knockout results in autophagy markers can be both increased or decreased in vivo depending on the age of the animals (Tong et al. 2012) shows that some autophagy markers are potentially regulated at stages downstream of LRRK2 itself. In other words, measurement of any given marker of macroautophagy could reflect both primary consequences of LRRK2 alterations and compensatory changes that follow.</p><p>Two independent exome sequencing studies of Swiss (Vilariño-Güell et al. 2011) and Australian families (Zimprich et al. 2011) with autosomal dominant Parkinson's disease identified a D620N mutation in the vacuolar protein sorting 35 (VPS35) gene. Other variants – P316S and L774M – have been identified for this gene, however, their pathogenicity is less clear as only D620N shows clear evidence of segregation in families (Vilariño-Güell et al. 2011; Zimprich et al. 2011; Ando et al. 2012).</p><p>VPS35 is a part of the retromer complex involved in endosomal–lysosomal retrograde transport of vesicles to trans-Golgi network (Seaman et al. 1997). The retromer has two subcomplexes: the cargo selection trimer consisting of Vps26, Vps29, and Vps35 and the membrane association sorting nexin dimer (Hierro et al. 2007). One of the well-studied types of cargo for retromer is a cation-independent mannose-6-phospate receptor (CI-MPR) that binds to the newly made lysosomal enzymes in the TGN and delivers them to the lysosome (Seaman 2004). After delivering lysosomal enzymes to lysosomes, the retromer returns CI-MPR back to the TGN.</p><p>Cathepsin D is a lysosomal enzyme that is modified by the mannose-6-phospate at the TGN (Fig. 2). This modification of cathepsin D is recognized by CI-MPR, which enables the delivery of the protease to lysosomes. Cathepsin D matures during the delivery and becomes active in lysosomes. Interestingly, cathepsin D is one of the main lysosomal endopeptidases responsible for the degradation of α-synuclein (Sevlever et al. 2008; Cullen et al. 2009). Moreover, marked increase in aggregated levels of α-synuclein have been observed in cathepsin D knockout mice (Qiao et al. 2008). In studies with transgenic flies expressing human wild-type α-synuclein, RNAi-mediated silencing of VPS35 resulted in the aberrant maturation of cathepsin D and the absence of cathepsin D led to the accumulation of α-synuclein in late endosomal and lysosomal compartments, accompanied by locomotor abnormalities in flies and mild eye disorganization (Miura et al. 2014). These data suggest that retromer may play an important role in the control of the degradation of α-synuclein and that depletion of VPS35 function may result in an increased load of synuclein.</p><p>Retromer containing the D620N VPS35 mutation incorrectly traffics CI-M6PR, resulting in improper processing, and increased secretion, of unprocessed cathepsin D (Follett et al. 2014). The impairment of cathepsin D processing was also confirmed in fibroblasts of patients carrying the same mutation (Follett et al. 2014). It is worth noting, that another study on pathogenicity of D620N in the rodent primary neurons and patient-derived human fibroblasts did not find any changes in localization of the retromer complex between endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, CI-M6PR (Tsika et al. 2014).</p><p>The D620N mutation does not disrupt interaction of VPS35 with any of the other retromer subunits (Follett et al. 2014; Tsika et al. 2014; Zavodszky et al. 2014) but selectively impairs the interaction between retromer and the WASH complex (McGough et al. 2014; Zavodszky et al. 2014), which functions to promote F-actin nucleation on endosomes and proper sorting of multiple cargo proteins (Derivery et al. 2009; Gomez and Billadeau 2009). Consequentially, the disrupted WASH-retromer interaction results in perturbed trafficking of the autophagy-related protein 9A (ATG9A), which is necessary for proper autophagy induction. ATG9A is retained more at TGN-golgi areas, and was not able to traffic to autophagic structures, leading to impairment in autophagy measured by decrease in LC3II levels resulted in increased levels of α-synuclein (Zavodszky et al. 2014). There is additional experimental evidence showing that retromer plays specific roles in autophagy. The autophagosome protein Atg9 cycles between the Golgi and endosomes under normal conditions but upon induction of autophagy Atg9 moves toward endosomes and then autophagosomes (Young et al. 2006). These data suggest that there are multiple links between retromer function and autophagy and, as discussed above, from autophagy to LRRK2.</p><p>Supporting this, MacLeod and colleagues have linked LRRK2 to retromer function in cells by showing that VPS35 can physically interact with LRRK2 (MacLeod et al. 2013). In fly models, over-expression of wild-type VPS35 rescued DA neuronal loss and shorter life span caused by expression of G2019S LRRK2 (MacLeod et al. 2013). A functional interaction between LRRK2 and VPS35 has been independently confirmed in lines expressing pathogenic mutations I2020T, Y1699C, and I1122V (Linhart et al. 2014). Again, locomotor deficits, shortened life span and abnormal eye phenotype were rescued by expression of two retromer subunits VPS35 or VPS26 (Linhart et al. 2014).</p><p>The cumulative evidence discussed here suggests that three genes, LRRK2, VPS35, and α-synuclein, along with risk factor genes such as Rab7L1 and GAK, have functional and/or physical links to each other. In our opinion, a likely nexus for these events is at the TGN, which a key vesicular environment that mediates overlapping function between the retromer and autophagy control. It is likely that the function of this set of genes is to regulate autophagy, suggesting that the pathway that is perturbed in these forms of Parkinson's disease is macroautophagy. A likely outcome, at least in neurons, is that there would be accumulation of α-synuclein as a downstream event, although there are other models that might also be reasonable.</p><!><p>There are many other genes and risk factor loci for PD, and it is not our intention here to try to link all of them to autophagy-related functions. However, there are two genes in particular that bear some further examination as they are linked to autophagy–lysosome function, which in turn is required for the execution of degradation in autophagy. However, the two examples discussed here, PD is only part of the clinical picture and we will emphasize that these are in some ways distinct from the autophagy genes discussed above.</p><p>Mutations in the gene encoding glucocerebrosidase (GBA) cause Gaucher's disease, a condition that is characterized by enlargement of multiple organs and, in a subset of cases, neurological problems. Gaucher's disease shows autosomal recessive inheritance and is related to the loss of normal function of GBA, which is to degrade glycosylceramide, an important lipid for cell membranes and as a source for ceramide for cell signaling. In patients, usually children, lack of GBA activity causes the accumulation of glycosylceramide in lysosomes and, as such, Gaucher's is the most common lysosomal storage disease (Baris et al. 2014).</p><p>Several years ago, it was noted clinically that grandparents of Gaucher's children had PD more often than would be expected by chance alone. Sequencing was used to identify heterozygous variants in the GBA gene in PD patients (Lwin et al. 2004). A multicenter analysis confirmed that these results generalize across cases, and that possession of a single copy of the defective GBA allele confers an ~5fold increase in risk of PD over lifetime (Sidransky et al. 2009). Similar estimates of risk have been seen in other studies (Lesage et al. 2011) and there is signal around the GBA gene in recent GWAS (Nalls et al. 2014).</p><p>These genetic results suggest that while full loss of GBA function is associated with a multisystem lysosomal storage disease, single mutant alleles are tolerated during development but increase the risk of a late-onset neurodegenerative disease. There has therefore been a great deal of interest in examining the links between GBA and other PD genes, particularly α-synuclein. An important study from Mazzulli et al. (2011) suggested not only that GBA dysfunction would impact α-synuclein accumulation via altered lysosomal processing, but also that the presence of a α-synuclein in an oligomeric form would limit the maturation of GCase, which similar to cathepsin D discussed above, requires the enzyme to transit from the ER to the TGN. Supporting this idea, iPSC lines from PD patients who have mutant GBA alleles have higher synuclein levels compared to controls (Schöndorf et al. 2014), as do cell lines edited to have diminished GCase activity (Bae et al. 2015).</p><p>There is an ongoing discussion as to whether the risk of PD is associated with lower GCase activity or whether gain of function mechanisms might contribute to disease risk. For example, increasing the expression of mutant forms of GBA was associated with increased amounts of α-synuclein, irrespective of the effects of the mutations on enzyme activity (Cullen et al. 2011). A similar dissociation between enzyme activity and levels of α-synuclein was noted in vivo by comparing mutant knockin and knockout GBA alleles in mice (Cullen et al. 2011). While these studies indicate that there are still important mechanistic aspects of the role of GBA in PD that need to be resolved, they also show that risk factor genes are difficult to place easily into loss of gain of function categories.</p><p>Along the same lines, there have been several attempts to link the function of the lysosomal enzyme ATP13A2 with α-synuclein. Mutations in ATP13A2 were first reported in a recessively inherited disease, Kufor–Rakeb syndrome (Ramirez et al. 2006). The phenotype of Kufor–Rakeb syndrome is complex, with multiple brain regions being involved leading to immobility, mutism, and dementia. However, mutations in ATP13A2 are also found in early onset parkinsonism cases without these additional features (Di et al. 2007), suggesting that the protein has one or more functions relevant to PD even if the overall clinical picture can be broader.</p><p>The ATP13A2 gene encodes a P-type ATPase normally initially localized to lysosomes, with some mutations preventing maturation through the ER-TGN and promoting degradation of the protein product (Ramirez et al. 2006). ATPases are known to transport metal cations, and so several studies have focused on the role of metal ions in ATP13A2-dependent phenotypes. In a range of organisms from yeast to mammals, it has been shown that loss of ATP13A2 sensitizes cells to excess divalent metal cations of various types (Gitler et al. 2009; Chesi et al. 2012; Podhajska et al. 2012; Ramonet et al. 2012; Kong et al. 2014; Park et al. 2014). Interestingly, some data suggest that ATP13A2 is expressed in components of the autophagy–lysosomal system other than the lysosomes themselves (Kong et al. 2014).</p><p>A picture therefore emerges that ATP13A2 mutations lead to an inability to regulate divalent metal cations in the lumen of various vesicular structures, including but not limited to lysosomes (Lopes da Fonseca and Outeiro 2014). Given that GBA is also involved in lysosomal function, it becomes reasonable to think that both GBA and ATP13A2 have common effects (Fig. 3). This would put both genes in a pathway that indirectly regulates α-synuclein clearance via the autophagy–lysosome system. Supporting this contention, patient fibroblasts carrying ATP13A2 mutations have been shown to have higher levels of α-synuclein (Tsunemi and Krainc 2014) and accumulation of α-synuclein may occur in one ATP13A2 knockout mouse model (Schultheis et al. 2013). It has further been suggested that part of the effects of ATP13A2 mutations on α-synuclein may be mediated via exocytosis (Kong et al. 2014).</p><p>Although elegant, this proposal is not fully supported by all the available data. For example, genome editing using zinc finger nucleases to remove ATP13A2 does not result in either lysosomal dysfunction or α-synuclein accumulation (Bae et al. 2014), despite the observation that manipulation of GBA does have the expected effects in the same cell line (Bae et al. 2015). In an ATP13A2 knockout mouse where lysosomal and protein trafficking deficiencies were noted, locomotor phenotypes were found to be independent of α-synuclein (Kett et al. 2015). In fact, in this in vivo model, which does show motor dysfunction and a variety of neuropathological phenotypes, no accumulation of α-synuclein was observed. A similar lack of dependency of ATP13A2 mutant induced neuropathology was noted in a rat model (Daniel et al. 2014). How these different observations will be resolved is not yet clear and will likely require confirmation or refutation of the key data.</p><p>Further complicating where to place ATP13A2, mitophagy can be impaired cells where ATP13A2 is knocked down with siRNA (Gusdon et al. 2012) or mutated at the endogenous level in patient fibroblasts (Grünewald et al. 2012). These studies might imply that ATP13A2 should be considered to be part of the PINK1/parkin/Fbxo7 pathway alluded to above. However, the available data suggest that the mitophagy phenotype in ATP13A2-deficient cells is secondary to an autophagy defect, presumably related to lysosomal dysfunction as, for example, the same effects can be achieved by siRNA against Atg7 (Gusdon et al. 2012).</p><p>Collectively, these observations suggest that while a substantial loss of lysosomal function is associated with lysosomal storage disorders, in some cases, parkinsonism can be part of the phenotype, but the phenotype tends to be broader than PD alone. The affected pathways at a cellular level are also broader for lysosomal enzyme deficiency than for recessive parkinsonism, but include defects in mitophagy as a consequence of the primary defects in the autophagy–lysosome system.</p><!><p>Predating the identification of genes, it was known that sporadic PD cases have pathologic features that might indicate dysregulation of autophagy. For example, Anglade and colleagues performed ultrastructural analysis of dopaminergic neurons in the substantia nigra of patients with PD, and found autophagic degeneration, accumulation of high density lysosome-like vacuoles, and presence of lipofuscin granules (Anglade et al. 1997). Similar results were seen by other groups (Chu et al. 2009; Alvarez-Erviti et al. 2010).</p><p>Postmortem studies in the substantia nigra pars compacta and amygdala of PD brains revealed significant reductions in expression levels of autophagy proteins LAMP2a and Hsc70 compared to age-matched controls or Alzheimer's disease samples (Chu et al. 2009; Alvarez-Erviti et al. 2010, 2013; Wu et al. 2011b; Murphy et al. 2015). Neuropathological examination of human brains derived from PD patients found alterations in the main CMA components, Hsc70 and LAMP2A using immunohistochemistry (Chu et al. 2009). In addition, this study found changes in other auto-lysosomal components, suggesting more generalized autophagy impairment, not specific to CMA (Chu et al. 2009). Similarly, using immunoblotting, Alvarez-Erviti et al. (2010) showed significant decreases in both Hsc70 and LAMP2A in the nigra and amygdala of PD brains compared to controls. An additional study correlated the loss of LAMP2 and Hsc70 proteins with the increased levels of α-synuclein seen in PD brains (Murphy et al. 2015). Mechanistically, several miRNAs that down-regulate CMA are significantly increased in substantia nigra compacta and amygdala PD brains compared to both age-matched controls and brain samples with AD (Alvarez-Erviti et al. 2013).</p><p>There is some evidence that these diminished autophagy functions may be a systemic effect rather than brain specific. For example, in one study, reduced LAMP2A and Hsc70 expression, along with impairment in fusion of autophago-some and lysosome has been noted in sporadic PD patients (Wu et al. 2011b). Others have confirmed the diminished Hsc70 expression but did not note changes in LAMP2A (Sala et al. 2014). Examination of cerebrospinal fluid (CSF) from PD patients showed significant decreased activity of multiple lysosomal hydrolases compared to age-matched controls (Balducci et al. 2007). Another study in the peripheral leukocytes of sporadic PD patients found significant reduction in activity of alpha-galactosidase A (GLA), one of the enzyme active in lysosomes (Wu et al. 2011a).</p><p>As discussed above, diminished function in the autophagy–lysosome system may contribute to the accumulation of α-synuclein. Interestingly, Gegg et al. (2012) found reduced glucocerebrosidase protein levels and enzyme activity in a range of affected brain regions in patients with sporadic PD. Supporting this observation GBA protein levels and enzyme activity were selectively reduced in brain regions that accumulate abnormal α-synuclein (Murphy et al. 2014).</p><p>Collectively, these observations suggest that, as well as being involved in familial PD, there may be some contribution of autophagy–lysosomal pathways to the more common sporadic form of the same disease. Whether the observed responses in sporadic disease indicate that autophagy is causal or a consequence of the disease process is less clear. However, it is reasonable to ask to what extent autophagy-related pathways contribute to the pathogenic mechanism relevant to the different forms of PD.</p><!><p>An ongoing debate in the PD field revolves around the extent to which we can integrate what we have found about genetic forms of the disease. There are really parts to this question: are all genetic forms of parkinsonism related to each other and what do they tell us, if anything about sporadic disease?</p><p>There are good reasons to distinguish between the different genes for parkinsonism. It has been argued persuasively, for example, that α-synuclein positive Lewy bodies are important for assignment of a disease to PD (Hardy and Lees 2005) but this position is complicated by the fact that not all LRRK2 cases have Lewy bodies despite being clinically homogenous (Cookson et al. 2008). Clinical information is also useful, and would suggest distinction between recessive and dominant genes, but as discussed for ATP13A2 and Fbxo7, there can be a wide range of symptoms for mutations in a single gene (Ramirez et al. 2006; Gündüz et al. 2014). In addition, because the number of cases, especially those with autopsy information, is small for the rarer inherited conditions, then defining pathways by patient information is a fraught process.</p><p>However, there is good evidence to think that at least some of the genetic forms of disease have relevance to sporadic PD. Strongest among these ideas is the observation that some of the known genes for inherited PD are in loci that are nominated by GWAS for risk of sporadic PD. This leads to the concept of pleomorphic risk loci, regions of the genome that contain multiple variants, some of which cause disease by changing amino acids whereas others affect disease risk because of differences in gene expression levels (Singleton and Hardy 2011). PD has multiple such loci, including α-synuclein and LRRK2 but to date none of the recessive genes has been shown to act as strong risk factors for sporadic PD. Therefore, for a subset of PD genes, but not all of them, it is reasonable to assume that there is a relationship with sporadic disease.</p><p>The functional data discussed above relating PD genes and proteins to forms of autophagy may be helpful in resolving these issues. It is clear that PINK1 and parkin are in a tightly related pathway linked to mitophagy and it is likely that this can be extended to Fbxo7 (Burchell et al. 2013). Therefore, for this form of parkinsonism there is reasonable evidence to support the idea that one form of autophagy is important in the disease process. Importantly, however, because mitophagy is a specialized form of autophagy it is not reasonable to infer that the disease in this case is a generalized defect in autophagy–lysosomal system. In addition, because some of the protein products of genes in the PINK1/parkin/Fbxo7 pathway have functions outside of mitophagy, it is possible that diminished mitophagy is not sufficient for parkinsonism.</p><p>It is also reasonably well established that LRRK2 is that it has physical and/or genetic partners that link it in to other forms of PD. Interactions with Rab7L1 and GAK (MacLeod et al. 2013; Beilina et al. 2014) suggest relevance to sporadic disease, as these are candidates from GWAS, and physical interaction with VPS35 (MacLeod et al. 2013) would provide a link to inherited PD. Furthermore, it has been shown that lack of LRRK2 limits the toxic effects of inflammation or α-synuclein over-expression in cells and animals (Lin et al. 2009; Daher et al. 2014; Skibinski et al. 2014). Therefore, LRRK2 appears to be a highly connected hub in the network of PD genes. Given the multiple links, it has been suggested previously that LRRK2 and α-synuclein each contribute to the toxicity seen in dopamine neurons in the disease (Taymans and Cookson 2010).</p><p>The crucially difficult question that next arises is whether the mitophagy defect in PINK1/parkin/Fbxo7 disease is functionally related to the regulation of autophagy that occurs as a result of mutations in LRRK2 and its interaction partners. To some extent this depends upon what the meaning of the word 'is' is. If it is the case that LRRK2 exerts an upstream effect on general autophagy, as might be inferred from the effects of mutations on autophagosome formation (Manzoni et al. 2013a,b), then it would be reasonable to infer that one could see defects in mitophagy as a secondary consequence. There is some evidence of such defects in cells expressing mutant LRRK2 (Su et al. 2015), although such results need confirmation and mechanistic development to be certain of their applicability to different models. At this time, we think that the distinction between PINK1/parkin and LRRK2 is sufficient to consider them separately but with the reasonable possibility that those pathways may intersect at the level of autophagic regulation of mitochondria. This will certainly be a hot topic area for the PD field in the next few years.</p><p>Along the same lines, the relationship between each of these genes and GBA and ATP13A2 also requires clarification. A reasonable interpretation of the data available to date is that loss of function of either of these genes results in a lysosomal storage disease that, as part of the spectrum, can include symptoms of PD. Again, the real question here is to what extent do we consider the effects of LRRK2 and interaction partners on autophagy regulation to be 'the same' pathway as lysosomal function. On the one hand, it is certainly reasonable to infer that a block in lysosomal function would result in limited ability to turn over clients by all forms of autophagy. On the other hand, the examples of parkin and LRRK2 suggest that in those causes of disease, the proteins are regulatory for forms of autophagy rather than essential to lysosomal function, which is an important distinction at least at the mechanistic level.</p><p>There are data that would be consistent with the concept that even though there are groupings of genes as outlined above, there are effects that cross any boundaries placed between them. For example, PINK1 and parkin are reported to influence diverse autophagic pathways including starvation responses (Parganlija et al. 2014), recognition of intracellular pathogens (Manzanillo et al. 2013) and perhaps most relevant to PD, α-synuclein turnover (Lonskaya et al. 2013). Conversely, expression of α-synuclein can influence mitochondrial morphology in the mouse brain (Chinta et al. 2010; Chen et al. 2015), as can expression of LRRK2 in cell culture models (Cherra et al. 2013; Su et al. 2015).</p><p>Our view at this time is that there are three broad groupings of PD genes; the mitophagy regulators, the effectors of autophagy–endosomal recycling and the lysosomal proteins. There are likely to be important higher level relationships between these groups and those are worth pursuing further experimentally in the future. Nonetheless, the discreteness of subsets of genes supports the idea that there are distinctions in disease mechanisms at the level of the individual causative mutations.</p><!><p>There are some key areas that must be explored in the near future. Most narrowly, additional mechanistic data are required. To give one example, exactly how LRRK2 influences autophagy is not well understood at this time. By analogy to parkin, it is possible that LRRK2 is not active basally but requires activation steps, which remain to be fully elucidated. Understanding mechanisms related to control of these genes are likely to provide important ways in to understanding pathways in more depth.</p><p>Mechanistic data are also important to clarify some of the genetic data that are currently available. Although most GWAS studies list genes that are near to the peak of statistical association, this gives a false sense of precision as any gene that is within a region of linkage disequilibrium remains a valid candidate until it can be disproved. Thus, while at some loci such as that around SNCA we can be reasonably certain of the best candidate, in other regions there are not such obvious 'smoking guns'. We have claimed recently that interactions between candidates, especially those that come from unbiased approaches, might be one way to rationally promote one gene over all others (Beilina et al. 2014), although whether that generalizes to all loci remains to be proven.</p><p>Importantly, the role of α-synuclein in different forms of PD needs to be clarified. The issue of Lewy bodies remains difficult to understand, but the available data still support α-synuclein as being required for neuronal damage (Lin et al. 2009; Daher et al. 2014; Skibinski et al. 2014). Perhaps, a better way to think about the role of α-synuclein is to consider Lewy bodies and contribution to cell death as separate hypotheses. If so, then testing whether α-synuclein is required for neurodegeneration in the context of other genes, such as ATP13A2 or VPS35 would be instructive.</p><p>A much more difficult question alluded to above is why changes in autophagy or its regulation would result in a neurodegenerative condition, much less one that affects the motor system prominently. Some of the genes nominated for risk for PD, specifically α-synuclein and tau, encode proteins with relatively restricted neuronal expression, which may contribute to expression of a brain disease. However, some proteins are also expressed in other cell types – LRRK2 is expressed in macrophages and microglia, for example (Moehle et al. 2012), leaving open the possibility that some of the neurodegenerative process in PD is non-cell autonomous. We therefore need consider that proteins expressed outside of neurons may also contribute to disease.</p><p>It should also be noted that while it is possible, with nuanced views, multiple genes to a series of related pathways, it is not yet clear if all PD genes can be placed in one of these categories. A full discussion of this complex problem is probably outside the scope of this review, but as an example we might consider the MAPT gene encoding tau that is a nominated gene for PD risk (Simón-Sánchez et al. 2009). Tau is a microtubule binding protein and could therefore be linked to autophagy either because vesicular transport depends on cytoskeletal transport (Yan 2014) and/or because some forms of tau are substrates for degradation by the autophagy–lysosomal system (Chesser et al. 2013). However, additional mechanism-based studies are required to distinguish whether risk factor variants in MAPT or other risk genes require alterations in autophagy for their actions relevant to PD pathogenesis.</p><p>At the end, of all these considerations comes the acknowledgement that despite the advances in understanding causes of disease there has been relatively little published on curing PD. While wanting to contribute to lessening symptoms or progression for PD patients is aspirational, an additional appeal of mechanism-based therapeutics is that they may further resolve some of the questions raised above. As a thought experiment, would a parkin-based drug work on LRRK2 disease? If so, then this would suggest that the different pathways alluded to above are in fact closely related.</p><!><p>Many significant discoveries about the causation of PD have occurred in the past two decades by studying the different genetic contributions to disease risk. More recently, it has become clear that at least some of the nominated genes are functionally related. Our working hypothesis is that there are strong relationships between subsets of these proteins but that there are distinct themes that relate to the regulation of autophagy. Further testing this hypothesis is critical for moving these observations toward clinical application.</p>

PubMed Author Manuscript

On the use of DFT+U to describe the electronic structure of TiO2 nanoparticles: (TiO2)35 as a case study

One of the main drawbacks in the density functional theory (DFT) formalism is the underestimation of the energy gaps in semiconducting materials. The combination of DFT with an explicit treatment of electronic correlation with a Hubbard-like model, known as DFT+U method, has been extensively applied to open up the energy gap in materials. Here, we introduce a systematic study where the selection of U parameter is analyzed considering two different basis sets: plane-waves (PWs) and numerical atomic orbitals (NAOs), together with different implementations for including U, to investigate the structural and electronic properties of a well-defined bipyramidal (TiO2)35 nanoparticle (NP). This study reveals, as expected, that a certain U value can reproduce the experimental value for the energy gap. However, there is a high dependence on the choice of basis set and, and on the +U parameter employed. The present study shows that the linear combination of the NAO basis functions, as implemented in FHI-aims, requires a lower U value than the simplified rotationally invariant approaches as 2 implemented in VASP. Therefore, the transferability of U values between codes is unfeasible and not recommended, demanding initial benchmark studies for the property of interest as a reference to determine the appropriate value of U. I. INTRODUCTIONTitanium dioxide, TiO2, nanoparticles involving a mixture of anatase and rutile polymorphs, in particular in the commercialized Degussa P25 form, constitute the most studied photocatalytic material and a model system for the mechanisms involved in photocatalysis. [1][2][3][4] The performance of TiO2 largely depends on its optical, electronic, structural, morphological and surface properties, 5-7 and one of the key properties of TiO2, especially in the anatase polymorph, is the formation of photogenerated charge carriers (holes and electrons), activated by the absorption of ultraviolet (UV) light. Indeed, the need for UV radiation constitutes one of the major bottlenecks towards developing efficient TiO2 photocatalysts that can work under sunlight as only ~5% of the incident solar spectrum corresponds to UV light. Hence, a major challenge in the development of competitive TiO2-based photocatalysts is reducing the energy gap to the visible (VIS) region. 8 In principle, the properties of TiO2 can be modulated by designing nanoparticles (NPs) with different sizes, shapes, crystallinities, and surface facets. 9-12 . However, to determine the relationship between structural and electronic properties of TiO2 nanoparticles, experimentally, is not a simple task. Alternatively, computational techniques provide a feasible, accurate, and unbiased approach to study such correlations and, consequently, can contribute to build connections between experiment and theory. 13 Density functional theory (DFT) 14,15 has been widely used to study the properties of different types of materials with high accuracy in the prediction of crystal structures and reasonable description of electronic structure features at a moderate computational cost 16 and

on_the_use_of_dft+u_to_describe_the_electronic_structure_of_tio2_nanoparticles:_(tio2)35_as_a_case_s

<!>II. MODELS AND COMPUTATIONAL METHODS<!>III. RESULTS AND DISCUSSION<!>(iv)<!>A. Structure Analysis<!>B. Energy Gap Analysis<!>IV. CONCLUSIONS

<p>with a well-established reproducibility. 17 Unfortunately, energy gaps computed using the popular local density approximation (LDA) and the generalized gradient approximation (GGA) are consistently underestimated by 30-100% 18,19 . The error arises from the inherent lack of derivative discontinuity and the delocalization error. [20][21][22] To overcome the drawbacks of LDA and GGA for estimating this electronic property, hybrid functionals, which include a part of the nonlocal Fock exchange, have been proposed and widely employed. 18,23,24 Depending on the type of basis set, the use of hybrid functionals can represent a significant increase in the cost of the calculations. Inspired by the Hubbard Hamiltonian, 25 Anisimov et al. 26 proposed to avoid the computational load inherent to hybrid functionals by implementing an empirical onsite Hubbard (U) correction to a selected atomic energy level, within standard DFT. The resulting method is often referred to as DFT+U, an unfortunate term as DFT is an exact theory.</p><p>DFT+U has been broadly used, especially after the contribution of Dudarev et al. 27 and is particularly useful in the description of the partially filled d-states of the transition metals -in the case of TiO2, the U-correction is applied to the Ti 3d orbital. 28,29 The DFT+U method combines the high efficiency of standard DFT with an explicit, albeit approximate and empirical treatment of electron on-site correlation, and constitutes one of the simplest approaches to describe the ground state of strongly correlated systems. 30 However, the choice of the appropriate U parameter value for each compound constitutes a challenge. This obstacle can be solved through (i) a linear response, fully consistent method, 31 or (ii) alternative routes based on comparison with experimental results for some physical property of interest such as magnetic moment, energy gap, redox potentials or reaction enthalpies. [32][33][34] For instance, the latter strategy has been employed in the study of electron transport in rutile phase, 35,36 of reduced forms of TiO2, 37,38 and ultrathin films of the rutile phase. 39 Nevertheless, the selection of the U parameter is not straightforward. Moreover, the choice of the appropriate form of the projector functions inherent to the method is also a concern, 40 especially after the work of Kick et al. 41 who recently implemented DFT+U with a numerical atomic orbital basis set. The authors showed that the value for U depends on the choice of projector function, which in turn depends on the type of basis set (atomic orbitals or plane waves) used. The aim of the current study is to evaluate the effect of the basis sets in the selection of the U value necessary to describe the electronic structure of semiconducting nanoparticles, taking a previously investigated, well-defined (TiO2)35 bipyramidal NP as a case study. 42</p><!><p>The well-defined bipyramidal stoichiometric (TiO2)35 anatase NP, which fulfills the requirement of a Wulff construction, 43 and was used in previous studies, 42 is selected for the present study (Fig. 1). This nanoparticle exposes the most favorable (101) facets only, as found in experiments. 7 Furthermore, its ~2 nm size is also appropriate to rationalize experimental results reported for TiO2 anatase NPs. 44 The calculations reported here have been carried out using two widely used codes, namely the Vienna Ab Initio Simulation Package (VASP) 45,46 and the Fritz Haber Institute ab initio molecular simulations (FHI-aims). 47 In both cases, the Perdew-Wang (PW91) exchangecorrelation functional 48 is used and spin-polarization is accounted for explicitly, although the final results do not exhibit any spin-polarization. The partially filled Ti3d states were consistently described by applying the Hubbard U correction 26 under the simplified rotationally invariant approach introduced by Dudarev et al. 27 In the following, we will refer to the resulting approach as PW91+U, which is more appropriate. The calculations carried out with VASP employ a plane waves (PWs) basis set with a kinetic energy cut-off of 396 eV. To account for the effect of inner electrons on the valence density we implement the projector augmented wave (PAW) method of Bloch 49 as implemented by Kresse and Joubert, 50 with 12 and 6 valence electrons for Ti and O atoms, respectively. The (TiO2)35 NP is included in a 20  20  40 Å supercell to give a vacuum gap of 11 Å in the x-and y-directions and 20 Å in the z-direction.</p><p>Γ-point sampling is used and the convergence criteria for the energy and forces are 10 -4 eV and 0.02 eV/ Å -2 , respectively.</p><p>On the other hand, the calculations carried out by the FHI-aims code include all electrons (AEs) and account for relativistic effects through the so-called zero-order regular approximation (ZORA) 51,52 proposed earlier by Chang et al. 53 A tier-1 light grid numerical atom-centered orbital (NAO) basis set has been used with a quality comparable to that of a TZVP Gaussian Type Orbital basis set for TiO2. 42 Here, for the implementation of Hubbard U correction, the projection functions for Ti3d states are introduced as an explicit linear combination of the NAO basis functions with the double-counting correction in the fully localized limit (FLL); see details in Ref. 41. The convergence threshold for the energy and is 10 -4 eV. Note that, hereinafter, the notation of PW and NAO is used to refer to the calculations performed with VASP and FHI-aims, respectively.</p><!><p>To provide a sound reference for the study, we first discuss the energy gap of fully relaxed anatase and rutile bulk phases as predicted from spin polarized DFT calculations with the PW91 GGA type density functional and using either PW or NAO basis sets. To avoid problems arising from a difference in the quality of the basis sets we increase the kinetic energy cutoff for the PW to 550 eV and used a more extended NAO basis set of tier-2 tight quality.</p><p>For rutile, the PW/NAO calculated band gap is 1.70/1.91 eV whereas for anatase the PW and NAO calculated band gaps coincide and amount to 2.10 eV. The difference in the rutile phase must be attributed to small differences in the optimized structure arising from the different treatment of the core electrons. In any case, the PW and NAO calculations for bulk rutile and anatase lead essentially to the same results with a deviation of at most 0.2 eV in the band gap.</p><p>Clearly, these calculated energy gaps are underestimated with respect to the experimental values, which are 3.0 and 3.2 eV, for rutile and anatase phases, respectively. [54][55][56] Hybrid functionals with an ad-hoc amount of non-local Fock exchange are known to provide a better estimate, as discussed for instance by Ko et al. 57 , while DFT+U can be tuned to recover the experimental band gap, but usually at the cost of a poorer description of other materials properties.</p><p>Next we focus on the representative (TiO2)35 anatase NP depicted in Fig. 1. The atomic structure of this NP has been obtained from a geometry optimization using both VASP and FHI-aims computational packages and PW91+U. However, to perform a rigorous comparison of the effect of U when using PW or NAO basis sets we consider four different situations which are as follows:</p><p>The structure is optimized in FHI-aims with PW91 (U=0) and single-point calculations are run with both FHI-aims and VASP at each U value, U=0-10 eV;</p><p>(ii)</p><p>The structure is optimized in VASP with PW91 (U=0) and single-point calculations are run with both FHI-aims and VASP at each U value, U=0-10 eV;</p><p>(iii) The structure is fully optimized in both FHI-aims and VASP at each U=0-10 eV.</p><!><p>Each structure obtained by FHI-aims (VASP) in (iii) is submitted to a single point calculation in VASP (FHI-aims) at the same U-value.</p><p>The first and second sets of calculations allow one to investigate differences in the description of the electronic structure that are not due to a difference in the atomic structure but to the different type of basis set and the implementation of the +U term. 41 The third set of calculations provides information about differences in the final optimized structure, and the effect of this optimization on the energy gap. Finally, the fourth set of calculations shows to what extent the fully relaxed atomic structure impacts on the electronic structure. In each of these data sets we can compare the results of the different set-ups by a linear fit of the data.</p><!><p>We start the discussion by analyzing the structural properties of the (TiO2)35 NP focusing mainly on its length and width (Fig. 2). The PW91 (U=0) fully optimized structures of the (TiO2)35 NP predicted by VASP and FHI-aims are almost the same. In both cases, the nanoparticle length, which is taken from the terminal atoms located in the apical region (see Fig. 1), is 19.61 Å. For the width of the NP, FHI-aims predicts a width that is 0.02 Å larger than VASP. Hence, in the absence of U, both types of basis set lead to the same structure, as expected. 17 Therefore, any difference in the PW91+U structure predicted by the two types of basis sets (codes) has to be attributed to differences in the implementation of U. Regarding the atomic structure, the main effect of U is to slightly increase nanoparticle length (Fig. 2a). The tendency is consistent, regardless of the basis set, up to U = 5 eV. When U is larger than 5 eV the lengths predicted by VASP and FHI-aims follow different trends. The analysis of the nanoparticle width presents some interesting features (Figs. 2b and 2c). Here the effect of U is different depending on whether the calculation is carried out with a PW or NAO basis set. When using NAO, the optimized NP width drops almost linearly with U up to U = 7 eV, whereas when using PW, the dependence with U is very small, almost negligible. We note that, when using PW, the trends are very stable along the interval of U. However, this is not the case when NAO basis set are employed, and the regular trend is broken at U = 7 eV. Note also that the breaking of the trend at U > 7 eV for the calculations with NAOs indicates that this value is too large to correctly describe correlation effects as it has an exceedingly large influence on the properties of the nanoparticle and induces unreasonable structural changes. Similar observations on the effect of U on the phase stability of TiO2 have been reported. 33 It is assumed that the large effect of U on the atomic structure predicted by the calculations using the NAO basis set arise from the more localized character of the atomic NAO Hubbard projectors as implemented in FHI-aims. 41</p><!><p>The analysis of the energy gap of the (TiO2) 35 anatase NP provides further interesting comparisons. The Kohn-Sham energy gaps, computed in the set-ups described in scenarios (i) and (ii), above, are shown in Fig. 3 and Table I. This data corresponds to two structures, each optimized with the respective codes, FHI-aims and VASP, at the PW91 (U=0) level. We begin by comparing the results of the single-point PW calculations performed on the FHI-aims (green) and VASP (blue) relaxed structures. At each U-value, the difference in computed energy gap between the two structures is negligible; in this case, the PW basis set implementation of +U is not sensitive to the geometry at which the electronic structure is computed.</p><p>This result contrasts with the NAO data: for each U-value, NAO calculations predict a larger energy gap for the FHI-aims structure, relative to the VASP structure. The energy gaps computed from single point NAO calculations over the FHI-aims relaxed structure (red) are positively offset by ~0.5 eV with respect to those values computed over the VASP relaxed structure (black). The change in the energy gap with increasing U is consistent, regardless of the atomic structure, as revealed by the slopes (a-values) of the red and black trendlines, presented in Table I; i.e. the 0.5 eV offset is maintained over the range of U-values. This result is interesting because, as discussed, both FHI-aims and VASP predict similar structures, vis length and width, at the PW91 (U=0) level. However, small differences in the atomic structures yield appreciable differences in the energy gaps computed with the NAO basis set, while no differences were shown with the PW basis set. This highlights that, to avoid misunderstanding interpretations in the analysis of the electronic properties, structural relaxation is crucial when using NAO basis set. It appears that the impact of U is greater with NAO, related to the localized projector functions. 41 It is also interesting to compare NAO and PW results when these calculations are performed on the same starting structure. For the FHI-aims relaxed structure, the energy gaps predicted by NAO (red) and PW (green) calculations are in agreement for small U-values, but the differences in the predicted gaps increase with increasing U. This is reflected in the slopes (a-values) of the trendlines fitted to the NAO (red) and PW (green) data, which are 0.103 and 0.075, respectively (see Table I). In this case, the energy gap varies to a greater extent in the NAO calculations, which consistently predict larger gaps with respect to the PW calculations.</p><p>Conversely, for the VASP relaxed structure, the energy gaps predicted by NAO (black) and PW (blue) differ over the entire range of considered U-values. For U = 0 eV, the PW-computed energy gap is larger than that computed with NAO by ~0.5 eV, but this difference decreases with increasing U, in accordance with the larger slope for the NAO data (0.106), with respect to that of the PW data (0.080). These results suggest that the differences observed in the computed Kohn-Sham energy gaps are not attributable to differences in the atomic structure, but rather to differences in the implementation of DFT+U for the NAO or PW basis set.</p><p>Finally, we note that each of the computational set-ups, with the exception of NAO calculations on the VASP relaxed structure (black), predict similar energy gaps of ~2.5 eV for U = 0 eV. For these three set-ups, the differences in the computed energy gaps are reasonable, i.e. within 0.15 eV, for U-values up to 4 eV. For U > 4 eV, the NAO basis set promotes a larger energy gap with respect to the PW basis set.</p><p>The data obtained from the calculations described in scenarios (iii) and (iv), above, are presented in Fig. 4 and Table II. We first look at the computed energy gaps for the structures optimized at each U-value in FHI-aims (red) and VASP (blue). The energy gaps computed with the NAO basis set increase from 2.5 eV to 3.8 eV as U increases from 0 eV to 10 eV. This monotonic increase with U is expected and is corroborated in the trendline data, shown in Table II.</p><p>Interestingly, the opposite trend is observed for the energy gaps computed for the structures that were fully relaxed at each U with the PW basis set: in this case, the energy gaps decrease monotonically with increasing U. As seen in our discussion of Figure 3, increasing the U-value in a PW calculation on a fixed structure yields a larger energy gap. Thus, here we must attribute the decrease in the energy gaps to effects arising from the structural optimization at each U. This result is surprising, not only because it is unexpected, but also because the changes in the PW-computed atomic structures over the range of U-values are modest (see Fig. 2), yet the impact on the electronic structure is significant, with states in the gap attributed to the presence of the low coordinated O atoms; see density of states plots in Fig. 5. In fact, for the VASP-relaxed PW91 (U=0) structure, a single-point PW calculation with U = 4 eV yields an energy gap of 2.76 eV whereas for the fully relaxed structure the energy gap is 2.35 eV. In other words, the emergence of the gap states occurs at lower U values in the PW calculations.</p><p>This is clearly seen in the density of states plots in Fig. 5 corresponding to the VASP and FHIaims calculations for U = 2 and 6 eV, respectively.</p><p>Performing a single-point PW calculation on the FHI-aims relaxed structures at each U-value produces the energy gaps represented with the green data points in Figure 4. Here we see that the data points agree with those computed with the NAO basis set (red) within 0.1 eV up to U = 4 eV, after which the differences increase. This is in agreement with the trendline data listed in Table II; the slopes for the NAO (red) and PW (green) basis sets are 0.136 and 0.095, respectively. Importantly, single-point PW calculations on the FHI-aims relaxed structures, at each U, predict an increase in energy gap with increasing U. This further confirms that the decreasing trend in energy gaps for the VASP-relaxed structures arises from structural effects.</p><p>The energy gaps computed with single-point NAO calculations on the VASP-relaxed structures, at each U, are shown with the black data points in Fig. 4. An outlier in this data is the energy gap computed for U = 0 eV, which is 2.02 eV. This value has been checked and the presence of an error in the calculation can be ruled out. Interestingly, for U = 1-10 eV, the computed energy gaps are consistently ~2.5-2.6 eV and this data shows no discernible increasing or decreasing trend. As seen in our discussion of single-point NAO calculations on both the FHI-aims and VASP PW91 (U=0) relaxed structures, the predicted energy gaps increase monotonically with increasing U. Once again, this suggests that subtleties in the structural optimization within the PW implementation of DFT+U, probably linked to the low coordinated O atoms at the NP edge, produce these effects in the electronic structure.</p><p>For the NAO calculations, consistent with the linear trends for the red data reported in the legends of Figures 3 and 4, the relaxation at each U value has a negligible effect, as expected, on the fitting offset with respect to the calculation at the PW91 (U=0) structure.</p><p>However, the fully relaxed calculations result in changes in the fitting slope. Thus, the opening of the energy gap is more pronounced for the fully optimized structures when employing the NAO basis.</p><p>This latter situation, where the NP structure is fully relaxed at each U in each code, is the most reasonable scenario to analyze the different behavior observed between basis sets because artifacts due to the use of a structure not optimized within the method/basis set are ruled out. First of all, the energy gaps between the PW and NAO basis set are shifted by 0.25 eV (see Fig. 3), which can be attributed to a different treatment of the effect of the core electrons and also relativistic effects. 58,59 The former are included explicitly in the calculations with the NAO basis set, whereas they are included through a frozen orbital type approach through the PAW in the calculations with the PW basis. Similarly, the relativistic effects are included explicitly at the ZORA level with the NOA basis and implicitly through the PAW description of the core electrons in the PW calculations. In principle, the most accurate results are obtained from the all-electron basis set implemented in FHI-aims. The most relevant results are found in the variation of the energy gap in response to increasing U. These are depicted in Figure 4 and the trends (Table II) reflected in the linear fittings with slopes of 0.136 and -0.028 for NAO and PW basis set, respectively. This result clearly shows the effect of U on the resulting energy gap does not only depend on the numerical value of this parameter but also on the projection of the Kohn-Sham states to determine the occupation numbers that enter the +U correction and the structural optimization, which, in turn, depend on the basis set used. Thus, the +U part of the exchange-correlation potential severely depends on the DFT code, as already shown by Kick et al. for some systems. 41 To clarify this issue, we comment on how results from the PW91+U approaches used In summary, the U value fitted to reproduce an experimental or hybrid functional calculated value using a given DFT code cannot be transferred to another code as it depends on the basis set used and, on the method employed to define the corresponding projectors.</p><p>Thus, for each materials system and DFT code, one should recompute suitable values for U through making initial benchmarks.</p><!><p>The effect of the DFT+U method on the structural and electronic properties of the (TiO2)35 NP is systematically investigated by two different basis sets, namely, plane-waves (PWs) and numerical atomic orbitals (NAOs), along with different approaches for the implementation of U value. In the absence of U, PW and NAO calculations report the same structure and, consequently, the structural variations observed by its inclusion are due to the different implementation of U based on a simplified rotationally invariant approach and a linear combination of the NAO basis functions, respectively. Interestingly, the analysis of the energy gap reveals that a certain U value can reproduce the experimental value, however, it depends on the basis set and on the employed U parameter. Therefore, the transferability of U values between codes is not to be recommended and requires initial benchmarks for the property of interest as a reference to find the appropriate value. This study clearly shows that the DFT+U implementation in a localized basis set code such as FHI-aims entails much lower values of U to reproduce results obtained with a plane wave basis set code such as VASP. II.</p><p>FIG. 5 Projected electronic density of states (PEDOS) of the full relaxed (TiO2)35 NP using PW and NAO basis sets for U=0, 2, 4, and 6 eV.</p>

ChemRxiv

A novel multicolor immunostaining method using ethynyl deoxyuridine for analysis of in situ immunoproliferative response

Immune responses are generally accompanied by antigen presentation and proliferation and differentiation of antigen-specific lymphocytes (immunoproliferation), but analysis of these events in situ on tissue sections is very difficult. We have developed a new method of simultaneous multicolor immunofluorescence staining for immunohistology and flow cytometry using a thymidine analogue, 5-ethynyl-2′-deoxyuridine (EdU). Because of the small size of azide dye using click chemistry and elimination of DNA denaturation steps, EdU staining allowed for immunofluorescence staining of at least four colors including two different markers on a single-cell surface, which is impossible with the standard 5-bromo-2′-deoxyuridine method. By using two rat models, successfully detected parameters were the cluster of differentiation antigens including phenotypic and functional markers of various immune cells, histocompatibility complex antigens, and even some nuclear transcription factors. Proliferating cells could be further sorted and used for RT-PCR analysis. This method thus enables functional in situ time-kinetic analysis of immunoproliferative responses in a distinct domain of the lymphoid organs, which are quantitatively confirmed by flow cytometry.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-015-1329-z) contains supplementary material, which is available to authorized users.

a_novel_multicolor_immunostaining_method_using_ethynyl_deoxyuridine_for_analysis_of_in_situ_immunopr

Introduction<!>Animals<!><!>Experimental design<!>Splenic lymphocyte isolation<!>Flow cytometric analysis<!>Cryosectioning and pretreatment<!>Multicolor fluorescence immunohistology using EdU<!>Multicolor enzyme immunohistology using BrdU<!>Correlation of EdU and BrdU in cell proliferation analysis<!>EdU staining correlates well with BrdU staining in immunohistology<!><!>Parallel analysis of FCM and immunohistology<!><!>Simultaneous detection of a transcription factor and cycling S-phase cells<!><!>Simultaneous detection of two different surface markers of cycling S-phase cells<!>Analysis of cellular interactions between two different cell types<!><!>Analysis of cellular interactions between two different cell types<!>Discussion<!>Simultaneous detection in situ of two different markers of cycling S-phase cells<!>In situ analysis of cellular interactions between two cell types<!>Parallel analysis of FCM and immunohistology<!><!>Conclusion<!>

<p>Immune responses induce activation and clonal expansion of antigen-specific lymphocytes with active DNA synthesis for cell division. Dendritic cells (DCs) undergo a crucial interaction with lymphocytes as professional antigen-presenting cells in the distinct domain of the secondary lymphoid organs. DCs form a cluster with antigen-specific lymphocytes and induce immunoproliferation, i.e., their differentiation and proliferation within the cluster (Matsuno et al. 1989; Saiki et al. 2001; Ueta et al. 2008). Therefore, in situ examination of phenotype and functional molecules of cycling cells and cellular interactions with DCs or stromal cells during the immune response should provide crucial information for understanding immunity in health and diseases.</p><p>We have long studied immunoproliferative responses in situ by using a multicolor immunoenzyme staining analysis (Matsuno et al. 1989, 2010; Saiki et al. 2001; Ueta et al. 2008) for a thymidine analogue, 5-bromo-2′-deoxyuridine (BrdU), and other parameters. However, because enzyme-developed color dyes tend to interfere with the following immunostaining step, only two-color analysis prior to BrdU staining could be performed at best and detection of two different markers on a single-cell surface was very difficult. Immunofluorescence staining has an advantage, because fluorescent dyes do not interfere with each other and four-color staining is possible by a standard protocol. However, BrdU immunofluorescence staining is also problematic, because it requires DNA denaturation steps for exposing antigen epitopes by strong acid or heating, resulting in decreased intensity of other fluorescent dyes. Therefore, a new method other than BrdU staining has been needed for a long time.</p><p>Although flow cytometry (FCM) enables quantitative analysis of multiple parameters of a proper cell subset, it requires single-cell suspensions and therefore cannot delineate the in situ localization or cellular interaction. In contrast, although immunohistology is the most practical method for in situ analysis, quantitative analysis of the immune response in the tissue sections is time consuming and not easy. Accordingly, parallel analysis of both immunohistology and FCM using one sample should provide good information about the immune response.</p><p>Recently, a thymidine analogue, EdU (5-ethynyl-2′-deoxyuridine), was described as a replacement for BrdU to directly measure de novo DNA synthesis of S-phase cycling cells using click chemistry (Salic and Mitchison 2008). Click chemistry is a method of covalently coupling an azide with an alkyne. Detection of EdU relies on the copper (I)-catalyzed click reaction with an azide-modified fluorescent dye to form a stable triazole ring. Because of the small size of the azide dye, no harsh denaturation steps are needed to gain access to the DNA (Salic and Mitchison 2008). Previous studies reported that EdU could be used for immunohistology (Salic and Mitchison 2008) or FCM (Diermeier-Daucher et al. 2009) as a thymidine analogue. Accordingly, EdU staining holds the potential to be applied in multicolor immunofluorescence including proliferating cells and double-membrane staining of a single cell, which is impossible with the standard BrdU method. In neuroscience research, a few publications report using triple immunohistogical staining for two neuronal peptide antigens and EdU (Guo et al. 2009). Because the tissues are prefixed with paraformaldehyde (PFA) and stable intracellular peptide antigens are targets of immunostaining, this method is not applicable for double-membrane staining of the cluster of differentiation (CD) antigens, which are mostly labile and easily denatured or masked by aldehyde fixatives.</p><p>In the present study, by applying our original multicolor immunoenzyme (Saiki et al. 2001; Ueta et al. 2008) and immunofluorescence (Sawanobori et al. 2014) staining methods, we have tried to develop a new method of simultaneous multicolor immunofluorescence staining using EdU for up to four colors for immunohistology and up to three colors for FCM. Two models for assessing the in vivo proliferative response of immune cells were examined. The first is the administration of a CD28 superagonist (CD28SA), reported to preferentially expand forkhead box P3 (Foxp3) gene—expressing naturally occurring regulatory T-cells (nTregs) (Beyersdorf et al. 2005). The second is the one-way graft-versus-host reaction (GvHR) by the transferring of parental congeneic T-cells to F1 hybrid rats (Matsuno et al. 2010). Here we show that lymphocyte markers, histocompatibility complex antigens, cell adhesion molecules, and even nuclear transcription factors in addition to EdU can be detected simultaneously by this method.</p><!><p>Inbred male Lewis (RT1AlBl) and PvG/c (RT1AcBc) rats were purchased by SLC Co. (Shizuoka, Japan). Congeneic PVG/c-RT7b/OlaHsd (RT1AcBcRT7b) rats and (PvG/c × Lewis) F1 hybrid rats were bred and maintained in the Laboratory Animal Center for Research (Dokkyo Medical University). All animals were reared under specific pathogen-free conditions and used at 8–10 weeks of age. Animal handling and care were approved by the Dokkyo Medical University Animal Experiments Committee and were in accordance with the Dokkyo University's Regulations for Animal Experiments and with Japanese Governmental Law (No. 105). No studies involving human participants are reported here.</p><!><p>Antibodies used in this study</p><p>#Own conjugation</p><p>aAbD serotec</p><p>bBiolegend</p><p>cCappel</p><p>dEbioscience</p><p>eJackson immunoresearch</p><p>fLife Technologies Corporation</p><p>gSigma</p><p>hECACC</p><p>iGenerously provided by Dr. Y. Sado</p><p>jGenerously provided by Dr. F. Kroese</p><p>kAlexa: Alexa Flour®</p><p>lThe tandem conjugate in Peridinin Chlorophyll Protein Complex and Cyanine 5.5</p><!><p>For the first experiment, Lewis rats received intravenous injection of a CD28 superagonist mAb (CD28SA, clone JJ316: 0, 0.25, 0.5, 1 mg/300 g body weight), and the spleens were collected 3 days later. In the second experiment, one-way systemic GvHR was induced by intravenous injection of T-cells of congeneic PVG/c-RT7brats into (PvG/c × Lewis) F1 hybrid rats, and the spleens were collected 1 and 2 days after injection. For a source of donor T-cells, thoracic duct lymphocytes (5.0 × 107 cells/rat) were used after thoracic duct cannulation, as reported (Zhou et al. 2008). In both experiments, recipient rats received an intravenous injection of a mixture of equivalent moles of BrdU (6 mg/200 g body weight, Sigma-Aldrich Japan, Tokyo) and EdU (5 mg/200 g body weight, Life Technologies Corporation) in phosphate-buffered saline (PBS) 1 h before killing. To avoid masking or loss of labile CD antigens by aldehyde fixatives, fresh cryosections without prefixation were employed. General anesthesia during animal procedures was provided using isoflurane (Mylan Inc., Tokyo, Japan) supplied by an isoflurane vaporizer (SN-487-OT; Shinano Manufacturing, Tokyo, Japan).</p><!><p>The harvested spleens were injected with Collagenase D (1 mg/mL, Roche Diagnostics GmbH, Mannheim, Germany) and DNase I (400 U/mL, Roche Diagnostics GmbH) in 3 mL Hank's buffered salt solution (HBSS) containing 5 % fetal calf serum, 1.2 mM CaCl2·2H2O, and 0.8 mM MgSO4·7H2O and were digested under gentle stirring for 30 min at 37 °C in a CO2 incubator (MCO-18AIC; Sanyo, Osaka, Japan). The collagenase digestion was stopped by adding 0.5 M EDTA solution and five volumes of cold PBS. The isolated splenocytes were filtered through a 200-μm nylon mesh and washed twice in PBS with 0.2 % bovine serum albumin (PBS-BSA) by centrifugation (himac CF16RX; Hitachi Ltd, Tokyo, Japan) at 280×g for 10 min at 4 °C. The splenic lymphocyte fraction was isolated in an OptiPrep discontinuous density gradient (15 and 11.5 %, Axis-Shield, Oslo, Norway) by centrifugation at 600×g for 24 min at room temperature (RT). With this approach, the upper layer cells of the 15 % OptiPrep were mainly lymphocytes; interface cells between 15 and 11.5 % OptiPrep were macrophages, and DCs. The lymphocyte fractions were washed once by centrifugation at 440×g for 10 min at 4 °C and used for FCM.</p><!><p>Splenic lymphocytes at 106 cells/100 μL PBS-BSA were incubated for 30 min at 4 °C with an optimal concentration of purified mouse mAbs to anti-rat CD antigens diluted and washed three times with PBS-BSA by centrifugation at 350×g for 5 min. The cells were incubated with PerCP/Cy5.5-conjugated anti-mouse IgG secondary antibody (Biolegend, San Diego, CA, USA) for 30 min at 4 °C in PBS-BSA with 1 % normal rat serum and rinsed three times with PBS-BSA. Cells then were incubated for 1 h at 4 °C with normal mouse IgG (20 µg/mL) in PBS-BSA for blocking additional mouse antibody binding. The next step was incubation with a purified second mAb directly conjugated with Alexa-647 for 30 min at 4 °C, followed by a wash. The EdU staining was performed at the final step. The cells were permeabilized with a permeabilization buffer set (00-5523-00, eBioscience San Diego, CA, USA) overnight (O/N) at 4 °C for intracellular staining and washed. EdU was visualized using the Click-iT kit for FCM according to the manufacturer's instructions. Cells were analyzed by FCM (FACSCalibur) with CellQuest Pro software (BD Biosciences).</p><p>In case of Foxp3 staining, the second mAb was omitted, and the cells were first permeabilized in the same manner as for EdU staining. Then, the cells were incubated with Alexa-647-conjugated anti-mouse/rat Foxp3 mAb (FJK-16s, eBioscience) in permeabilization buffer for 30 min at 4 °C and washed at least three times with the same buffer. The EdU staining was performed without the permeabilization step.</p><p>In the first experiment, TCRαβ+CD25highEdU+ or EdU− cells were further isolated by FACSAria (BD Biosciences) sorting. Then Foxp3 message was examined in both cell groups by reverse transcription polymerase chain reaction (RT-PCR, Model TP600, Takara Bio, Inc., Shiga, Japan) at 30 cycles (for 10 s at 98 °C, 30 s at 60 °C, and 60 s at 72 °C). Primers were as follows: Foxp3, forward primer, 5′-CGG GAG AGT TTC TCA AGC AC-3′; reverse primer, 3′-GGA GCT CTT GTC CAC TGA GG-5′; GAPDH (glyceraldehyde-3-phosphate dehydrogenase: Internal control), forward primer, 5′-AGA CAG CCG CAT CTT CTT GT-3′; and reverse primer, 3′-CTT GCC GTG GGT AGA GTC AT-5′.</p><!><p>We applied the processing method for the immunoenzyme (Saiki et al. 2001; Ueta et al. 2008) and immunofluorescence (Sawanobori et al. 2014) staining methods in our laboratory to the EdU immunofluorescence staining. Fresh cryosections were cut using a LEICA CM1850 (Leica Microsystems, Ontario, Canada) and were processed as described in the "Recommended protocols."</p><!><p>For both experiments, three- to four-color immunofluorescent staining was performed as precisely described in supplemental online materials for either phenotype (TCRαβ, etc.) of proliferating cells (EdU), nuclear transcription factor (Foxp3), tissue frameworks (type IV collagen), and DCs (CD103, etc.) in the spleen cryosections. Multicolor images were captured using an Axioskop2 Plus fluorescent microscope (Carl Zeiss, Jena, Germany) with an AxioCam MRm camera and AxioVision software (Carl Zeiss). Filters used were Filter Set 49 for Alexa-350, 17 for Alexa-488, 32 for Alexa-647 or -680 (Carl Zeiss), and XF407 for Alexa-594 (Omega Optical, Brattleboro, VT, USA), respectively. This filter combination had negligible crossing over of emitted lights between filters. We assigned pseudocolors to each channel to make merged images more comprehensible by maximizing contrast using AxioVision software (Carl Zeiss).</p><!><p>For the second experiment, the spleen cryosections were triple enzyme-immunostained for donor lymphocytes (RT7b congeneic marker, blue), type IV collagen (brown), and proliferating cells (BrdU, red) as previously described (Saiki et al. 2001; Ueta et al. 2008).</p><!><p>To confirm that EdU-positive (EdU+) cells and BrdU+ cells were the same proliferating cell population, triple immunofluorescent staining for type IV collagen, EdU, and BrdU was performed. After the blocking solution, sections were incubated for 1 h ~O/N at RT with a rabbit anti-mouse type IV collagen Ab and washed. Sections were incubated with aminomethylcoumarin (AMCA)-conjugated anti-rabbit IgG for 1 h and washed. Then, EdU was stained using the Click-iT® kit. For DNA denaturation, sections were treated for 10 min at 89 °C by a Microwave processor (MI-77, Azumaya, Tokyo, Japan) with a Retrivagen kit (BD Biosciences) and cooled to RT. After being washed and blocked, sections were incubated with Alexa-647-conjugated anti-BrdU mAb for 1 h at RT. Sections were mounted with coverslips and were examined under a fluorescence microscope.</p><!><p>The Click-iT® kit resulted in intense and clear EdU staining with a low signal-to-noise ratio for all fluorochromes tested: Alexa-488, -594, and -647. The protocol from the manufacturer was easy and reproducible and applicable not only for FCM but also for immunohistology.</p><p>The immunostaining of the spleen in the first experiment showed a massive proliferative response in the white pulp at 3 days after CD28SA stimulation (Supplementary Fig. 1). Double immunofluorescent staining for EdU and BrdU showed superimposition of EdU+ nuclei on almost all BrdU+ nuclei (Supplementary Fig. 1). To note, in some cells, the intensities of both stainings were different where BrdUhigh cells are EdUlow or vise versa, which was also shown previously (Salic and Mitchison 2008).</p><!><p>Assessment of proliferating lymphocytes in the spleen activated by CD28SA. a FCM analysis of the splenic lymphocytes for lymphocyte markers and EdU at day 3 after CD28SA injection (0.5 mg/rat). Note increase in TCRαβ+, CD4+, or CD25+ EdU+ cells but not CD8β+EdU+ cells compared to control. b Dose response of splenic lymphocytes to CD28SA. Absolute number of EdU+ proliferating cells with different lymphocyte markers/spleen, showing 1.0 mg group induces the highest response. c Triple immunofluorescent staining for lymphocyte markers (indirect staining with Alexa-680-conjugated anti-mouse IgG, green), EdU (Alexa-594-conjugated azide, red) and type IV collagen (indirect staining with AMCA-conjugated anti-rabbit IgG, white). Day 3 after CD28SA injection (0.5 mg/rat). Pseudocolors were assigned using AxioVision software. Scale bar 100 µm. The splenic PALS area with the central arteries is depicted by type IV collagen. Proliferating cells with different markers are shown as green cells with red nuclei (inset of lower panel)</p><!><p>Multicolor fluorescence immunohistology of rats receiving 0.5 mg of CD28SA showed that many TCRαβ+, CD4+, and CD25+ cells but only a few CD8β+ cells had EdU+ nuclei in the T-cell area of the splenic white pulp, i.e., the periarterial lymphocyte sheath (PALS) (Fig. 1c). Therefore, EdU staining enables the parallel examination of the proliferative response of activated cells both quantitatively by FCM and qualitatively by immunohistology of tissue sections.</p><!><p>FCM for proliferating Tregs induced by CD28SA. a Three-color FCM analysis of the splenic lymphocytes for lymphocyte markers, Treg transcription factor (Foxp3), and EdU at day 3 after CD28SA injection (0.5 mg/rat). b Dose response of Foxp3+Tregs with different markers to CD28SA. Absolute number of EdU+ proliferating Tregs with different lymphocyte markers/spleen, showing 0.5–1.0 mg induces an intense response</p><p>FACSAria sorting and immunohistological analysis for proliferating Tregs. a RT-PCR analysis of TCRαβ+CD25highFoxp3+ EdU+ or EdU− Tregs isolated by FACSAria sorting. b Four-color immunofluorescent staining for CD25 (indirect staining with Alexa-680-conjugated anti-mouse IgG, red), Foxp3 (Biotin-labeled mAb plus Alexa-488-conjugated streptavidin, green), EdU (Alexa-594-conjugated azide, blue), and type IV collagen (indirect staining with AMCA-conjugated anti-rabbit IgG, white). Pseudocolors were assigned using AxioVision software. Splenic PALS area at day 3 after CD28SA injection (0.5 mg/rat). Proliferating CD25+Foxp3+ Tregs are depicted as red cells with light blue nuclei (inset and white arrows) in the CD28SA spleen but few in the control spleen. Scale bar 100 μm</p><!><p>Multicolor fluorescence immunohistology of the same spleen showed that many CD25high cells with Foxp3+EdU+ nuclei were more frequently observed in the PALS than in the control (Fig. 3b). The results were in agreement with those of the FCM study.</p><p>We thus could directly demonstrate proliferation and expression of some transcription factors of Tregs by FCM and immunohistology. Foxp3+EdU+ proliferating Tregs could be further isolated and used for RT-PCR analysis.</p><!><p>Migration and proliferation of donor lymphocytes in GvHR. a Three-color immunoenzyme staining of the spleen after GvHR induction for donor lymphocytes (RT7b+, blue), type IV collagen (brown), and BrdU (red). Note migration of donor lymphocytes into the PALS on day 1 and their proliferation on day 2 (inset). Scale bar 100 μm. b Four-color immunofluorescence staining of the spleen for TCRαβ or CD8β (Alexa-647-conjugated mAb, blue), donor lymphocytes (indirect staining with Alexa-488-conjugated anti-mouse IgG, green), type IV collagen (indirect staining with AMCA-conjugated anti-rabbit IgG, white), and EdU (Alexa-594-conjugated azide, red). Pseudocolors were assigned using AxioVision software. Merged images are explained schematically in the right side of the panels. Note proliferation of TCRαβ+ (light blue cell with red nucleus, inset) but not CD8β+ donor cells (no light blue cell with red nucleus, inset). Scale bar 100 μm. c FCM for donor lymphocytes, TCRαβ, and EdU of the spleen, showing increase in proliferating TCRαβ+ donor T-cells on day 2. d Absolute number of EdU+ proliferating donor lymphocytes with different lymphocyte markers/spleen, showing TCRαβ+CD4+ donor T-cells actively proliferate on day 2</p><!><p>Second, four-color immunofluorescence staining of the same rat spleens was performed. As expected, many EdU+RT7b+ donor lymphocytes were TCRαβ+ (Fig. 4b) and CD4+ (not shown), but only a few of them were CD8β+ (Fig. 4b). The FCM analysis of the spleen cells of the same rats showed that TCRαβ+ and CD4+ donor T-cells were mostly EdU− on day 1 but that many of them became EdU+ on day 2 (Fig. 4c, d). In contrast, CD8β+ donor T-cells were very few in number and rarely became EdU+ on day 2 (Fig. 4c, d), confirming the immunohistological finding (Fig. 4b).</p><!><p>For analysis of cellular interactions in vivo, direct observation of different types of cells being activated through the interactions is crucial. We previously reported that early cluster formation of donor T-cells with host DCs and the proliferative response of these T-cells within the cluster represent the direct pathway of allosensitization in the allograft response (Saiki et al. 2001; Ueta et al. 2008). To reveal either two different surface markers of cycling S-phase cells or host DCs in the cluster, four-color immunofluorescence staining of the same rat spleens on day 2 was performed.</p><!><p>Phenotype of proliferating donor lymphocytes that cluster with host MHCII+ putative DCs (APCs). Four-color immunofluorescence staining of the spleen of day 2 after GvHR induction for TCRαβ or CD8β (Alexa-647-conjugated mAb, blue), donor lymphocytes (Alexa-488-conjugated mAb, green), host MHCII (indirect staining with Alexa-350-conjugated anti-mouse IgG, magenta), and EdU (Alexa-594-conjugated azide, red). Pseudocolors were assigned using AxioVision software. Scale bar 100 μm. Merged images are explained schematically in the right side of the panels. Note proliferation of TCRαβ+ (a light blue cell with red nucleus, inset and white arrows) but not CD8β+ donor T-cells (b green cell with red nucleus is CD8β−EdU+, inset and white arrows) that cluster with host MHCII+ cells</p><p>Phenotype of host MHCII+ putative DCs that cluster with proliferating cells. Three-color immunofluorescence staining of the spleen of day 2 after GvHR induction for host MHCII (Alexa-647-conjugated mAb, blue), CD205, CD103, or CD11c (indirect staining with Alexa-594-conjugated anti-mouse IgG, green), and EdU (Alexa-488-conjugated azide, red). Pseudocolors were assigned using AxioVision software. Scale bar 100 μm. Merged images are explained schematically in the right side of the panels. Note host MHCII+ cells that cluster with proliferating cells (red nuclei) are either CD205+ (a), CD103+ (b), or CD11c+ (c) (light blue, inset and white arrows)</p><p>Cluster formation of host DCs with EdU+ proliferating donor lymphocytes. Four-color immunofluorescence staining of the spleen of day 2 after GvHR induction for host MHCII (Alexa-647-conjugated mAb, blue), CD205 or CD103 (Alexa-594-conjugated mAb, green), donor lymphocytes (indirect staining with Alexa-350-conjugated anti-mouse IgG, yellow) and EdU (Alexa-488-conjugated azide, red). Pseudocolors were assigned using AxioVision software. Scale bar 100 μm. Merged images are explained schematically in the right side of the panels. Note host MHCII+ cells that cluster with donor proliferating cells (yellow cells with red nuclei) possess DC markers, CD205+ (a) and CD103+ (b) (light blue, white arrow). Central yellow ring in b is the central artery, which emits nonspecific fluorescence</p><!><p>The results show that EdU staining enables analysis of cellular interactions in situ by simultaneous visualization of different surface markers of cycling S-phase cells or stromal cells.</p><!><p>In this study, by taking advantage of the unique characteristics of EdU, we have developed a new method that allows examination of immune responses with lymphocyte proliferation both functionally and morphologically in situ as well as in vitro. The four-color immunofluorescence staining using EdU for immunohistology or three-color for FCM and the simultaneous application of both staining approaches for one target tissue have not been reported so far. EdU staining correlated well with BrdU staining of spleen sections activated by the CD28SA stimulation (Supplementary Fig. 1). This outcome confirms previous indications that EdU can be used for immunohistology of cycling S-phase cells as a thymidine analogue (Salic and Mitchison 2008). The presence of some BrdUhighEdUlow or BrdUlowEdUhigh cells might be due to the competitive uptake of both nucleosides by a single cell.</p><!><p>Because of the small size of the detection reagent and elimination of DNA denaturation steps, EdU staining allows the multicolor immunofluorescence of at least four colors including two different markers on a single-cell surface, which has been impossible by the standard BrdU method. In this way, any cells of specific phenotypes can be identified in situ, e.g., T-cells (Fig. 1), Tregs (Fig. 3), donor T-cells (Fig. 4b), and host DCs (Figs. 6, 7) and their functional molecules can be further studied, e.g., expression of CD25 (Fig. 3) and CD103 (Figs. 6, 7). Because these markers are mostly labile surface CD antigens, our method using fresh cryosections can provide a superb staining result for these antigens compared to the prefixation method used in the neuroscience field (Guo et al. 2009). Furthermore, some nuclear transcription factors can be simultaneously detected, e.g., Foxp3 in CD4+CD25highEdU+ cells (Fig. 3). These results show that this method enables functional time-kinetic analysis of immune responses of a certain cell type in a distinct domain of the lymphoid organs. An example is the Treg proliferative response occurring in the splenic PALS at day 3 after CD28SA stimulation (Figs. 2, 3).</p><!><p>Concerning the cluster formation between proliferating T-cells and DCs, the standard three-color immunoenzyme staining using BrdU could at best show only clusters of BrdU+ cells and host MHCII+ putative DCs (Saiki et al. 2001; Ueta et al. 2008). In the GvHR study, we could identify cluster formation between EdU+CD4+ donor T-cells and host MHCII+ putative DCs (Fig. 5), and the latter were further confirmed as host DCs, being either CD103+, CD205+ (Figs. 6, 7), or CD11c+ (Fig. 6). Therefore, the present method could allow analysis of cellular interactions in situ more precisely by the simultaneous visualization of different specific markers or functional molecules of cycling S-phase cells or stromal cells.</p><p>This cluster represents a site of antigen presentation by DCs to T-cells and proliferation and differentiation of activated T-cells (Saiki et al. 2001; Ueta et al. 2008), making it one of the most crucial structures when the afferent limb of the immune response occurs. Therefore, we propose that EdU staining can provide an exclusive method for clarifying essential cellular interactions in the immune response in vivo.</p><!><p>Flow cytometry (FCM) analysis indicated that cycling S-phase cells could be detected by EdU staining at a higher signal-to-noise ratio due to click chemistry (Salic and Mitchison 2008) than ordinary BrdU staining that requires an antigen–antibody reaction. EdU staining also has enabled the parallel examination of the proliferative response of activated cells both by immunohistology of tissue sections and by FCM of cells derived from the same lymphoid organs of one animal. Thus, the immunoproliferative response was examined qualitatively by immunohistology of tissue sections, which then could be quantitated by FCM.</p><p>In this way, we could demonstrate not only the dose response of Tregs to CD28SA (Figs. 1, 2), but also the time kinetics and quantification of donor T-cell migration and proliferation in the GvHR (Fig. 4). In addition, some nuclear transcription factors detected by immunohistology were also detected by FCM and some proliferating cell subsets could be further isolated and used for RT-PCR analysis. Accordingly, this method can become a powerful tool for the objective and precise analysis of cellular interactions during the immune response in situ.</p><!><p>A flow diagram of sequential steps for the present method. A1–A5, B1–B3, and C1–C11 correspond to the numbers in detailed working protocols described in supplemental materials</p><!><p>We have demonstrated a newly developed method that enables the functional time-kinetic analysis of immunoproliferative responses in vivo, including activation, proliferation, and cellular interactions in a distinct domain of the lymphoid organs, which are quantitatively confirmed by FCM. RT-PCR of proliferating cells can be analyzed further. This method is as easy and reproducible as standard immunofluorescence methods and would be applicable not only for the immune response but also for other studies examining cell and tissue growth, such as hematopoiesis and organogenesis.</p><!><p>Supplementary material 1 (DOCX 98 kb)</p>

PubMed Open Access

Energetic basis and design of enzyme function demonstrated using GFP, an excited-state enzyme

The last decades have witnessed an explosion of de novo protein designs with a remarkable range of scaffolds. It remains challenging, however, to design catalytic functions that are competitive with naturally occurring counterparts as well as biomimetic or non-biological catalysts. Although directed evolution often offers efficient solutions, the fitness landscape remains opaque. Green fluorescent protein (GFP), which has revolutionized biological imaging and assays, is one of the most re-designed proteins.While not an enzyme in the conventional sense, GFPs feature competing excited-state decay pathways with the same steric and electrostatic origins as conventional groundstate catalysts, and they exert exquisite control over multiple reaction outcomes through the same principles. Thus, GFP is an "excited-state enzyme". Herein we show that rationally designed mutants and hybrids that contain environmental mutations and substituted chromophores provide the basis for a quantitative model and prediction that describes the influence of sterics and electrostatics on excited-state catalysis of GFPs.As both perturbations can selectively bias photoisomerization pathways, GFPs with fluorescence quantum yields (FQYs) and photoswitching characteristics 1-4 tailored for specific applications could be predicted and then demonstrated. The underlying energetic

energetic_basis_and_design_of_enzyme_function_demonstrated_using_gfp,_an_excited-state_enzyme

<!>INTRODUCTION<!>RESULTS AND DISCUSSION<!>Applications, Generalizations, and Implications for Design. This model allows us<!>CONCLUSIONS

<p>landscape, readily accessible via spectroscopy for GFPs, offers an important missing link in the design of protein function that is generalizable to catalyst design.</p><!><p>Numerous methods have been employed in developing GFPs with desired behaviors [5][6][7][8][9][10][11][12][13][14][15][16][17] , including directed evolution and high-throughput screening of mutant libraries [5][6][7][8][9] that optimize brightness. Machine learning has afforded redder and brighter GFPs 10,11 , and de novo protein design has reduced the size of GFP 12 . Unfortunately, the former lacks physical insight, and the latter does not factor in structure-FQY relationships, leading to a FQY (~ 2%) substantially below those of GFPs derived from Aequorea victoria (avGFP; FQY ~ 80%). Only through further substantial screening and chromophore modification were brighter versions (FQY ~ 23%) obtained 13 .</p><p>Photoswitching, the ability to toggle between strongly and weakly fluorescent states through irradiation 18,19 , is another useful function that facilitates super-resolution imaging and optogenetic applications 20,21 . One of the most common photoswitching mechanisms is photoisomerization (Figure 1A), an excited-state bond-rotation pathway that competes with fluorescence emission. Due to this competition, selecting for an efficient photoswitchable protein is difficult via high-throughput screens; past efforts have relied on naturally occurring photoisomerizable GFPs as starting points 14 and/or painstaking combinations of rational design and screening [15][16][17] . A physical framework capturing the protein environmental factors that control the FQY and photoisomerization in GFPs is necessary to guide more efficient designs, and this is intimately related to the challenge of catalyst design. Potential energy surface (PES) for the GFP chromophore along the isomerization coordinate. After excitation from the cis ground state (indigo arrow), the chromophore can either fluoresce (kfl) or decay by isomerization through excited-state barrier crossing (kiso) and conical intersections (trajectory not shown) or by other nonradiative pathways (kother) back to the ground state. Isomerization can either occur about the phenolate bond (P bond; kP, phenolate ring flip) or the imidazolinone bond (I bond; kI, cis-trans isomerization), with opposite directions of electron flow. The relative barrier heights (EP and EI) depend on steric and electrostatic factors of the environment around the chromophore 22 , catalyzing one pathway over the other. (B) The driving force of the chromophore 𝛥𝜈̅ is defined as the relative energy between the P (left) and the I (right) resonance forms in a given environment. In all proteins studied in this work, the P form is consistently lower in energy 23 , defined as a positive driving force. (C) Marcus-Hush model explaining shifts in transition energy 𝜈̅ 𝑎𝑏𝑠 depending on the electrostatic influence of the protein environment on the chromophore's ground and excited states 23 . (D) The chromophore and its local environment within Dronpa2. R66, S142, and T159 are the residues mutated in this work, while tyrosine analogues in place of Y63 are used to introduce substituents into the phenolate ring of the chromophore 22 .</p><p>In earlier work, we discovered that the FQY of the anionic GFP chromophore embedded in the fixed native protein environments of Dronpa2 or superfolder GFP can be modulated through the introduction of electron-donating and -withdrawing substituents 22 . The FQY exhibits a peaked trend when correlated with transition energy (Figure 2A; now converted into driving force, vide infra); the shift in transition energy reflects the extent of electronic perturbation conferred by the substituents. This observation reveals two competing nonradiative photoisomerization pathways (Figure 1A), with the probability of each influenced by the electrostatic interaction between the protein environment and the electron flow within the chromophore during photoisomerization 24,25 . Because the twisting about the two exocyclic bonds (the P and I bonds) in the excited state is associated with opposite electron flow directions (Figure 1A), electrostatics can cause bond-selective photoisomerization of the chromophore, complementing the more commonly argued role of steric hinderance in suppressing chromophore (photo)isomerization 3,26,27 . The relative barrier heights EP and EI determine the outcome, and control of these barrier heights is analogous to conventional concepts in catalysis.</p><p>To quantify this electrostatic perturbation, we use the driving force Δ𝜈̅ (Figure 1B) 23,28 , which is the relative energy between the P and I resonance forms of the chromophore. Δ𝜈̅ is obtained from the observed transition energy (absorption peak maximum) 𝜈̅ 𝑎𝑏𝑠 through the Marcus-Hush treatment 23,28 :</p><p>where V0 (= 9530 cm -1 23 ) is the electronic coupling between the two resonance forms.</p><p>With respect to the wild-type environment or chromophore, any decrease or increase in Δ𝜈̅ caused by modifications results in a red or blue shift, respectively (Figure 1C). The driving force can be perturbed through either direct modification of the chromophore or through changes in the protein environment, so it can serve as an ideal quantity to reflect the electron distribution of the chromophore 23 , unify both sources of perturbations 29 , and connect to the underlying theme of electrostatic catalysis.</p><!><p>2.1. Tuning Electrostatics with Mutants and Hybrids. Figure 1D shows the chromophore environment of Dronpa2, which exhibits a balance between emission and photoisomerization. To isolate the electrostatic effects, residues immediately surrounding the chromophore were replaced with amino acids that minimized differences in size. The S142A mutation causes a red shift by destabilizing the P form through removal of a hydrogen bond to the phenolate oxygen (Figures 1B, 1C, S1A, and S2A). The blue-shifted R66M mutant results from I-form destabilization via the removal of the favorable electrostatic interaction between the arginine and the imidazolinone oxygen (Figures 1B, 1C, S1A, and S2B). Within an isosteric T159 mutant series (T159M, T159Q, T159E), T159M is the most red-shifted (by 15 nm compared to wild type), while increasing polarity and/or charge causes a blue shift in T159Q/E; the glutamine and glutamate in T159Q and T159E mutants, respectively, replace S142 as the primary hydrogen bonding partner to the phenolate oxygen and preferentially stabilize the P form (Figures S1A and S2C-S2F).</p><p>We next measured the FQYs (Table S1) and plotted them against the corresponding driving forces (eq 1) to determine the electrostatic effect on photoisomerization (Figure 2A). S142A and R66M have a decreased FQY along with strong red-and blue-shifted peak maxima, respectively, recapitulating the peaked trend for chromophore variants (Figure 2B). In contrast, the isosteric T159 mutant series displays a linear correlation with peak maximum, rendering Dronpa (T159M) an outlier of the trend. We attribute this to an increased steric effect for the isosteric series in conjunction with the electrostatic mechanism (vide infra). Nevertheless, we still find that the FQY can be tuned electrostatically through environmental mutations.</p><p>To circumvent the confounding steric effect, we created hybrids by introducing substituted chromophores into environmental mutants. We first chose one red-shifted (S142A) and one blue-shifted (T159E) mutant with the wild-type Dronpa2 chromophore.</p><p>We then introduced electron-donating or -withdrawing chromophore substituents to the P ring, which would be predicted to either respectively enhance or compensate for the electronic effect of the mutant with respect to wild-type properties. For example, as the S142A mutation destabilizes the P form, an "enhancing" chromophore modification would be electron-donating and push the electronic properties of the chromophore (driving force and FQY) even further from wild type. A "compensating" modification with an electronwithdrawing group would stabilize the P form, countering the mutational effect and creating a more wild-type-like chromophore (Figure 2C). Note that the same substituent can act as enhancing or compensating in different environmental contexts according to electrostatic FQY tuning.</p><p>For the hybrids, we can quantitatively predict the optimal substituent, within the range available 22 , to pair with a given mutant based on driving force additivity (Table 1).</p><p>Each point mutant has a driving force, to which a fixed value is added or subtracted based on the chromophore substituent, obtained from the difference between the driving force of Dronpa2 with a natural and substituted chromophore 23 . For the compensating hybrids, the optimal substituents to bring the driving force of S142A and T159E close to wild type are 2,3-F2 and 3-OCH3, respectively. For the enhancing hybrids, we chose substituents with low steric bulk but that still provide a large perturbation to the driving force: S142A/3-CH3 and T159E/2,3-F2. The observed absorption peak maximum for each hybrid agrees well with the predictions (Table 2; Figures S1B and S1C): incorporation of electrondonating and -withdrawing substituents leads to the predicted red and blue shift, respectively. Figure 2D shows the correlation between FQY and driving force for the Dronpa2 hybrids. Both enhancing hybrids (S142A/3-CH3 and T159E/2,3-F2) have a decreased FQY, pushing the values further from wild type as anticipated from electrostatic FQY tuning. Remarkably, both compensating hybrids (S142A/2,3-F2 and T159E/3-OCH3) have an increased FQY compared to the respective mutant with the unsubstituted chromophore, bringing the values closer to the wild-type value. This observation implies that the electronic effect of the chromophore substituent successfully compensates for the electrostatic perturbation caused by the environmental mutation.</p><p>Either the chromophore substituents (2,3-F2 or 3-OCH3) or the environmental mutations (S142A or T159E) alone each cause a decrease in FQY compared to the wild-type Dronpa2, so the observation of an increased FQY in these compensating hybrids suggests cooperativity ("reciprocal sign epistasis") 8,30 between deleterious perturbations that cannot otherwise be explained without electrostatic FQY tuning. The FQY φfl is the ratio between the intrinsic spontaneous emission rate kfl and the total excited-state decay rate constants 31 (Figure 1A):</p><p>where kiso and kother denote the total rate constant for excited-state isomerization and other nonradiative pathways, respectively; 𝜏 is the fluorescence lifetime. We can then dissect the temperature, electrostatic, and steric dependence of each term to understand how the chromophore's FQY is influenced by its environment. kfl is minimally tunable through electrostatics as evidenced by the nearly constant transition dipole moment across different GFP mutants 23,32 ; steric effects are irrelevant since emission is a Franck-Condon process. The only way the protein environment can tune the FQY is through modulating the competing nonradiative decay pathways. kfl is estimated to be (3.5 ns) -1 33 , so any nonradiative process much slower than this value cannot tune FQYs.</p><p>kother arises from both direct internal conversion and intersystem crossing, but the latter is much less competitive than other excited-state processes 34 . Accordingly, we can approximate kother with a single rate constant from direct internal conversion kIC due to vibrational wavefunction overlap between the ground and excited electronic states, which is relatively temperature insensitive (see Section S6 of ref. 22 and Section S11 of ref. 30).</p><p>To obtain kIC, we examine a GFP mutant series in which the threonine at position 203 is replaced with aromatic side chains that π-π stack with the chromophore P ring and can be varied in electron richness. The corresponding FQYs are nearly constant around 77% despite the modified electrostatic interaction (Figure S3 and Table S2). Steric hinderance by the aromatic ring overwhelms electrostatics and renders kiso uncompetitive; the remaining 23% of excited-state decay can be ascribed to internal conversion; kIC is (12 ns) -1 and imposes an upper limit for GFP's FQY of approximately 80% 35 , close to that of avGFP. Extensive mutational studies also demonstrate that avGFP is indeed located at the local maximum of the fitness landscape for brightness 8 . Any approach that slows excited-state isomerization down to tens of nanoseconds is sufficient to maximize FQY.</p><p>In contrast with other processes, excited-state isomerization requires crossing over an energy barrier along with significant electronic and nuclear motion (Figure 1A), so the isomerization rate kiso is almost solely responsible for the temperature, electrostatic, and steric dependence of FQY 22 . The associated barriers are typically > 3 kcal/mol for GFPs 22 , and the corresponding rate constants are comparable with kfl (ns timescale). The rapid intramolecular vibrational energy redistribution (ps timescale) 31,39 right after excitation renders the system thermally equilibrated before emission and isomerization, so the assumption for Arrhenius behavior, also common for ground-state catalysis, is met for isomerization. A pre-exponential factor A and an energy barrier E can thus be assigned for each isomerization pathway:</p><p>where kiso is then approximated with a single Arrhenius expression when we measure the excited-state energy barrier E of Dronpa2 variants using the temperature dependence of their fluorescence lifetimes 22 . If AP and AI are close in value, A should be close to both AP and AI, and the measured excited-state barrier height E can well approximate the lesser of the two barriers, EP or EI (Figure 1A). A is 10 3 -10 5 ns -1 22 , agreeing well with the value estimated from transition state theory ( 𝑘𝑇 ℎ ~ 10 13 s -1 ). This suggests that when the excitedstate barrier exceeds 9 kcal/mol (i.e., kiso being 1% of kfl at 300 K), as for the π-π stacking GFP mutants (Figure S3), no further increase in FQY can be seen as it reaches the upper limit. We now replot the excited-state barriers from Dronpa2 variants (Figure 3B in ref.</p><p>22) against the corresponding driving forces to better understand the electrostatic effect (Figure 3A). Linear fits to the electron-donating and -withdrawing substituent data exhibit slopes of +0.6 and -0.7, reflecting the electrostatic sensitivity of EP and EI, respectively.</p><p>These slopes are about equal in magnitude (~ 0.65 within experimental errors) and opposite in sign; the signs agree well with a model treating the chromophore as an allylic anion 22 . Analogous to electrostatic enzyme catalysis 40,41 , this electrostatic sensitivity originates from chromophore charge redistribution during photoisomerization interacting with the protein environment (Figure 1A), effectively an excited-state enzyme that selectively catalyzes either P-or I-bond rotation. We expect these slopes in Figure 3A to be directly transferable to different environments around the chromophore, since the driving force is the only parameter responsible for the electrostatic sensitivity of the entire PES 22 :</p><p>𝐸 𝑃 = 0.65Δ𝜈̅ + 𝐶 𝑃 and 𝐸 𝐼 = −0.65Δ𝜈̅ + 𝐶 𝐼 (4) where the steric effects, including the intrinsic barrier to bond isomerization in the absence of any external steric constraint, can be separated out in terms of empirical constants CP and CI (y-intercepts of red and blue lines in Figure 3B, respectively). We can then rewrite eqs 2 and 3 to explicitly show the electrostatic and steric dependence of the FQY:</p><p>Two factors mediate excited-state pathway selection: sterics, which acts upon large scale nuclear motion of two rings during isomerization, and electrostatics, which interacts with electronic redistribution during isomerization (or driving force). The electrostatic influence of the red fluorescent protein environment on the corresponding chromophore's FQY is also extensively discussed by a recent paper 42 , while our physical model treats electrostatics differently and explicitly incorporates the steric component (see Section S2 in Supporting Information). According to eq 5, FQY is a nonlinear function of Δ𝜈̅ , and thus the linear additivity of driving force does not translate to an additivity of FQY, as observed from the compensating hybrids (Figure 2D and Table 2). Cooperativity between mutations, a phenomenon that renders protein design and even directed evolution challenging 29,43 , could similarly be partly explained by a nonlinear function (i.e., FQY) encoding two (or more) pathways dependent on an additive underlying parameter (i.e., driving force) 23 . Steric effects CP and CI serve as an alternative tuning mechanism for the excited-state barriers EP and EI, preventing the FQY from being completely tied to color via electrostatics, as is the case for other photophysical properties 23 . If CP equals CI, there should be no preference for either isomerization pathway when Δ𝜈̅ = 0, corresponding to a maximum FQY (eq 5; Figure 3B, case 1). Since Δ𝜈̅ = 0 also corresponds to the reddest possible absorption (eq 1), a combination of these two equations would suggest that the redder the chromophore, the higher the FQY by varying Δ𝜈̅ . However, we observe an apex in the trend that is not centered at Δ𝜈̅ = 0 (Figure 3A), suggesting that CP is not identical to CI. Intuitively, the volume-demanding I twist experiences more steric hinderance than the P twist within the protein environment since the I ring is covalently anchored.</p><p>With eq 4, we can explain the apex position in the FQY (or excited-state barrier)</p><p>vs driving force plot (Figure 3B). The sign of the driving force is defined positive when the P form is more stable than the I form, which is the case for all proteins studied so far 23 (Figure 1B). With zero differential sterics from the protein environment (CP = CI; dashed lines) and zero driving force, the negative charge of the anionic chromophore is maximally delocalized and both exocyclic bonds are equally probable to twist upon excitation. This corresponds to the largest possible barrier when CP = CI, and the apex is located at Δ𝜈̅ = 0 (Figure 3B, case 1). When the driving force becomes positive (right side of Figure 3B), electron density is reduced at the I bond (i.e., more single-bond character) upon excitation, and the I twist becomes more favorable 44 (Figure 3B, case 2). If the I ring is anchored inside the protein, CI becomes larger than CP (yellow arrows and solid lines in Figure 3B).</p><p>Consequently, the apex shifts along the x-axis and lies at a positive driving force, as observed in Figure 3A, and it also increases along the y-axis due to the resulting constriction on bond rotation (Figure 3B, case 3). At that apex, the driving force from electrostatic influences matches the apex shift caused by differential steric interactions.</p><p>However, when the steric effects are large enough to render kiso uncompetitive with kfl (Figure S3), the maximally allowed FQY is reached, and the apex for FQY cannot be detected. Note that the driving force at the apex is determined from the differential sterics (CI -CP), while the barrier heights are affected by the absolute sterics (CI or CP), so it is possible to have an apex location at zero driving force when steric hinderance to the P twist is comparable with I ring anchoring (Figure 3C).</p><!><p>to quantitatively evaluate the contributions of sterics and electrostatics to excited-state catalysis. From Figure 3A, wild-type Dronpa2 sits at the apex among all Dronpa2 variants.</p><p>As its FQY (~ 50%) is far from the maximally allowed 80%, this implies that the corresponding driving force (23.6 kcal/mol) offsets the differential sterics, so we can estimate the differential sterics as 31 kcal/mol (23.6 × 2 × 0.65, Figures 3C and 3D). For superfolder GFP, the apex (the monochlorinated variant, Figure 2A) lies at a driving force of 19.9 kcal/mol and approaches the FQY limit of 80% 22 . The corresponding differential sterics is 26 kcal/mol (= 19.9 × 2 × 0.65). Combined with the fact that GFP has a higher apex FQY than Dronpa2, we can infer that the overall steric contribution should be higher for GFP than Dronpa2, but the differential sterics is also 5 kcal/mol smaller (= 31 -26) for GFP, leading to an apex located at a smaller driving force than Dronpa2 (Figure 3D). This is explained by a tighter β-barrel for GFP compared to Dronpa2, resulting in a more sterically hindered P twist (Figure 4A). Moreover, since the unmodified chromophore in Therefore, both steric and electrostatic (to a lesser extent) effects work together in the GFP barrel to promote chromophore fluorescence, while Dronpa2 exhibits a higher photoisomerization efficiency (Figure 5A). For the Dronpa2 T159 isosteric series, the lengthened side chain creates more steric bulk to P twist and shifts the apex to a smaller driving force and higher FQY (Figure 2B), explaining why T159M appears as an outlier to the peaked trend.</p><p>This analysis can also explain why the de novo designed mFAPs (Figure 4B) failed to recapitulate avGFP's high FQYs (Figure 4C) 12 and more generally how an understanding of the energy landscape can provide guidance for the design of functional proteins. Original mFAPs utilize the same difluorinated chromophore as the RNA mimic Spinach (Figure 4D) 45 to encourage chromophore deprotonation, but fluorines lower the I-twist barrier as electron-withdrawing substituents 22 . In Spinach, π-π stacking with Gquadruplexes effectively inhibits isomerization (Figure 4D) 45,46 , leading to a FQY of 72%.</p><p>In mFAPs, however, the chromophore is neither anchored to the protein as in avGFP (Figure 4C) nor motionally restricted. M27W is present in mFAP1 and mFAP2 to interact with the I ring via a hydrogen bond (Figure 4B), but this interaction is not sufficient to restore the maximal FQY. To further increase the FQYs, this analysis suggests the addition or removal of fluorines from the chromophore's I or P rings, respectively, and the introduction of aromatic amino acids near the chromophore's P ring to encourage π-π stacking interactions. In fact, the newly installed -CF3 group on the I ring and L104H likely explains the much-improved FQY (23%) of chromophore-bound mFAP10 13 .</p><!><p>GFP is both green and fluorescent, while the free GFP chromophore in water is neither, so it is tempting to ascribe this drastic change in properties to the protein environment. However, the chromophore's ability to be green and fluorescent is already encoded in its PESs (i.e., energy landscape), and these properties can also be elicited using non-protein environments 3,27 . An analogous example is the relationship between an enzyme and its substrate. The availability of different reaction pathways and the potential for pathway selection, existing for numerous ground-state and excited-state enzymes [47][48][49][50] , are already inscribed in the PES(s) of the chromophore/substrate, illustrated by diverse examples in Figure 5. The protein environment can only stabilize the transition state of one particular pathway over another that is otherwise suppressed; it cannot than create new reactions. Therefore, to rationally design enzymes that are superior at catalyzing a reaction, it is important to sample a wide range of perturbations to substrates (or chromophores capable of structural change) and the environment's steric or electrostatic influences on the energetics of non-productive yet competitive pathways rather than only those that exhibit more desirable phenotypes 51 . Only when those less desirable cases are understood can we mechanistically deduce why the more productive pathway is not taken, guiding future design efforts to optimize the desired function. Dronpa2 (Figure 3D). (B) Y(M210)F mutant (purple) of Rhodobacter sphaeroides photosynthetic reaction center reveals that tyrosine at M210, which stabilizes the first intermediate, is in part responsible for the unidirectional excited-state electron transfer of wild type (orange) 52,53 . (C) Wild-type Fe(II)/2-oxoglutarate (2OG)-dependent halogenases (orange) chlorinate their substrates, but their intrinsic hydroxylating power can be unleashed upon mutation (purple) 54,55 . The default (blue) and the side pathways (red for all and green for panel A) are shown on the right and left for each panel, respectively. Energies are not drawn to scale.</p>

ChemRxiv

A Novel SNP in a Vitamin D Response Element of the CYP24A1 Promoter Reduces Protein Binding, Transactivation, and Gene Expression

The active form of vitamin D (1\xce\xb1,25(OH)2D3) is known to have antiproliferative effects and has been implicated in cancers of the colon, breast, and prostate. These cancers occur more frequently among African Americans than Caucasians, and individuals with African ancestry are known to have approximately two-fold lower levels of serum vitamin D (25(OH)D) compared with individuals of European ancestry. However, epidemiological studies of the vitamin D receptor (VDR) have shown inconsistent associations with cancer risk, suggesting that differences in other genes in the pathway may be important. We sought to identify functionally significant polymorphic variants in CYP24A1, a gene that is highly inducible by 1\xce\xb1,25(OH)2D3 and that encodes the primary catabolic enzyme in the pathway. Here we report the identification of six novel SNPs in the human CYP24A1 promoter, including one at nucleotide -279 occurring within the distal vitamin D response element (VDRE2). Our experiments demonstrate that the VDRE2 variant results in decreased protein binding and transactivation in vitro, and reduced expression of CYP24A1 in cultured primary human lymphocytes provides evidence for an effect in vivo. This variant was only observed in our African American population, and represents a first step toward understanding differences in disease risk among racial/ethnic groups.

a_novel_snp_in_a_vitamin_d_response_element_of_the_cyp24a1_promoter_reduces_protein_binding,_transac

1. Introduction<!>2.1. Subjects<!>2.2. DNA isolation and sequencing to screen for CYP24A1 promoter polymorphisms<!>2.3. DNA isolation and SNP Genotyping for the VDRE2 polymorphism<!>2.4. Plasmid construction and site-directed mutagenesis<!>2.5. Cell culture, transient transfection, and luciferase assays<!>2.6. Electrophoretic mobility shift assays (EMSA)<!>2.7. Quantitative Real-Time PCR of CYP24A1 and VDR<!>3.1. Identification and genotyping of polymorphisms in the CYP24A1 promoter<!>3.2. Differential transactivation of wild-type and polymorphic CYP24A1 promoter constructs<!>3.3. Impaired VDR/RXR\xce\xb1 binding to the polymorphic VDRE<!>3.4. Decreased induction of CYP24A1 expression by 1\xce\xb1,25(OH)2D3 in a heterozygous polymorphic individual<!>4. Discussion

<p>The biologically active form of vitamin D (1α,25(OH)2D3) has multiple roles in the body, including regulation of intestinal calcium absorption, maintenance of bone mineral density, and modulation of cell growth and apoptosis [1]. Genomic actions of 1α,25(OH)2D3 are mediated through ligand-binding to the vitamin D receptor (VDR), which forms a heterodimer with retinoid x receptor alpha (RXRα) and subsequently binds to vitamin D response elements (VDRE) to either enhance or repress transcription of various genes. These response elements are typically comprised of two conserved hexameric half-sites separated by a three nucleotide spacer, referred to as a DR3 type element. Although it is known that the sequence of a VDRE can have a strong influence on the degree of protein binding, particularly at the fifth position in the half-site [2], previous studies have focused on synthetic variations of response elements and not naturally occurring sequences [3].</p><p>It has been known for some time that there are significant racial/ethnic differences in serum vitamin D status, with individuals of African ancestry having approximately two-fold lower levels than those with European ancestry [4-6]. Low levels of serum vitamin D (25(OH)D) have been associated with cancers of the colon, breast, and prostate, as well as an increased risk of cardiovascular disease [7-10]. Compared with whites, African Americans are more frequently diagnosed with these cancers and are at a greater risk for cardiovascular disease [11-13], indicating the possibility that polymorphic variants in the vitamin D pathway could be influencing these disparities. There are several well-known variants in the vitamin D receptor (VDR), but epidemiological studies have shown inconsistent associations with disease outcomes, particularly among different racial/ethnic groups [14-19]. Taken together, this suggests that other variants or combinations of variants within the pathway may underlie the differences in serum vitamin D and disease risk.</p><p>The CYP24A1 gene has very low basal expression, but is strongly upregulated by 1α,25(OH)2D3 through two VDRE in the proximal promoter region [20, 21]. The resulting 24-hydroxylase enzyme catalyzes the first step in the catabolic pathway, converting 1α,25(OH)2D3 into the less active intermediate 1,24,25(OH)3D3 [22]. Through activation of this and other negative feedback loops, 1α,25(OH)2D3 can regulate its own metabolism. We hypothesized that there were unidentified SNPs in the promoter of the CYP24A1 gene that could alter expression of the 24-hydroxylase enzyme and impact the rate at which an individual can metabolize 1α,25(OH)2D3. Population studies to date have sequenced only a small number of individuals, particularly individuals of African ancestry, so it is possible that significant polymorphic variants in this region have yet to be identified.</p><p>In the work presented here, we report the identification of six novel SNPs in the human CYP24A1 promoter, including one that occurs in the fifth position of the distal vitamin D response element (VDRE2). We demonstrated that this VDRE2 polymorphism results in impaired receptor protein binding and decreased transactivation in vitro, and more importantly, we provide evidence that the variant can lead to decreased expression of the CYP24A1 gene in a heterozygous polymorphic individual.</p><!><p>Healthy individuals who classified themselves as at least 50% Caucasian or at least 50% African American were recruited at Pennsylvania State University General Clinical Research Center during the winter and spring months. The 100 total participants were matched on age (within 2 years), sex, and opposite self-reported race. Each participant donated a venous blood sample that was used to prepare dried blood spot cards and for Ficoll separation with Ficoll-Paque PLUS (GE Healthcare) to obtain lymphocytes.</p><!><p>Genomic DNA for SNP discovery was isolated from dried blood spot cards from 20 randomly selected African American participants in our study using the QIAamp DNA Micro Kit (QIAGEN). A 677 bp region of the proximal CYP24A1 promoter (-618 to +59) including two known VDRE was PCR-amplified using Phusion DNA polymerase (New England Biolabs) and the following primers: 5′-GTGTCAAGGAGGGTAGATGAGATG-3′ (forward) and 5′-TTGCTCAAGTTAAGAAAGTCTCCTC-3′ (reverse). The desired PCR products were gel-isolated using the QIAquick Gel Extraction Kit (QIAGEN), run on agarose gels to verify DNA recovery, and sequenced using the forward PCR primer at the University of Pennsylvania School of Medicine DNA Sequencing Facility by automated cycle sequencing. Polymorphisms were identified by BLAST alignment with the wild-type sequence for human chromosome 20 (GenBank accession number NT_011362) and by visual inspection of printed chromatograms. Some SNPs were also confirmed by restriction fragment length polymorphism analysis. Resulting sequence variants were compared to data from NCBI (build 36, dbSNP b126) and the HapMap project (www.hapmap.org, release 22).</p><!><p>Genomic DNA was isolated from frozen lymphocytes of all 100 participants with the QIAamp DNA Mini Kit (QIAGEN). A custom TaqMan SNP Genotyping assay (Applied Biosystems) was designed for the newly identified VDRE2 polymorphism and subsequently tested on samples of known genotype to conduct an internal validation of the assay. Quantitative Real-Time PCR and allelic discrimination were conducted at the Functional Genomics Core Facility at the Penn State College of Medicine using 10 ng genomic DNA per assay. Genotypes were successfully determined for 99% of the samples with a reliability rate of 100% when a random 10% sample was again genotyped in a separate experiment. Samples were also genotyped for the presence or absence of the M1T polymorphism in VDR (rs10735810) using a predesigned TaqMan assay (Applied Biosystems, assay ID# C_12060045_20) with similar success.</p><!><p>The hCYP24p-Luc construct was generated by first PCR-amplifying and gel-isolating a 677 bp region of the proximal CYP24A1 promoter (-618 to +59) as described above. The purified product was then subjected to a second round of amplification using two nested primers designed to introduce Xho I and Hind III restriction sites for subcloning: 5′-TGCTCgAGTTAAGAAAGTCTCCTCTTC-3′ (forward) and 5′-GGACCAaGCtTTTATGGAGACAGA-3′ (reverse). The new PCR product of 603 bp (-617 to -15) was digested with the above restriction endonucleases to expose the cohesive ends and was gel-purified as before. The pGL3-Promoter vector (Promega) was similarly digested and gel-purified, then ligated overnight with the CYP24A1 promoter fragment and transformed into chemically competent E. coli. Sequencing was done to confirm the orientation and integrity of the newly created hCYP24p-Luc construct, which contains the proximal CYP24A1 promoter (-611 to -25) driving expression of firefly luciferase under the control of two VDRE: GAGTCAgcgAGGTGAgcgAGGGCG at -169 to -145 (VDRE 1) and GAGTTCaccGGGTGT at -289 to -274 (VDRE2).</p><p>To create the hCYP24pV2SNP-Luc construct, we used the hCYP24p-Luc plasmid as template for site-directed mutagenesis with the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) as described by the manufacturer. Briefly, the primers 5′-CGAAGCACACCCGGTGGACTCCGGGCTT-3′ (sense) and 5′-AAGCCCGGAGTCCACCGGGTGTGCTTCG-3′ (antisense) were annealed to the hCYP24p-Luc plasmid to allow synthesis of the mutated promoter with Pfu DNA polymerase, followed by digestion of the parental plasmid by Dpn I, transformation into XL1-Blue chemically competent cells, and screening of the resulting colonies by restriction endonuclease digestion. Positive clones were verified by sequencing, and the new construct was identical to the wild-type promoter with the exception of a single base pair change in VDRE2 (GAGTTCaccGGGTGT was mutated to GAGTCCaccGGGTGT) to reflect the polymorphism identified at -279 during sequencing of our human subjects.</p><p>The r24OHase-Luc construct, a generous gift from Dr. H. DeLuca at the University of Wisconsin-Madison, contains the proximal 942 bp of rat 24-hydroxylase (CYP24A1) promoter with two VDRE cloned into pMAMneo-Luc vector upstream of firefly luciferase. This was used as a positive control for induction by 1α,25(OH)2D3 in transfection experiments.</p><p>Expression constructs for VDR and RXRA were generated from an existing human cDNA pool made by reverse-transcription of human liver RNA, a gift from Dr. Philip Lazarus at Penn State University. Coding sequences for both VDR and RXRA were specifically amplified with the following primers: 5′-GGTCTGAAGTGTCTGTGAGACCTC-3′ (VDR-forward), 5′-ACAAACAGCAACTCCTCATGGCTG-3′ (VDR-reverse), 5′-GGGCATGAGTTAGTCGCAGA-3′ (RXRA-forward), and 5′-AAACAGGCCAGGCAGAGAAG-3′ (RXRA-reverse). Amplified cDNAs were gel-isolated and sequenced to verify that they were wild-type, then TA-cloned separately into the pcDNA3.1/V5-His-TOPO vector (Invitrogen) and transformed into chemically competent E. coli. Multiple transformed colonies were screened for the presence and orientation of the cDNA inserts, and positives were confirmed by bidirectional DNA sequencing. The resulting constructs (pcDNA3.1-VDR and pcDNA3.1-RXRA) contain the wild-type coding sequences with native stop codons, and expression is driven by the CMV promoter.</p><!><p>Human breast cancer cells (MCF-7) and human lung carcinoma cells (H1299) were maintained at 5% CO2 in MEM supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen).</p><p>H1299 cells were seeded in 6-well plates and allowed to attach for at least 18 hours. Prior to transfection, cells were rinsed once with PBS to remove traces of serum and replaced with serum-free, antibiotic free MEM. Lipofectamine 2000 reagent (Invitrogen) was used as described by the manufacturer to form DNA complexes containing 0.6 μg luciferase reporter construct (r24OHase-Luc, hCYP24p-Luc, or hCYP24pV2SNP-Luc), 0.6 μg of each expression vector (pcDNA3.1-VDR and pcDNA3.1-RXRA), and 0.2 μg pRL-SV40 vector (Promega) to control for transfection efficiency. Whenever necessary, pUC19 (carrier DNA) was added to bring the total amount of DNA per well to 2 μg. Four hours post-transfection, complexes were removed by aspiration and one rinse with PBS prior to the addition of complete MEM and 100 nM 1α,25(OH)2D3 (Alexis Biochemicals) or vehicle (EtOH). After 16 hours of treatment, cells were harvested by trypsinization, centrifuged for 5 min. at 100 × g to pellet, resuspended thoroughly in passive lysis buffer (Promega), and stored at -70°C. MCF-7 cells were transfected in a similar manner using a total of 4 μg DNA per well, keeping the same plasmid ratios.</p><p>To enhance cell lysis prior to the luciferase assay, samples were thawed for 3 minutes in a room temperature water bath following removal from storage at -70°C. Thawed lysates were centrifuged at 4°C for one minute at high speed in a microcentrifuge to pellet cellular debris and the supernatant was transferred to a new microcentrifuge tube on ice. The Dual Luciferase Assay Kit (Promega) was used according to the manufacturer's instructions and samples were measured using a dual-injector luminometer (Pharmingen). Data were expressed as a ratio of firefly to Renilla luciferase units.</p><!><p>To prepare the probes for gelshift assays, the following sets of complementary oligonucleotides containing the desired response element were synthesized with a 5′ biotin label as such (sequences given for the plus strand only, and VDRE is in uppercase): wild-type VDRE2 from hCYP24A1 promoter, 5′-cgaagcACACCCggtGAACTCcgggctt-3′; and polymorphic VDRE2 from hCYP24A1 promoter, 5′-cgaagcACACCCggtGGACTCcgggctt-3′. (Identical oligos were also synthesized without the 5′ biotin label for competitive binding reactions.) Double-stranded DNA probes were made by boiling 150 pmoles of each complementary oligo in the presence of 50 mM NaCl for 5 minutes, then slowly cooling to room temperature overnight to allow annealing. Binding reactions were set up using 100 ng recombinant human VDR protein (BIOMOL) and/or 100 ng recombinant human RXRα (OriGene) in binding buffer (10 mM Tris, 50 mM KCl, 1 mM DTT; pH 7.5) with 1 mM EDTA, 5% glycerol, 1 μg BSA, and 0.1 μg poly (dI-dC) in the presence or absence of 1 μM 1α,25(OH)2D3. In some cases, binding reactions were set up with additional salt for a total of 150 mM KCl. After a 30 min preincubation, 25 fmol biotinylated dsDNA probe was added to each reaction and incubated for an additional 20 min at room temperature. For competitive binding studies, 5 pmol unlabeled competitor DNA was added at the preincubation step to be in 200-fold molar excess of the labeled probe. Reactions were separated on 6% native polyacrylamide gels, transferred to Biodyne B membranes (Pierce), and crosslinked using a UV Stratalinker (Stratagene). Detection was performed using the LightShift Chemiluminescent EMSA Kit (Pierce) according to the manufacturer's instructions.</p><!><p>Primary lymphocytes from one subject with the CYP24A1 VDRE2 polymorphism and one subject wild-type for VDRE2 were matched on age (+/- 3 years), male sex, and race. The cells were rapidly thawed and placed in culture medium (RPMI, 10% heat-inactivated fetal bovine serum, L-glutamine, and pen/strep, all from Invitrogen) and allowed to recover overnight in an incubator (37°C, 5% CO2). The cells were then counted by use of trypan blue exclusion dye and a hemacytometer and each subject's cells were divided equally into two wells of a 6-well plate in culture medium supplemented with 2 μg/ml PHA-M and 20 ng/ml TPA (both from Sigma) for activation. After 14 hours, either 100 nM 1α,25(OH)2D3 (Alexis Biochemicals) or vehicle (ethanol) was added to each set of lymphocytes and allowed to incubate an additional 6 hours. Total RNA was isolated from 5×105 cells using the Absolutely RNA Microprep Kit (Stratagene) as described by the manufacturer. The quality of RNA yield was determined at the Functional Genomics Core Facility at Penn State College of Medicine on an Agilent BioAnalyzer with the Total RNA Nano assay (Agilent Technologies, Inc.). Approximately 250 ng of total RNA from each sample were used as template for cDNA synthesis with SuperScript II Reverse Transcriptase and oligo(dT)12-18 (Invitrogen) as described by the manufacturer. The resulting cDNA was template for Real-Time PCR using prevalidated TaqMan Gene Expression Assays for human CYP24A1, VDR, and GAPDH (Applied Biosystems assay ID#s Hs00167999-m1, Hs01045840_m1, and Hs99998805_m1, respectively), and the relative quantification of treated versus control lymphocytes was determined using SDS software and the ΔΔCT method.</p><!><p>Direct sequencing results yielded 8 SNPs in the 500 nucleotide segment of proximal CYP24A1 promoter among African Americans. Of these, 6 were novel low-prevalence polymorphisms currently not listed on NCBI or HapMap (Table 1 and Figure 1); all were identified only in heterozygotes. The SNP at -279 relative to the transcription start site occurs in the fifth position of the hexameric repeat in VDRE2 in three of the twenty samples sequenced. After genotyping all 100 participants from our study (African Americans and Caucasians) for the VDRE2 polymorphism with the custom SNP genotyping assay, we were able to identify one more heterozygous individual in our African American population and no homozygous polymorphic individuals (4/50 heterozygotes, or 4% allelic prevalence). The SNP was not observed in any of the 49 individuals successfully genotyped in the Caucasian population.</p><!><p>It is known that the 24-hydroxylase gene has very low basal expression and that strong induction by 1α,25(OH)2D3 is mediated through two vitamin D response elements (VDRE) in the proximal promoter region. [20, 21] To test for differences in inducibility of the wild-type and polymorphic variant VDRE2 by 1α,25(OH)2D3, we transiently transfected H1299 cells with three different luciferase reporter constructs: 1) r24OHase-Luc [23], which contains the rat 24-hydroxylase promoter and was used to test for induction by 1α,25(OH)2D3 (data not shown), 2) hCYP24p-Luc, which contains approximately 600 bp of the wild-type proximal hCYP24 promoter, and 3) hCYP24pV2SNP-Luc, which is identical to the previous construct with the exception of a single base pair change to create the SNP identified at -279 in VDRE2. Each reporter plasmid was transfected either alone or co-transfected with expression constructs for VDR and RXRA. The wild-type human 24-hydroxylase promoter was induced 8-fold by 1α,25(OH)2D3 even in the absence of coexpressed VDR and RXRα, and the polymorphic human construct was induced about 5.5-fold (Figure 2A). The same experiment was performed in MCF-7 cells, and while little to no induction was seen without the coexpression of VDR and RXRα, the activation was still markedly higher with the wild-type human construct than the polymorphic variant at 16-fold and 6-fold, respectively (Figure 2B).</p><!><p>It has been shown previously that VDR and RXRα heterodimerize and bind to both VDRE in the CYP24A1 promoter [20, 21, 24-26]. The nucleotide at the fifth position in the hexameric repeat of the VDRE can have a dramatic effect on VDR/RXRα binding [2]. To determine whether the polymorphism we observed in the African American population would have an effect on protein binding to the response element, we utilized EMSA to test binding to both the wild-type and polymorphic sequences for VDRE2. The DNA probes used were comprised of the wild-type or polymorphic VDRE2 and surrounding sequence (-295 to -268) and were incubated with proteins and 1α,25(OH)2D3 as indicated (Figure 3A). As predicted, VDR and RXRα bound the wild-type VDRE as a heterodimer, but binding to the polymorphic VDRE was markedly reduced. The presence of 1α,25(OH)2D3 enhanced these interactions, but was not required for binding. To confirm the results seen with our polymorphic sequence, we ran a second EMSA using competitive binding reactions with either unlabeled wild-type or unlabeled polymorphic VDRE2 probes in a 200-fold molar excess of the labeled DNA probe. Reactions were also run using both 50 and 150 mM KCl, as others have shown a difference in binding relative to salt content [27],[28]. The results (Figure 3B) demonstrate that high salt conditions decreased binding, as the wild-type DNA-protein complex formation was greatly reduced and the polymorphic DNA-protein complex did not form under high salt conditions. More importantly, the degree of binding to the polymorphic probe was much less than that of the wild-type probe under any conditions. When included, unlabeled wild-type DNA was able to effectively out-compete the labeled wild-type DNA for binding under both low and high salt conditions, while the unlabeled polymorphic DNA had little to no impact on the ability of VDR and RXRα to bind the wild-type sequence. Taken together, the results from Figures 2 and 3 provide strong evidence that the polymorphic variant identified in VDRE2 may impact the regulation of CYP24A1 expression in vivo.</p><!><p>To determine whether the presence of a single copy of the VDRE2 polymorphism would be enough to impact CYP24A1 gene expression, we treated primary lymphocytes from two individuals (one heterozygous polymorphic and one wild-type) with 0 or 100 nM 1α,25(OH)2D3 for 6 hours and isolated RNA to synthesize cDNA for Real-Time PCR. TaqMan gene expression assays were performed to determine the relative quantification of both CYP24A1 and VDR in comparison to the endogenous control GAPDH. The basal levels of CYP24A1 expression in untreated samples were similar for both wild-type and polymorphic (data not shown). The fold induction by 1α,25(OH)2D3 in the heterozygous polymorphic individual was approximately half that of the wild-type individual with mean relative expression values of 9.4 and 17.5, respectively (Figure 4). Expression of VDR was essentially unchanged with treatment for either sample. These preliminary results suggest that the presence of a single copy of the VDRE2 allelic variant may be sufficient to impair induction of CYP24A1 gene expression in heterozygous individuals, and that a homozygous polymorphic individual would likely demonstrate a more dramatic reduction in inducibility.</p><!><p>There is limited epidemiological research on polymorphic variants in CYP24A1. Studies of selected SNPs report no association with prostate cancer [29] or postmenopausal breast cancer risk in Caucasians [19]. However, a major drawback of most current SNP research is the lack of accompanying functional data, as the significance of the variants being tested is often unknown. It is also widely assumed that most important genetic variants have already been identified, and while somewhat true, it is likely that many low-prevalence polymorphisms have yet to be discovered. The proximal promoter region of CYP24A1 is highly GC-rich (approximately 71% in the first 550 nucleotides) and is challenging to sequence, which could explain the relative lack of known polymorphic variants in that region. The NCBI dbSNP database lists 31 variants in the 5 kb immediately upstream of the transcriptional start site, but only two of these have been genotyped and validated by the HapMap project [30]. In this study, we have identified six novel SNPs in the CYP24A1 proximal promoter, including a naturally-occurring polymorphism in a vitamin D response element. This polymorphic variant appears to occur with low frequency (∼4%) among healthy African Americans, although it is possible that the polymorphism is also present in the Caucasian population at a lower prevalence and was not represented in our sample set. Here we have demonstrated that the change in VDRE2 sequence has functional impacts on protein binding, transactivation, and gene expression.</p><p>Transfection of H1299 cells with CYP24A1 promoter constructs revealed that induction by 1α,25(OH)2D3 was consistently 30% lower using the polymorphic construct compared with the wild-type. In MCF-7 cells the difference was even more striking, with induction of the polymorphic construct being 63% lower than wild-type. Mobility shift assays illustrated reduced protein binding to the polymorphic versus the wild-type response element under multiple conditions, including low and high salt content. Competitive binding reactions also provided compelling evidence for the impact of the polymorphic variant, as the unlabeled polymorphic probe was shown to be largely ineffective at outcompeting the labeled wild-type probe for binding of the VDR-RXRα heterodimer. Prior studies have shown the fifth position of the hexameric repeat to be particularly important in influencing protein:DNA interactions [2]. Consistent with those results, the VDRE polymorphism we observed in the fifth position also has a detrimental impact on binding.</p><p>The transfection and binding assay results strongly indicate that the VDRE2 polymorphism may cause functional impairment in individuals that are homozygous polymorphic. Of greater interest is the potential impact in a heterozygous individual and the severity of the effect. Analysis of CYP24A1 gene expression in primary lymphocytes suggest there may be a moderate decrease in the ability of heterozygous individuals to upregulate CYP24A1 expression, and we hypothesize that the difference in a homozygous polymorphic individual would be more pronounced. These results are limited by the small and finite number of samples in our laboratory, and since we did not identify any individuals who were homozygous for the SNP in our study, we were unable to test its impact in vivo.</p><p>It is unclear why a lack of induction of the CYP24A1 promoter constructs was observed in MCF-7 cells in the absence of cotransfected expression constructs for VDR and RXRα, as previous reports have shown a response using a different 24-hydroxylase promoter construct without the addition of exogenous protein [31]. We did not explore this possibility, but as our cells are from a higher passage they may have a diminished response to 1α,25(OH)2D3 treatment due to low expression of VDR [32], as the addition of exogenous protein restored sensitivity to 1α,25(OH)2D3.</p><p>In our EMSA binding studies we observed that higher salt content was inhibitory towards complex formation, regardless of which DNA probe was used. This is in contrast with a previously published report showing enhanced binding with higher salt [27], although direct comparison to this study is not possible because the binding reactions were dissimilar. It is also difficult to draw definitive conclusions from the previous experiments, as the low salt reactions were done only in the absence of ligand and using less protein. Human physiological sodium concentrations are at or above 140 mM [33], so the reactions done at high salt (150 mM) are likely to be more reflective of the conditions in vivo.</p><p>One potentially interesting question not addressed in the current study is the effect of variants in the vitamin D receptor in combination with changes in the vitamin D response element. The VDR has a highly prevalent start codon polymorphism (SNP ID: rs10735810, historically identified by its RFLP as FokI), with the variant allele having a frequency of greater than 55% in each of the four major populations genotyped by HapMap [30]. This particular SNP results in a VDR protein that is three amino acids shorter in length due to a nonsynonymous alteration of the first codon that causes translation to initiate at a second (in-frame) Methionine. There have been conflicting results from studies of the two VDR forms (short and long), with some reporting no difference in transactivation or ligand-binding [16], and another finding that the short form interacts more efficiently with the transcription factor TFIIB [34]. More recently, a third group demonstrated that the short form of VDR is better at activating immune cells and can enhance transcription through some factors, but showed no difference in transactivation through a classical DR3 type VDRE [35]. It is possible that the effects vary from one response element to another; the authors of the latter study used a luciferase reporter construct driven by three copies of the mouse osteopontin VDRE, and thus the results may not necessarily extend to all positively regulated VDRE. As the functional significance of the shorter form of the VDR protein has not been conclusively determined, the CYP24A1 expression assays in this study were conducted using lymphocytes from two individuals with different VDRE2 genotypes but the same VDR genotype (both homozygous for the short form) for ease of comparison. Unpublished observations from our lab suggest that the short form of the VDR may have a greater capacity to induce expression of CYP24A1 in comparison to the long form. Further studies are needed from multiple individuals who are both wild-type and polymorphic for the CYP24A1 VDRE2 variant and possess the three different VDR genotypes (homozygous short-form, heterozygous, and homozygous long-form) in order to determine the true differences in CYP24A1 expression and the effect of the different VDR forms. It also remains to be seen whether the decrease in gene expression translates to a measurable decrease in 24-hydroxylase protein.</p><p>The importance of the vitamin D pathway in cancer etiology is an emerging field, and while it has been well-established that 1α,25(OH)2D3 has anti-proliferative properties against many cancer cell lines [26, 36, 37], the exact mechanisms of action are still being determined. Results from several recent reports would suggest that the vitamin D pathway is dysregulated in certain cancers, including changes in CYP24A1. For example, one group has shown by immunohistochemistry that the cellular localization of CYP24A1 protein changes dramatically in malignant colon tissues, with a significant elevation in the amount of cytoplasmic protein compared with normal colonic epithelium [38]. A separate group has also recently demonstrated that certain antineoplastic agents can selectively destabilize CYP24A1 mRNA transcripts [39]. This effectively increased the half-life of 1α,25(OH)2D3 by decreasing the amount of enzyme present for its inactivation. Further studies are needed to see whether the previous in vitro work can be recapitulated in vivo, but show promise in enhancing current strategies in chemotherapy by targeting the vitamin D pathway.</p><p>In summary, our study identified a naturally-occurring polymorphism in a vitamin D response element of the human CYP24A1 gene that appears to impair receptor protein binding, decrease transactivation, and decrease expression of the CYP24A1 gene in vivo. Further molecular studies are needed to determine the extent of the effect in humans, both alone and in combination with other defects in vitamin D pathway genes. These results are a first step towards identifying the causes of differential cancer susceptibility among both individuals and racial/ethnic groups. By evaluating the contribution of multiple, small genetic changes, future studies will increase our understanding of how they interact to affect an individual's lifetime risk of disease and should lead to more informed epidemiological research.</p>

PubMed Author Manuscript

Phosphonic acid: preparation and applications

The phosphonic acid functional group, which is characterized by a phosphorus atom bonded to three oxygen atoms (two hydroxy groups and one P=O double bond) and one carbon atom, is employed for many applications due to its structural analogy with the phosphate moiety or to its coordination or supramolecular properties. Phosphonic acids were used for their bioactive properties (drug, pro-drug), for bone targeting, for the design of supramolecular or hybrid materials, for the functionalization of surfaces, for analytical purposes, for medical imaging or as phosphoantigen. These applications are covering a large panel of research fields including chemistry, biology and physics thus making the synthesis of phosphonic acids a determinant question for numerous research projects. This review gives, first, an overview of the different fields of application of phosphonic acids that are illustrated with studies mainly selected over the last 20 years. Further, this review reports the different methods that can be used for the synthesis of phosphonic acids from dialkyl or diaryl phosphonate, from dichlorophosphine or dichlorophosphine oxide, from phosphonodiamide, or by oxidation of phosphinic acid. Direct methods that make use of phosphorous acid (H3PO3) and that produce a phosphonic acid functional group simultaneously to the formation of the P–C bond, are also surveyed. Among all these methods, the dealkylation of dialkyl phosphonates under either acidic conditions (HCl) or using the McKenna procedure (a two-step reaction that makes use of bromotrimethylsilane followed by methanolysis) constitute the best methods to prepare phosphonic acids.

phosphonic_acid:_preparation_and_applications

Introduction<!><!>Phosphonic acids: properties and applications<!><!>Phosphonic acids: properties and applications<!><!>Phosphonic acids: properties and applications<!><!>Phosphonic acids: properties and applications<!>Hydrolysis of phosphonates with hydrochloric acid<!><!>Hydrolysis of phosphonates with hydrochloric acid<!><!>Hydrolysis of phosphonates with hydrochloric acid<!><!>Hydrolysis of phosphonates with hydrochloric acid<!><!>Hydrolysis of phosphonates with hydrochloric acid<!><!>Hydrolysis of phosphonates with hydrochloric acid<!><!>Hydrolysis of phosphonates with hydrochloric acid<!><!>Hydrolysis of phosphonates with hydrochloric acid<!>Catalytic hydrogenolysis<!><!>Catalytic hydrogenolysis<!><!>Catalytic hydrogenolysis<!>McKenna’s method – use of bromotrimethylsilane<!><!>McKenna’s method – use of bromotrimethylsilane<!><!>McKenna’s method – use of bromotrimethylsilane<!><!>McKenna’s method – use of bromotrimethylsilane<!><!>McKenna’s method – use of bromotrimethylsilane<!><!>McKenna’s method – use of bromotrimethylsilane<!><!>McKenna’s method – use of bromotrimethylsilane<!><!>McKenna’s method – use of bromotrimethylsilane<!><!>McKenna’s method – use of bromotrimethylsilane<!><!>Dealkylation with boron reagents<!><!>Other conditions for dealkylation of phosphonate<!><!>From dichlorophosphine (R–PCl2) or dichlorophosphine oxide (R–P=OCl2)<!><!>From dichlorophosphine (R–PCl2) or dichlorophosphine oxide (R–P=OCl2)<!><!>Phosphonic acid from phosphonodiamide RP=O(NR2)2<!><!>Phosphonic acid from phosphonodiamide RP=O(NR2)2<!><!>Phosphonic acid from phosphonodiamide RP=O(NR2)2<!><!>Direct method from phosphorous acid H3PO3<!>Moedritzer–Irani reaction<!><!>Moedritzer–Irani reaction<!><!>Reactivity of phosphorous acid (H3PO3) with imine, nitrile and carbonyl groups<!><!>Reactivity of phosphorous acid (H3PO3) with imine, nitrile and carbonyl groups<!><!>Reactivity of phosphorous acid (H3PO3) with imine, nitrile and carbonyl groups<!><!>Preparation of phosphonic acid by oxidation of phosphinic acid<!><!>Preparation of phosphonic acid by oxidation of phosphinic acid<!><!>Miscellaneous<!><!>Miscellaneous<!><!>Miscellaneous<!><!>Conclusion

<p>Phosphonic acid is a functional group featuring two hydroxy moieties, one P=O double bond and one P–C bond. This functional group was incorporated in a broad diversity of molecules and polymers to introduce specific properties including water solubility, coordination or supramolecular properties. Several books, book chapters or reviews have been focused on the construction of the P–C bond [1], on the description of the different classes of phosphorus-containing functional groups [2], on specific applications (hybrid materials [3], surface modification [4], oil industry [5]) or dedicated to a family of compounds (e.g., aminophosphonic acids [6], organometallic phosphonic acids [7]). However, no recent review was focused on the different methods that can be employed to prepare phosphonic acids which is a function needed to address numerous applications that are summarized in the first part of this review (section 2). Then, we review the principal methods that can be employed to prepare phosphonic acids. The most frequently applied methods start from phosphonates (section 3 and Figure 1). However, other possibilities exist: the hydrolysis of dichlorophosphine or dichlorophosphine oxide (section 4), the hydrolysis of phosphonodiamide (section 5), the direct methods that make use of phosphorous acid (H3PO3) to create the P–C bond simultaneously to the formation of phosphonic acid (section 6) or the oxidation of phosphinic acid (section 7). The last section (section 8) includes additional miscellaneous methods to prepare phosphonic acids. Of note, the biosynthesis of phosphonic acid, which is a dynamic field of research aiming to discover new bioactive compounds [8], will not be detailed in this review which is focused on the chemical way of producing phosphonic acids. Of note, this review is not an exhaustive list of all the phosphonic acids synthesized over the last 20 years but a selection of examples that aim to illustrate the most relevant methods that can be employed to produce phosphonic acids.</p><!><p>Summary of the synthetic routes to prepare phosphonic acids detailed in this review. The numbers indicate the corresponding sections of this review.</p><!><p>In the solid state, a phosphonic acid function possesses one P–O bond which is shorter than the two others and that can be attributed to the P=O double bond (as an example for methylphosphonic acid, Figure 2, the P=O bond length is 1.4993(11) Å, the two other P–O bond lengths are 1.5441(11) Å and 1.5443(12) Å and the P–C bond is 1.7586(17) Å). The bond angles at the phosphorus atom are ranging from 103.46(8)° to 112.86(7)° for methylphosphonic acid, indicating a distorted tetrahedral geometry around the phosphorus [9]. This function is very stable but under some oxidative conditions (e.g., Mn(II) with O2) the rupture of the P–C bond can occur to produce phosphate [10]. The phosphonic acid function possesses two acidic protons featuring, for instance, when R is an aromatic moiety, a first pKa ranging from 1.1 to 2.3 and the second acidity which features a pKa ranging from 5.3 to 7.2 [11]. The values strongly depend on the electronic properties induced by the substituent R. It must be noted that due to the high polarity of the phosphonic acid function, the purification of phosphonic acids is quite difficult except by recrystallization for the solid samples. The purification by chromatography on silica gel requires very polar eluents (e.g., CHCl3/MeOH/H2O 5:4:1, v/v/v) [12] and further purification by HPLC with a C18 grafted column. Due to these difficulties of purification, it must be noted that in many cases the purification occurs on the precursors of the phosphonic acid. As an example, dialkyl phosphonates (the diester derivatives of phosphonic acid) can be easily purified by chromatography on silica gel and diverse clean and efficient methods can be applied to produce phosphonic acid from phosphonates without the need of intense purification in the final step. Indeed, the purification is limited to remove the solvent and volatile reagents that are frequently used in excess (see section 3). Due to the acidity of phosphonic acid this function is deprotonated in water and this property was used to increase the water solubility of organic compounds [13–14], polymers [15–16] or ligands for coordination chemistry [17]. It must be noted that for similar compounds, that only differ by the replacement of a carboxylic acid group with a phosphonic acid moiety, the log POW values (partition coefficient between octanol/water solution) are decreased by about 1 log unit (meaning that the phosphonic acid derivatives when placed in an octanol/water mixture the compound's concentration in the aqueous phase is 10 times the concentration of the corresponding carboxylic acid analogue) [11]. Meanwhile, the phosphonic acid derivatives are more acidic when compared to their carboxylic acid equivalents (1.9 to 2.9 units of pKa below for the first acidity of phosphonic acid derivatives) [11]. This property was used to design Brönsted acid catalysts that were used for the depolymerization of cellulose [18] and the synthesis of dihydropyrimidine derivatives [19]. The monosodium salt of phosphonic acids were also employed as organocatalysts for Michael addition [20]. The capacity of phosphonic acids to increase the solubility of organic compounds in water was employed to develop water soluble catalysts [21], or to improve the water solubility of drug-chelate supramolecular assemblies (e.g., calixarene) [22–23]. The water solubility of phosphonic acid is strongly improved when the phosphonic acid is deprotonated (basic media). It is not rare to observe, when preparing NMR tubes with D2O as solvent that phosphonic acid appeared weakly soluble whereas the addition of K2CO3 induced its solubilization. Interestingly the increase of the number of phosphonic acid functional groups on a molecule is not systematically associated with an increase of its water solubility as exemplified with triphenylphosphine functionalized either by two or three phosphonic acid sodium salts; the former being more water soluble than the later [24]. This behavior is associated to the fact that the trisphosphonic acid sodium salt was already solvated at the solid state.</p><!><p>Chemical structure of dialkyl phosphonate, phosphonic acid and illustration of the simplest phosphonic acid: methylphosphonic acid.</p><!><p>The high polarity of the phosphonic acid function and its ability to be ionized in water render this group attractive to design the polar head group of anionic amphiphilic compounds [25–26] or polymeric-based amphiphilic derivatives [27]. This type of compound was used as surfactant to stabilize colloidal solutions of nanocrystals [28], to prepare oil/water emulsions [29], or to prevent corrosion [30–32]. The ionic interactions between phosphonic acid and lipophilic amine produced catanionic supramolecular aggregates that were assessed as HIV inhibitors [26,33].</p><p>Many phosphonic acids are present in nature and their biosynthesis involves, as one of the key steps, the isomerization of phosphoenolpyruvate to phosphonopyruvate which is catalyzed by phosphopyruvate mutase [34]. Recent studies indicate that up to 25% of the phosphorus available in the ocean would consist of phosphorus species featuring one P–C bond [35] pointing out the importance of such type of compounds in natural biochemical processes [36]. Phosphonic acid mimics the phosphate group, which is omnipresent in nature, but also the tetrahedral transition-state intermediate encountered for instance during the hydratation of carbon dioxide by carbonic anhydrase [37] or during the hydrolysis of amide [38]. The main difference between phosphate and phosphonic acid arises from the higher stability (resistance towards enzymatic degradation) of the P–C bond when compared to the P–O bond present in phosphate. Accordingly, phosphonic acid was used for numerous applications in biology and medicine to mimic the phosphate group leading to antiretroviral drugs (e.g., tenofovir (1)) [39], isoprenoid biosynthesis inhibitors [40–41], antibiotics (e.g., fosfomycin (2)) [42], tyrosine phosphatase inhibitors 3 [43], antimalarial 4 [44], antihypertensive drugs (e.g., K4 5 and K26 6) [45] or the anti-osteoporosis compounds alendronate (7) [46] and zoledronate (8) [47] (Figure 3A). In some cases, the bio-active phosphonic acid is generated in vivo from a phosphonate pro-drug [48] as exemplified by the formation of 10 from 9 which permits to improve the pharmacokinetic properties (Figure 3B) [49]. Some phosphonic acid-containing compounds were also used for their herbicidal properties as exemplified by glyphosate (11) [50] (Figure 3C). Finally, in the field of immunotherapy, γδ T cells, at the difference to αβ T cells, respond to non-peptidic antigen or to the alterations in the expression of normal cell surface components. The subset of the human γδ T cells that express the Vγ9Vδ2 receptor recognize and present non-peptide antigens including prenyl pyrophosphate antigens also identified as phosphoantigens. These Vγ9Vδ2 T cells are involved during bacterial or protozoan infections but play also a role in tumor immunity [51]. The synthesis of prenyl pyrophosphate analogues was assessed as a strategy to activate Vγ9Vδ2 T cells [52]. These analogues are mainly composed of pyrophosphate compounds but some phosphonic acids such as compound 12 was identified as an activator of Vγ9Vδ2 T cells [53–54] (Figure 3D).</p><!><p>Illustration of some phosphonic acid exhibiting bioactive properties. A) Phosphonic acids for biomedical applications ; B) phosphonic acid generated in vivo from a phosphonate pro-drug; C) chemical structure of glyphosate known for its herbicidal properties; D) chemical structure of a phosphoantigen containing phosphonic acid functional groups.</p><!><p>Phosphonic acid is a function that is readily involved in hydrogen bonds thus generating auto-assembling supramolecular structures [55] or, when a second organic partner possessing basic groups is present (e.g., amine), mixt supramolecular materials [56–58]. This supramolecular behavior was also involved in the development of organic materials exhibiting proton conduction [59], or organo-gel properties [60]. In the field of analytical chemistry, molecules functionalized with phosphonic acid groups were used to prepare the solid phase for immobilized metal affinity chromatography (IMAC). Such types of solid phases were applied to enrich carbohydrate from extracts [61] or as chiral selectors immobilized on silica for chiral cation exchange chromatography [62] (Figure 4). Porphyrin tetra-functionalized with phosphonic acid groups was used as a chemosensor for trinitrotoluene (TNT) [63].</p><!><p>Illustration of the use of phosphonic acids for their coordination properties and their ability to be involved for the synthesis of hybrid materials or to interact with metal-oxide surfaces.</p><!><p>The third important feature of phosphonic acid arises from its coordination properties. Indeed, the three oxygen atoms can be engaged in coordination or iono-covalent bonds. These coordination properties were used to design molecular hybrid materials [64] also identified as metal organic framework (MOF) or coordination polymers that are synthesized by reaction with a metallic salts (e.g., copper [65], lanthanides [66]) under hydrothermal conditions [67–68] or by insertion of phosphonic acid in low dimensional inorganic materials like layered double hydroxide (LDH) [69] or layered simple hydroxide (LSH) [70]. These materials prepared from phosphonic acids were assessed for numerus applications, including nuclear fuel stewardship and separations of actinides [71], porosity [72–74], bactericidal properties [75], inorganic salt release [76], luminescence [77–78], protonic conduction [79–80], materials with magnetic properties [81–82], or were spread off in polymers to produce nanocomposites [83–84]. Of note, despite the non-chiral nature of compound 13 (Figure 4), it produced, when associated with copper salt, a homochiral non-centrosymmetric crystalline hybrid material [85]. The coordination properties of phosphonic acid were also applied to design tetraazamacrocyclic compounds functionalized with phosphonic acid pendant arms. As an example, ((1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(methylene))tetraphosphonic acid, DOTP, compound 14, Figure 4), was assessed to complex indium [86] or terbium [87] with the aim to develop bone targeting and dosimetry. It was also employed to complex 89Y and applied as pH sensitive NMR probe [88], or as organic precursor of crystalline manganese, nickel [89] or lanthanide-containing hybrid materials [90]. Such type of chelating agents was also designed to develop responsive contrast agents for magnetic resonance spectroscopy [91]. Acyclic diamine compounds (e.g., 15, Figure 4) possessing phosphonic acid groups as additional coordination sites or the tetraazamacrocyclic compound 14 were also reported as a chelator of 177Lu which was explored as a radiotracer that accumulates in bones [92]. These coordination properties were also applied to water treatment and to selectively extract lanthanide from water solutions [93], for nuclear waste treatment [94] or as sequestration or decorporation agents [95]. The high affinity of phosphonic acid to calcium ions is another feature of the coordination properties of phosphonic acids that triggered biomedical applications. This coordination property for calcium cations was further enhanced by placing two phosphonic acid moieties and one hydroxy group around a methylene unit (hydroxymethylenediphosphonic acid). Such type of molecular fragment was incorporated in the structure of drugs used to reduce bone resorption that occurs in bone metastastis or osteoporosis (alendronate (7), zoledranote (8), Figure 3). This affinity for calcium was also used to develop bone targeting which was assessed for therapy [96] and imaging [97–98]. Finally, the phosphonic acid group was also employed to immobilize organic or organometallic molecules on the surface of metal oxide [99–100] (Al2O3 [101], TiO2 [102–103], SnO2 [104], Fe3O4 [105], ZnO [106]), CdSe quantum dots [107] and used as anchoring group for dye-sensitized solar cells [108–110] or for the immobilization of organocatalyst [111–112]. Phosphonic acid was also used for coating superparamagnetic iron oxide (e.g., magnetite) assessed as contrast agent in magnetic resonance imaging [113] or to conceive a red/ox catalyst that can be magnetically separated from the reaction media [114]. The immobilization of phosphonic acid can be also achieved by supramolecular interaction between the support and the phosphonic acid moiety leading to heterosupramolecular structures [115]. Some phosphonic acids were also considered for their flame retardant properties [116].</p><p>All these applications, that cover a very broad panel of scientific topics, point out the great interest of phosphonic acids.The next sections report the most important and efficient methods to produce phosphonic acids.</p><!><p>The most general method to prepare phosphonic acids from phosphonates is to use concentrated HCl solution (35–37% in water; ≈12 M) at reflux for 1 to 12 h as shown in Figure 5. At the end of the reaction the excess of HCl and water can be readily eliminated by distillation and finally, the last traces of water can be removed by azeotrope distillation with toluene. Phosphonic acids, that can be very hygroscopic, can be further dried by leaving the product in a desiccator over P2O5 but, usually, they do not require further purification. When further purification is required, purification can be achieved by recrystallization in polar solvents (e.g., acetonitrile [106], methanol/acetone [117], water [55]), or in concentrated HCl [118] (acidic media reduces the water solubility of phosphonic acids). When recrystallized in aqueous solution, the phosphonic acid can co-crystallize with water due to the presence of hydrogen bonds between the phosphonic acid moiety and H2O [119].</p><!><p>Hydrolysis of dialkyl phosphonate to phosphonic acid under acidic conditions.</p><!><p>As depicted on Figure 6, the selected phosphonic acids prepared by the hydrolysis of phosphonate with concentrated HCl water solution present only few other functional groups. This methods was applied to prepare the alkylphosphonic acid 16 [120], arylphosphonic acid 17 [121] or 18 or block co-polymers possessing pendant arms functionalized with phosphonic acids (compound 19 [122]). The hydrolysis with 20% HCl solution (≈6 M) was also occasionally applied to coordination complexes bearing a phosphonate function as exemplified with compound 20 [123].</p><!><p>Examples of phosphonic acids prepared by hydrolysis of dialkylphosphonate with HCl 35% at reflux (16–19) or 20% HCl solution (20).</p><!><p>HBr was used more occasionally to hydrolyze phosphonates [124–126]. Regarding the nature of the alkyl chains of dialkyl phosphonates, it must be noted that most of the time these chains are methyl, ethyl, isopropyl or, occasionally, n-butyl [127]. This observation is likely explained by the synthetic methods employed to prepare phosphonates that frequently made use of the Arbuzov reaction [128] involving the commercially available trimethyl, triethyl or triisopropyl phosphite [129]. To check the achievement of phosphonate hydrolysis, phosphorus NMR is a method of choice and can also eventually detect side reactions. Indeed, with some substrates it was found that the P–C bond can be cleaved when treated with concentrated HCl as first reported by Redmore et al. [130]. For instance, hydroxynaphthylphosphonate 21 when treated with HBr induced partial rupture of the P–C bond to yield phosphoric acid which is difficult to remove from the expected phosphonic acid 22 (Figure 7A) [131]. The same side reaction was observed in the course of the acidic hydrolysis of 4-hydroxybenzenephosphonate 23 into phosphonic acid 24 (Figure 7B). However, the regioisomer diethyl 3-hydroxyphenylphosphonate can be converted to phosphonic acid 25 with HCl 35% at reflux without any P–C bond cleavage (unpublished result). The diphosphonic acids 26 and 27 were also prepared by hydrolysis with HCl 35% without P–C bond cleavage. These results indicated that the mesomeric effect induced by the phenol function likely explained the difference of stability of compounds 23 and diethyl 3-hydroxyphenylphosphonate in refluxing HCl 35% solution. Of note, if classical heating at 100 °C is usually employed some reaction under microwave activation was reported to reduce the reaction time up to only 7 minutes [132].</p><!><p>A) and B) Observation of P–C bond breaking during the hydrolysis of phosphonate with concentrated HX water solutions; C) examples of compounds prepared by hydrolysis of dialkyl phosphonate in presence of a 35% HCl–water solution without the observation of a P–C bond cleavage.</p><!><p>For the mechanism of hydrolysis of phosphonate by hydrochloric acid, some works dedicated to assess the hydrolysis of trimethyl phosphate are instructive and can likely be extrapolated to phosphonate. When trimethyl phosphate (TMP) is placed in 4 N HCl solution, infrared analysis showed a shift of the P=O band from 1273 cm−1 to 1236 cm−1 [133]. When the HCl concentration was further increased to 12 N, this band disappeared. The titration of trimethyl phosphate with HCl was also monitored by 31P NMR and the results are consistent with a protonation at the oxygen atom that is doubly bonded to the phosphorus atom leading to a phosphorus centered cation (the protonated trimethyl phosphate was characterized by a pKa of −3.6). These results, extrapolated to phosphonate, suggest that the protonation of the phosphonate would occur on the oxygen atom which is doubly bonded to the phosphorus atom. This protonation likely yields the intermediate I (Figure 8) which exists as two mesomeric forms. Then, two competitive mechanisms can occur. Intermediate I can lose a carbocation according to a SN1 mechanism whereas the second way could consist in a nucleophilic substitution (SN2) involving chloride ions as nucleophilic species to produce the intermediate II. Then, a repetition of this mechanism yields phosphonic acid. The preponderant route is likely governed by the stability of the carbocation and the steric hindrance around the electrophilic carbon atom of intermediate I (Figure 8).</p><!><p>Mechanism of the hydrolysis of dialkyl phosphonate with HCl in water.</p><!><p>Di-tert-butyl phosphonate belongs to the precursors of phosphonic acid that requires milder acidic conditions to be converted into phosphonic acid. The mechanism likely occurs following the SN1 pathway as reported above. This type of di-tert-butyl phosphonate was used for the synthesis of phosphonic acid analogues of peptides like protein-tyrosine kynases (PTKs) [134] or to prepare gadolinium complexes used as magnetic resonance imaging (MRI) contrast agents [135–136]. The introduction of the di-tert-butyl phosphonate was achieved by the reaction between di-tert-butyl phosphite and either benzyl halide [137] (Michaelis–Becker reaction) or aldehyde [134] (Pudovik reaction) or using tris-tert-butyl phosphite (Abramov reaction) [135]. The elimination of the t-Bu moieties can be efficiently achieved in presence of trifluoroacetic acid (TFA). As exemplified in Figure 9, the use of TFA induces the formation of the phosphonic acid 29 concomitantly with the formation of carboxylic acid from the tert-butyl ester. The final compounds were purified by removing the excess of TFA (72.4 °C at 1 bar) [138], using HPLC [137] or by precipitation in diethyl ether [136].</p><!><p>Hydrolysis of bis-tert-butyl phosphonate 28 into phosphonic acid 29 [137].</p><!><p>All the precedent examples illustrate the hydrolysis of dialkyl phosphonate in phosphonic acid under acidic conditions. Similar protocols (concentrated HCl or HBr) can be used to prepare phosphonic acid from diphenyl phosphonate (Figure 10A). The use of a mixture of HBr in acetic acid was also reported [139–140]. It must be noted that the reaction work-up is usually slightly different since the treatment with propylene oxide that acts as an acid scavenger, can be applied during the purification step. The preparation of phosphonic acid from diphenyl phosphonate was reported to prepare phosphohomocysteine 30 [141], the arginine mimetic 31 that was developed as a potent metallo-aminopeptidase inhibitor [142] or the aminophosphonic acid 32 [143] (Figure 10B).</p><!><p>A) Hydrolysis of diphenyl phosphonate into phosphonic acid in acidic media. B) Examples of phosphonic acids prepared by this method.</p><!><p>The mechanism of hydrolysis of diphenyl phosphonate in acidic conditions is likely different to the one occurring with dialkyl phosphonates (Figure 8). For the hydrolysis of diphenyl phosphonate it is likely that after protonation of the phosphonate, water acts as a nucleophile and subsequently phenol is eliminated (Figure 11). The repetition of this sequence produces phosphonic acid.</p><!><p>Suggested mechanism occurring for the first step of the hydrolysis of diphenyl phosphonate into phosphonic acid.</p><!><p>Of note, the treatment of phosphonate with nucleophilic basic reagents like NaOH [144], LiOH [145] or NaHCO3 [146] is not suitable to produce phosphonic acid. Indeed, these conditions yield phosphonic acid mono-esters. This monohydrolysis takes place with dialkyl phosphonate or diphenyl phosphonate [146]. For dimethyl phosphonate, the monohydrolysis can be also achieved using NaI as nucleophile in acetone [147] or butanone as solvent [148].</p><!><p>Dibenzyl phosphonates are readily synthesized using dibenzyl or tribenzyl phosphite. This type of phosphonate offers an alternative to the use of acidic conditions to prepare phosphonic acid since the benzyl moieties can be removed by hydrogenolysis (Figure 12). Palladium on charcoal, which is the most used method to prepare phosphonic acid from dibenzyl phosphonate [149], was the catalyst used to prepare the phosphonic acids 33 [150], 34 [151] and 35 [152] (Figure 12). Of note, mono-hydrolyzed phosphonate can be obtained when a monoalkyl-monobenzyl phosphonate is hydrogenolyzed [150,153].</p><!><p>A) Hydrogenolysis of dibenzyl phosphonate to phosphonic acid. B) Compounds 33, 34 and 35 were prepared by hydrogenolysis on Pd/C from dibenzyl phosphonates.</p><!><p>The synthesis of phosphonic acid from diphenyl phosphonates can be prepared under acidic conditions (see above) but can be also achieved in presence of Adam's catalyst (PtO2) and hydrogen as exemplified by the preparation of the compounds 36 [154] and 37 [155] (Figure 13).</p><!><p>A) Preparation of phosphonic acid from diphenyl phosphonate with the Adam's catalyst. B) Compounds 36 and 37 were prepared with this method.</p><!><p>It is also worth noticing that phosphonic acid can be prepared by the hydrogenolysis of diallyl phosphonates. For this purpose the use of the Wilkinson catalyst (ClRh(PPh3)3) was reported [156].</p><!><p>As reported above, the hydrolysis in acidic media requires harsh conditions (most of the time concentrated HCl solution in water at reflux) that finally limit its application when the molecules possess sensitive functional groups. The need of a milder method to prepare more functionalized phosphonic acid derivatives was therefore required. In some aspect the selection of benzyl or tert-butyl phosphonate can be suitable since specific mild conditions can be applied to prepare phosphonic acids as discussed above. Nevertheless, as the simplest way to prepare phosphonate involves trialkyl phosphite, the subsequent dealkylation of dialkyl phosphonate under mild conditions was of a great importance. The group of C. E. McKenna reported in 1977 the use of bromotrimethylsilane as a reagent that permitted an efficient transesterification of dialkyl phosphonate to bis-(trimethylsilyl) phosphonate. It is worth noticing that these silylated phosphonates produced quantitatively phosphonic acid derivatives after water or alcohol (methanol, ethanol) treatment [157]. In this initial work, NMR data indicated that silylation of dimethyl or diethyl phosphonate with bromotrimethylsilane was almost quantitative. Other studies indicated that this reaction can be also applied to diisopropyl phosphonate, or di-tert-butyl phosphonate leading to the conclusion that all dialkyl phosphonates can be dealkylated by the transesterification followed by a methanolysis or hydrolysis subsequent step. As suggested in the initial works of McKenna, the mechanism occurs by an oxophilic substitution on the silicon atom whereas bromide acts as a leaving group to produce the intermediate I (Figure 14). This intermediate is then dealkylated following a similar path than that occurring with the Arbuzov reaction to produce the intermediate II [158]. The repetition of this mechanism produced de disilylated intermediate III. The hydrolysis of this intermediate III produced phosphonic acid, trimethylsilanol and hexamethyldisiloxane that are two volatile side products. The methanolysis of the intermediate III is even a better choice to transform III into phosphonic acid since the methoxytrimethylsilane is also volatile and methanol used in excess is also more volatile than water. It must be noted that an experimental proof (using 17O- and 18O-enriched diethyl phenylphosphonate) has reported that the terminal oxygen doubly bonded to the phosphorus atom was indeed the nucleophilic atom that attacks the silicon atom [159].</p><!><p>Suggested mechanism for the preparation of phosphonic acid from dialkyl phosphonate using bromotrimethylsilane.</p><!><p>Regarding the mechanism of this reaction, the oxophilic silylation involving the oxygen atom which is doubly bonded to the phosphorus atom, thus producing the intermediate I, is also supported by the works of Bartlett et al. [160] (Figure 15A) that show that the treatment of the phosphonate-thiophosphonate 38 with iodotrimethylsilane produced, after methanolysis, only the phosphonic acid 39. Of note, the thiophosphonate functional group is not hydrolysed. The need of the nucleophilic halide anion is another feature of this reaction. Indeed the treatment of TMSOTf on a phosphonate did not induce the dealkylation likely due to the absence of nucleophilic species [161].</p><!><p>A) Reaction of the phosphonate-thiophosphonate 37 with iodotrimethylsilane followed by methanolysis. B) Illustration of the selectivity of BrSiMe3towards dialkyl phosphonate versus diaryl phosphonate. C) Use of bromotrimethylsilane to prepare the 3-butenyltrisphosphonic acid pentasodium 2,4,6-trimethylpyridinium salt 42.</p><!><p>It is worth noticing that the nucleophilic attack of bromide only occurs on alkyl chains as exemplified by a work of Pohjala et al. [162] (Figure 15B) The authors showed that the diphosphonate 40 featuring both phenyl and alkyl substituents treated with chlorotrimethylsilane and sodium iodide in CH3CN yielded, after methanolysis and treatment with NaOH, compound 41 resulting from the hydrolysis of the diethyl phosphonate moiety. Finally, it must be noted that BrSiMe3 selectively induced the silylation of phosphonate without affecting ketone, amide, halogenoalkane or alkyne [163]. The preparation of phosphonic acid from phosphonate with bromotrimethylsilane was also applied to methylene-tris-phosphonic acids [164], and to substituted hexaethyl 1,1,1-tris-phosphonate as illustrated with the reactivity of compound 42. This reaction occurred in presence of collidine to prepare, after treatment with sodium hydroxide, the collidinium salt 43 [165] (Figure 15C).</p><p>The mechanism of dealkylation with bromotrimethylsilane, that produces a bis-silylated phosphonate intermediate, thus incited to use tris(trimethylsilyl) phosphite as nucleophilic phosphorus species to produce after methanolysis phosphonic acid [166] or hydroxymethylene bis-phosphonic acid as illustrated in Figure 16 with the synthesis of the hydroxymethylenebisphosphonic acid 45 [167].</p><!><p>Synthesis of hydroxymethylenebisphosphonic acid by reaction of tris(trimethylsilyl) phosphite with acyl chloride.</p><!><p>The trans-esterification of phosphonate with bromotrimethylsilane to produce silylated phosphonate requires at least two equivalents of BrSiMe3 per phosphonate function. Usually an excess is engaged in the reaction to guarantee a full conversion of dialkyl phosphonate into the disilylated phosphonate. The same reaction is also observed with the use of iodotrimethylsilane (Me3SiI) [168]. However, with chlorotrimethylsilane the reaction is not really efficient as indicated in the initial work of McKenna since only a partial conversion was observed after several days of reaction. Nevertheless, the use of a mixture of chlorotrimethylsilane with NaI in acetonitrile can be used to achieve the silylation of dialkyl phosphonates that can be then transformed in phosphonic acids after hydrolysis or methanolysis. Following these experimental conditions, the efficacy is likely explained by the in situ generation of iodotrimethylsilane that result from a halogen exchange reaction [169]. This method (ClSiMe3 + NaI in acetonitrile) is currently less employed likely due to the need to remove NaI from the final phosphonic acid after the step of hydrolysis or methanolysis. It is worth noticing that when NaI, LiBr or KI was used alone in anhydrous solvents (acetone, MeCN or butanone) under heating (80–100 °C) a selective monodeprotection of the dialkyl phosphonates (alkyl = methyl or ethyl) was observed. The sodium or lithium salts being formed in high yields (87–97% yields) [170]. This procedure was applied to prepare a molecular receptor of lysine residue [171]. Following this procedure, the monodealkylation can be explained by the formation of a sodium salt that due to electronic factors prevent the second dealkylation. However, the protonation of this anionic compound 46 by ion exchange procedure yields 47 that can then be fully dealkylated with NaI in acetone to produce the phosphonic acid disodium salt 48 [172] (Figure 17).</p><!><p>Synthesis of the phosphonic acid disodium salt 48 by reaction of mono-hydrolysed phosphonate 47 with NaI.</p><!><p>Bromotrimethylsilane is nowadays the gold standard reagent to produce phosphonic acid from dialkyl phosphonate under mild conditions (usually at room temperature). This reaction can be achieved in a non-protic solvent including CH2Cl2 [173], acetonitrile [174], chloroform [175], DMF [176], pyridine [177] or collidine [178]. The use of BrSiMe3 simultaneously as reagent and solvent was also reported [120]. The direct production of sodium salts of phosphonic acid is readily achieved by adding 2 N NaOH/water solution to the silylated phosphonate [178]. This two-step sequence: 1. bromotrimethylsilane; 2. methanolysis or hydrolysis was used for the synthesis of numerus phosphonic acids including heterocyclic compounds that are too sensitive to be prepared by the transformation of phosphonates under acidic conditions. As an illustration, the thiophene diphosphonic acid 49 [173], the pyridine oxide 50 [179], the furane phosphonic acid 51 [174], the bipyridine bis thiophene phosphonic acid 52 [180] or the α-aminophosphonic acid 53 that was assessed as inhibitor of the human farnesyl pyrophosphate synthase (hFPPS) [181] were prepared from their corresponding diethylphosphonates (Figure 18). Nucleotide analogues were also prepared by this methodology as exemplified by the compound 54 [176] or 55 [182]. For these last two examples, the bromotrimethylsilane induced the silylation of diethyl phosphonate but also the phosphoramidate and the phosphinate functional groups. Other phosphonic acids possessing different functionalities including phosphine 56 [183], trimethylsilyl 57 [184], diazo 58 [185] or styrene 59 [186] moieties were also prepared efficiently using bromotrimethylsilane followed by methanolysis.</p><!><p>Phosphonic acid synthesized by the sequence 1) bromotrimethylsilane 2) methanolysis or hydrolysis. The notation R indicates the nature of the alkyl chains present on the phosphonate precursors of these phosphonic acids.</p><!><p>This two-step methodology (1. BrSiMe3; 2. MeOH or H2O) was also applied to prepare macromolecules functionalized with phosphonic acid functional groups. For instance, a cyclodextrine derivative (compound 60 [177], Figure 19), amphiphilic compounds that were used for self-assembled monolayers (e.g., compound 61 [187]), polymers (PEG derivative 62 [188] or functionalized polyethylene 63 [189]) were reported. The McKenna's procedure was also applied to prepare polyphosphonic acids as exemplified by the compounds 64 [120], 65 [190], 66 [191] and the dendrimers 67 [192].</p><!><p>Polyphosphonic acids and macromolecular compounds prepared by the hydrolysis of dialkyl phosphonate following the McKenna's method. The notation R indicates the nature of the alkyl chains present on the phosphonate precursors of these phosphonic acids.</p><!><p>The use of BrSiMe3 was also applied to diverse organometallic compounds as exemplified in Figure 20. This procedure was applicable to compounds featuring CpFe(CO)2 68 [193] or ferrocenyl 69 [194–195] moieties but also to palladium or platinium pincer complexes 70 [196] or arene–chrome carbonyl derivatives 71 [197]. It must be noted that when the organometallic complex possessed halogen–metal moiety, then Me3SiBr can induce halogen exchange [198].</p><!><p>Examples of organometallic complexes functionalized with phosphonic acids that were prepared by the hydrolysis of dialkyl phosphonate according to the McKenna's method. The notation R indicates the nature of the alkyl chains present on the phosphonate precursors of these phosphonic acids.</p><!><p>The use of bromotrimethylsilane to prepare phosphonic acid from dialkyl phosphonate is rarely associated with undesired side reactions. Nevertheless, G. David et al. have reported in a study dedicated to the synthesis of methacrylate monomers functionalized with phosphonic acids the occurrence of side compounds [199]. The treatment of the bis-phosphonate 72 with TMS-Br followed by methanolysis, induces a cleavage of the carboxylic esters simultaneously with the formation of the phosphonic acid 75 (Figure 21). The authors have shown, using model compounds, that the hydrolysis is triggered by the acidity of the phosphonic acid. Interestingly, the addition of aqueous ammonia during methanolysis of the disilylated phosphonate produced the expected phosphonic acid 73 without any cleavage of the carboxylic ester function.</p><!><p>Side reaction observed during the hydrolysis of methacrylate monomer functionalized with phosphonic acid.</p><!><p>A second example reported by Pailloux et al. shows that depending on the reaction time for the hydrolysis of the silylated phosphonate 76 (Figure 22) either the expected phosphonic acid 77 or the phosphonic acid 78 resulting from the opening of the benzoxazole fragment (after 3 days in contact with water), were produced [200]. This side reaction is likely explained by the sensitivity of the product to acidic condition coming from the acidity of the phosphonic acid function. During the hydrolysis the precipitation of the phosphonic acid 77 occurred; a rapid filtration (5 minutes) led to isolate the phosphonic acid 77 in 70% yield. However, a prolonged contact time with water (3 days) yields the phosphonic acid 78 that features a benzoxazole ring opening. It can be concluded form these studies (Figure 21 and Figure 22) that the side reactions is not due to bromotrimethylsilane but to the sensitivity of the product to acidic conditions that are induced by the acidity of the phosphonic acid functional group.</p><!><p>Influence of the reaction time during the hydrolysis of compound 76.</p><!><p>Dealkylation of dialkyl phosphonates could also be performed using C6H11BCl2 [201] or BBr3 [202] under mild and efficient conditions as reported by Mortier et al. The reaction can be applied to dialkyl phosphonates (R = Me, Et, iPr, t-Bu) and proceed first at −30 °C and then at 70 °C in an aprotic solvent (toluene) for 6 hours (Figure 23). The authors reported the formation of a primary adduct R3B···O=PR(OR')2 which was subsequently decomposed as borophosphonates oligomers through a bis-bromodealkylation [203]. The phosphonic acids were finally obtained after methanolysis with quantitative conversion and yields up to 95%. Interestingly, this procedure is selective to P–O dealkylation and compatible with the presence of other groups such as allyl, ketone, primary alcohol, phthalimide, ester or thioether as illustrated with the transformation of the phosphonate 79 to the phosphonic acid 80 (Figure 23). The best results were obtained when 0.9 equivalents of BBr3 per phosphonate function was used.</p><!><p>Dealkylation of dialkyl phosphonates with boron tribromide.</p><!><p>The dealkylation of diethyl phosphonate groups present in amino acids or peptides requires mild conditions to avoid the reactivity with the other functional groups. If Me3SiBr can be used to dealkylate peptides functionalized with diethyl phosphonate groups [204], other mild conditions, developed in the context of peptide chemistry, were reported. It is based on the use of trimethylsilyl trifluoromethanesulfonate (TMS-OTf) as silylating agent, trifluoroacetic acid (TFA) as acidic reagent, and dimethylsulfide (DMS) and m-cresol to avoid side reactions (from SN1 or SN2 mechanism) [204] that can involve the functional groups present in the amino acid or peptide. These conditions were applied to prepare the compound 82 from 81 [205–206] (Figure 24).</p><!><p>Dealkylation of diethylphosphonate 81 with TMS-OTf.</p><!><p>Aryl- and to a lesser extent alkylphosphonic acids have been prepared by the hydrolysis under mild conditions of aryldichlorophosphine or aryldichlorophosphine oxide. The hydrolysis is often performed under basic conditions using aqueous sodium hydroxide (5% NaOH); the phosphonic acid being lately isolated by acidification with hydrochloric acid. For example, the sterically hindered phenylphosphonic acid 85 was prepared in a two-step procedure starting from the dichlorophenylphosphine 83. Ph–PCl2 was first oxidized into the phenylphosphine oxide 84 with sulfuryl chloride in CCl4 and then hydrolysis occurred with aqueous NaOH to afford the corresponding arylphosphonic acid 85 in good yield (65% yield, 2 steps) as shown in Figure 25 [207].</p><!><p>Synthesis of substituted phenylphosphonic acid 85 from the phenyldichlorophosphine 83.</p><!><p>More recently, the synthesis of o-trifluoromethylbenzenephosphonic acid (87) was prepared by hydrolysis of the phenyldichlorophosphine oxide 86 with NaOH in acetonitrile (75% yield, Figure 26). The hydrolysis in acidic conditions (10% HCl) was also reported and produced the phosphonic acid with similar yield (75–85%). However, hydrolysis under acidic conditions required an extended reaction time [208].</p><!><p>Hydrolysis of substituted phenyldichlorophosphine oxide 86 under basic conditions.</p><!><p>Phosphonodiamides are commonly used as precursors of phosphonic acids especially for the synthesis of aminophosphonic acids (α [209–210], β [211] or γ [212]) and also for the synthesis of nucleoside analogues [213]. The use of these intermediates is likely explained by the possibility to use chiral diamine that can act as a chiral auxiliary to control the chirality of the carbon atom in α-position of the phosphorus atom as exemplified by the alkylation of the phosphonodiamide 88 (Figure 27). Furthermore, phosphonodiamide can be purified by different methods including chromatography and their transformation to phosphonic acid is easily achieved by hydrolysis under acidic conditions as illustrated by the reaction of compound 89 that produced the α-amino-phosphonic acid 90 after acidic hydrolysis [210] (Figure 27). Similar acidic hydrolysis was used to produce the imidazolyl phosphonic acid 91 [214], 92 [215], the chiral phosphonic-carboxylic acid 93 [216] or the cyclopropylphosphonic acid 94 that was assessed as inhibitor of the glutamate metabotropic receptors [217].</p><!><p>A) Illustration of the synthesis of chiral phosphonic acids from phosphonodiamides. B) Examples of phosphonic acid prepared by acidic hydrolysis of phosphonodiamides.</p><!><p>The synthesis of phosphonic acid via phosphonodiamide intermediates started by the use of the nucleophilic bis(dialkylamino)chlorophosphine. This possibility is illustrated by the recent works reporting the reaction of bis(diethylamino)chlorophosphine (95) with the acetal 96 in the presence of a Lewis acid to yield the phosphonodiamide 97. Then the nucleophilic addition of adenine and the hydrolysis of the phosphonodiamide function in phosphonic acid produce compound 98 [213] (Figure 28A). A second example corresponds to the nucleophilic addition of N-heterocyclic phosphine 99 to a nitroalkene (phospho-Michael reaction) as shown in Figure 28B. The thiourea unit in 99 plays a crucial role by assuming intramolecular interaction with the nitro function that finally led to the formation of the phosphonodiamidate 100. The reduction of the nitro function and the hydrolysis under acidic conditions of the phosphonodiamide produced the phosphonic acid 101 [211]. As a last example, lithiated diaminophosphine borane 102 was added to imine to produce the bis(diethylamino)phosphone borane 103 (Figure 28C). Then acidic hydrolysis conditions and likely air oxidation produced the phosphonic acid 104 [209].</p><!><p>A) Illustration of the synthesis of the phosphonic acid 98 from phosphonodiamide 97. B) Use of cyclic phosphonodiamide 100 to prepare phosphonic acid. C) Illustration of the use of lithiated bis(diethylamino)phosphine borane complex 102 to prepare phosphonic acid.</p><!><p>Phosphonodiamide, which is more robust than phosphonate towards some nucleophilic species like a phosphide anion (R2P-Li), was used to prepare triarylphosphine functionalized with three phosphonic acid groups (Figure 29) [24]. The sequence requires the preparation of the 4-fluorophenylphosphonodiamide 107 which is prepared by nucleophilic addition of an aryllithium intermediate (from 105) on chlorophosphadiamide 106. In the last step, the tris(phenylphosphonodiamide)phosphine 108 was hydrolysed into the tris-phosphonic acid 109 using water and HCl up to pH 1. Of note, the same reaction that engaged diethyl 4-fluorophenylphosphonate instead of the 4-fluorophenylphosphonodiamide 107 is inefficient (3% yield) due to the monohydrolysis of the phosphonate.</p><!><p>Synthesis of tris(phosphonophenyl)phosphine 109.</p><!><p>The direct methods correspond to the methods where the formation of the P–C bond yields simultaneously phosphonic acid. These reactions mainly use phosphorous acid (H3PO3) as source of phosphorus. The main difficulty with such type of reaction is the purification of the product since crystallization, precipitation and dialysis constitute the possible methods of purification. The purification by chromatography requires, due to the high polarity of phosphonic acids, reversed-phase chromatography and is therefore limited to preparative RP-HPLC [218]. Despites these difficulties, some reactions are very efficient as reported below.</p><!><p>The reaction of an amine (primary or secondary amine), formaldehyde (aqueous or solid paraformaldehyde) and phosphorous acid in acidic media produces amino-bis(methylenephosphonic acid, Figure 30A,B). This reaction, is also known as the Moedritzer–Irani reaction since it was first reported by these authors in 1966 [219]. When primary amine is used, the reaction produces a bis-phosphonic acid since the secondary amine that is generated in situ is more reactive than the starting primary amine [220]. In consequence, the stoichiometry must be adapted and 2 equivalents of both formaldehyde and phosphorous acid must be used. The reaction is also applicable to secondary amine and produce mono-aminomethylene phosphonic acid (Figure 30B). The yield of this reaction is variable as illustrated in Figure 30C. The compound 110 was obtained in 20% from diaminocyclohexane [221]. The compound 111 was prepared from a bis(α-aminomethylene diphosphonic acid) and was isolated in 42% yield. This compound was assessed as a scale inhibitor [222]. As a last example, the compound 112 was prepared from diaminoethane using microwave heating. The final product was isolated in 63% yield [223].</p><!><p>Moedritzer–Irani reaction starting from A) primary amine or B) secondary amine. C) Examples of phosphonic acids prepared by the Moedritzer–Irani reaction.</p><!><p>The Moedritzer–Irani reaction was also applied to introduce phosphonic acid functions on polymer or dendrimers possessing reactive amine. Accordingly, phosphonic acids were introduced on polyethyleneglycol 113 [188], polyethylene imine 114 [224] or chitosan 115 [225]. The functionalization of polyacrylamide obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization was also recently reported to produce 116 (Figure 31). However, the conditions of the Moedritzer–Irani reaction induced the hydrolysis of the polymers side chains [226].</p><!><p>Phosphonic acid-functionalized polymers prepared by Moedritzer–Irani reaction.</p><!><p>The phosphorous acid function reacts with imine in the absence of solvent to produce α-amino-phosphonic acid as exemplified by the synthesis of the α-amino-phosphonic acid 118 from the imine 117 (Figure 32) [227]. It is worth noting that the reaction of H3PO3 with some enamine (e.g., 1-morpholinocyclohexene) gives a reduction of the enamine to amine. This is likely why this reaction was seldomly used.</p><!><p>Reaction of phosphorous acid with imine in the absence of solvent.</p><!><p>The reaction of phosphorous acid on nitrile in presence of methanesulfonic acid followed by the addition of POCl3 or PCl3 [222] is a method that produces in one step the aminomethylene bis-phosphonic acid [228] (Figure 33A). This methodology was applied to prepare the compound 119 [229] that was assessed as HIV-1 integrase inhibitor. Compound 120 [230] was prepared simply by the refluxing acetonitrile with phosphorous acid at 130 °C for 12 hours. 120 was a member of a series of compounds developed as a potential inhibitor of the farnesyl pyrophosphate synthase. The reaction also occurs with amide (Figure 33B) as exemplified for the synthesis of compounds 121 [231], 122 [232] or 123 [231].</p><!><p>A) Reaction of phosphorous acid with nitrile and examples of aminomethylene bis-phosphonic acids. B) Reaction of phosphorous acid with amide and examples of compounds prepared by this reaction.</p><!><p>The nitrile group can be replaced in this reaction with a carboxylic acid function. In that case the final product is a hydroxymethylenebis-phosphonic acid (Figure 34). This reaction was optimized to produce, for instance, compounds 124 [233–234] or 125 [229]. This reaction is not further detailed herein as it was recently reviewed [235].</p><!><p>Reaction of carboxylic acid with phosphorous acid and examples of compounds prepared by this way.</p><!><p>Phosphinic acid derivatives (also identified as phosphonous acid) are prepared by reaction of hypophosphorous acid (Figure 35) on alkene or alkyne (hydrophosphonation) [236], by its addition on aldehyde or imine [237] or by hydrolysis of alkyl or aryldichlorophosphine (RPCl2) [238] (for a review see reference [239]). Then, these phosphinic acids constitute a suitable precursor to produce phosphonic acids by oxidation (Figure 35).</p><!><p>Synthesis of phosphonic acid by oxidation of phosphinic acid (also identified as phosphonous acid).</p><!><p>The oxidation of phosphinic acid is readily achieved in the presence of DMSO [26] and a catalytic amount of iodide as exemplified by the synthesis of compound 127 by oxidation of 126 [240] (Figure 36A). This method was also applied to prepare 1-aryl-1-hydroxymethylphosphonic acid [241]. HgCl2 [242] or bromine water are also efficient for the oxidation of phosphinic acid. As an example, the phosphinic acid 128 (Figure 36B) which is prepared by the addition of hypophosphorous acid on imine, was converted quantitatively in α-amino phosphonic acid 129 with bromine water [243] (Figure 36B). HgCl2, despite its toxicity and environmental hazard is nevertheless an efficient reagent for the oxidation of phosphinic acid [244]. Diiodide in the presence of acid (HI) in water/ethanol solution yields the oxidation of the phosphinic acid 130 without oxidizing the thioether function [245] (Figure 36C). The Atherton–Todd conditions [246] (CCl4, NEt3, H2O) in the presence of water was also applied for the oxidation of phosphinic acid [247]. The oxidation of the phosphinic acid into phosphonic acid can be achieved with air or by a catalytic process involving the presence of palladium salts [236,248]. For instance, Kafarski et al. reported an efficient procedure that consisted in transforming the H-phosphinic acid into trivalent trimethylsilyl esters by reaction with hexamethyldisilazane followed by the oxidation with air followed by methanolysis [249]. A similar method that used a silylated intermediate and oxygen as oxidant was reported by Piettre et al. to produce alkyl-α,α-difluorophosphonic acid [250]. Ozone was also reported as oxidizing agent for the synthesis of phosphonic acid as exemplified by the synthesis of the diphosphonic acid 133 that was prepared from the phosphinic acid 132 [251] (Figure 36D).</p><!><p>Selection of reaction conditions to prepare phosphonic acids from phosphinic acids.</p><!><p>If the most frequently used methods to prepare phosphonic acid have been reported above, others exist. In a non-exhaustive way of presentation, few of them are presented below.</p><p>Barton et al. reported a procedure to prepare phosphonic acids from carboxylic acids that made use of white phosphorus (P4) [252]. First, the carboxylic acid is esterified with N-hydroxy-2-thiopyridone in the presence of DCC to produce 134 (Figure 37). This compound 134 was added to P4 solubilized in THF and was stirred for 30 minutes before replacing THF by DME. Then, H2O2 was added portion-wise and the solution was heated at reflux to produce the phosphonic acid 135. For some compounds, the reaction with H2O2 occurred at rt and then SO2 is added to complete the oxidation reaction. The work-up is, however, tedious because some excess of P4 must be removed without any contact with oxygen. This procedure was applied to natural compounds including lipophilic carboxylic acid (e.g., linoleic acid) or amino acid (e.g., L-2-amino-4-phosphonobutyric acid).</p><!><p>Synthesis of phosphonic acid from carboxylic acid and white phosphorus.</p><!><p>Red phosphorus is much easier to handle due to its polymeric nature that renders this compound much stable but also less reactive. Its reaction with benzaldehyde was reported to produce an α-hydroxy-phosphinic acid intermediate that was converted to phosphonic acid in the presence of HI in an aquous organic media at reflux (Figure 38). However, the benzylphosphonic acid (136) was formed simultaneously with phosphoric acid thus requiring a purification step [253].</p><!><p>Synthesis of benzylphosphonic acid 136 from benzaldehyde and red phosphorus.</p><!><p>More recently, the phosphonation of graphite was reported. First, a mechanochemical cracking yielded carboradical intermediates that were reacted with red phosphorus and then oxidized in the presence of air to produce graphene phosphonic acid 137 [116] (Figure 39). This material which is water soluble was used as a non-toxic flame-retardant.</p><!><p>Synthesis of graphene phosphonic acid 137 from graphite and red phosphorus.</p><!><p>Phosphonic acid is a functional group of interest for many current fields of research that include the development of bio-active compounds, medical imaging, material sciences or surface chemistry.</p><p>The most frequently used method to prepare phosphonic acids is stirring dialkyl phosphonates with concentrated HCl in aquous solution at reflux. Despite these drastic conditions, this protocol is a method of choice to prepare phosphonic acids that are stable within acidic media and that are thermally stable. The advantage of this methodology is that it can be applied on large scale and, as exemplified in this review, was applied to a huge variety of compounds. According to this method, the purification step is rendered simple by the fact that the excess of reagents and side products (alkyl halide) are easily removed under vacuum. For the molecules featuring acid sensitive functionalities, the McKenna's method, that starts by the reaction of dialkyl phosphonate with bromotrimethylsilane to produce a bis-silylated phosphonate under mild conditions (usually at 20 °C), is a method of choice. This intermediate (silylated phosphonate) is then quantitatively converted into phosphonic acid by hydrolysis or methanolysis. The reaction has a very broad scope and the rare examples where side reactions were observed are actually due to the acidity generated by the final compound (phosphonic acid). According to the McKenna's method the purification is also simple since the excess of reagent can be easily removed under vacuum.</p><p>Beside these two main methods to prepare phosphonic acids other direct methods can be selected. In that case the P–C bond is formed simultaneously with the production of a phosphonic acid functional group. In this way, the Moedritzer–Irani reaction is a well-documented and an efficient procedure. The limitation of this procedure can arise from the purification step which is mainly limited to crystallization.</p><p>Other methods are available as reported in this review; however, their use has a less broad scope but can be applied to prepare some specific phosphonic acids.</p>

PubMed Open Access

Modular microfluidic system for on-chip extraction, preconcentration and detection of the cytokine biomarker IL-6 in biofluid

The cytokine interleukin 6 (IL-6) is involved in the pathogenesis of different inflammatory diseases, including cancer, and its monitoring could help diagnosis, prognosis of relapse-free survival and recurrence. Here, we report an innovative microfluidic approach that uses the fluidization of magnetic beads to specifically extract, preconcentrate and fluorescently detect IL-6 directly on-chip. We assess how the physical properties of the beads can be tuned to improve assay performance by enhancing mass transport, reduce non-specific binding and multiply the detection signal threefold by transitioning between packed and fluidization states. With the integration of a full ELISA protocol in a single microfluidic chamber, we show a twofold reduction in LOD compared to conventional methods along with a large dynamic range (10 pg/mL to 2 ng/mL). We additionally demonstrate its application to IL-6 detection in undiluted serum samples.

modular_microfluidic_system_for_on-chip_extraction,_preconcentration_and_detection_of_the_cytokine_b

<!>Materials and methods<!>Microfluidic setup.<!>Off-chip optimization of bead-based assay. Bead grafting optimization.<!>Mode of injection<!>Conclusion

<p>Biomarkers are considered as objective quantizers of biological processes and particularly pathophysiological processes; they can be used for patient diagnosis or prognosis as well as to monitor disease progression or patient response to treatment. Biomarkers provide guidance through the development of new medicines [1][2][3] and are pivotal to decipher molecular or cellular mechanisms involved in pathologies. The increased interest for biomarkers has been accompanied by the emergence of a wide range of bioanalytical developments such as mass spectrometry or high throughput screening.</p><p>Among the different biomarkers (cellular, molecular, vesicular), proteins have significantly demonstrated their potential and many of them are analysed and quantified for clinical diagnosis of diseases from asthma and allergies 4,5 through infections 6 and cancer 7 . Cytokines are small proteins involved in cell signalling often used as indicators for disease monitoring 8 such as in tumor progression 9 , liver diseases 10,11 or hepatic inflammations and fibrosis 12 . In particular, interleukin 6 (IL-6) is involved in the response of the human immune system to infection and cellular injury 13,14 , being secreted by T cells and macrophages into the serum in case of acute and chronic inflammation. Recently, it has been suggested that coronaviruses may activate dysregulated host immune responses. Exploratory studies have suggested that interleukin-6 (IL-6) levels are elevated in cases of complicated COVID-19 15,16 . Thus, a quantitative analysis of cytokines in bodily fluids, and IL-6 in particular, can benefit the monitoring of a wide range of diseases. The current standard methods to detect and analyse cytokines are immunoassays, typically in the form of ELISA, microarrays and bead-based assays 17 . While immunoassays can be highly specific and sensitive, cytokine detection by batchwise immunoassay remains challenging due to their very low concentrations in biological samples down to sub pico or femto-molar concentration [18][19][20] .</p><p>The potential benefits of microfluidics are multi-fold: decrease analysis time, improve bioassays sensitivity, reduce sample and reagent volumes, decrease costs and miniaturize and integrate complex protocols. Impressive results of IL-6 detection in microfluidic systems have already been published relying on glass capillary 21 , modified controlled-pore glass packet 22 or carbon nanotube forests 23 . But while several microfluidic systems have already OPEN 1 Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168 Paris, France. 2 Institut Pierre-Gilles de Gennes, Paris, France. 3 LAAS-CNRS, Université de Toulouse, CNRS, INSA, 31400 Toulouse, France. 4 Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, 92296 Châtenay-Malabry, France. * email: [email protected]; [email protected] shown their efficiency for cytokine detection and quantification, there is still a need for new technologies and methods that can tackle the challenge of precise detection in complex matrices at low cost.</p><p>Here we approach this challenge by improving current immunoassay-based protocols in an integrated bead-based microfluidic format. Immunoassays have been widely implemented in microfluidic [24][25][26][27] , initially as a miniaturization of conventional microtiter plate ELISA, the antibody being grafted at the surface of the microchannel 28,29 . To further improve the specific surface of interaction and consequently the surface to volume ratio, solid supports have been inserted in microdevices starting with mechanical trapping of micrometric polystyrene beads functionalized with antibodies 30 . The interest of using microbeads as solid support was exemplified by Teste et al. demonstrating theoretically and experimentally that the kinetics of target analyte capture is improved by using micro-and nano-magnetic particles compared to standard microtiter plates 31,32 . Since then, other strategies have been investigated leveraging electrokinetic and magnetic forces [33][34][35] , in particular in the form of droplet immunoassays combined with magnetics beads [36][37][38][39] . Previously, we developed the microfluidic magnetic fluidized bed, a beads-based microfluidic technology based on a homogeneous suspension of magnetic beads inside a microfluidic chamber 40 . A balance of drag and magnetic forces on the beads results in physical properties similar to those of a macroscale fluidized bed. The resulting high surface to volume ratio, constant mixing and compatibility with commercial and functionable beads make it attractive for bioanalysis integration. The porosity of the bed of beads plays a key role in the efficiency of the system, as it could affect the sample residence time and diffusion distances to the solid phase. This was demonstrated in a wide range of applications: bacteria analysis in raw samples 41,42 , detection of histone modifications 43 and as a miniaturized platform for extracorporeal circulation 44 . However, sensitive protein detection requires relatively complex multi-step protocols, challenging to integrate in a single device.</p><p>Here we leverage the fluidized bed as a tool able to perform all the steps of an ELISA protocol for IL-6 detection: specific extraction, preconcentration, enzymatic binding and detection in a single microfluidic chip. We optimize and evaluate the performance of each step and study how the unique physical features of the mobile solid phase can be tuned to improve assay performance. We do this by adjusting the porosity of the system and the arrangement of the beads according to the molecular diffusion constant and the characteristics of the enzymatic reaction. Finally, we compare the results of our optimized system to the performance of current standard protocols for IL-6 quantitation.</p><!><p>Reagents and chemicals. The washing buffer was prepared with Tris HCl (200 mM, Sigma Aldrich), Bovine Serum Albumin "BSA" (1%, Sigma Aldrich) and Tween 20 (0.1%, Sigma Aldrich). The pH was adjusted at 7.5. The washing buffer was stored at − 4 °C.</p><p>The enzymatic substrate MUP (Methylumbelliferyl phosphate, Thermofisher Scientific) was dissolved in the washing buffer at 10 mM, pH adjusted at 8.0. The substrate was kept at − 20 °C.</p><p>Tosylactivated beads (DynabeadsTM M-280 Tosylactivated, Thermofisher Scientific) were grafted following the Dynabeads datasheet with anti-human IL6 antibodies (Thermofisher Scientific from the kit CHC1263): they were shaken and incubated with anti-IL6 antibodies, Tris buffer and ammonium sulfate (3 M) at 37 °C for 18 h. Then, the beads were washed and resuspended in PBS with BSA 0.1% at a final concentration of 5 mg/mL. The detection antibodies and enzyme were provided by this same kit.</p><p>Chip fabrication. The microfluidic chip was described in previous publications 40,45 . It consists of an elbow channel leading to a diamond-shape chamber, with an opening angle of 13°. The height of the chamber and channel was set at 50 µm, and the total volume of the PDMS chamber was 0.6 µL.</p><p>Microfluidic chips were designed using a micro-milled mold. These molds were machined in brass pieces of 5 cm × 5 cm. The designs were a positive replica of the chip. The chips were fabricated by pouring polydimethylsiloxane (PDMS, Sylgard184, Dow Corning) into the molds (concentration 1:10) and were bonded by oxygen plasma. A surface treatment of PDMA-AGE 0.5% 46 was incubated inside the chip chamber for 2 h then rinsed with distilled water and dried with compressed air.</p><!><p>The liquid flow was produced by a pressurization of the sample reservoir using a pressure controller (MFCSTM, Fluigent) allowing to reach a range of pressures from 0.1 mbar to 1 bar, translated in flowrates between 0.1 and 3 µL/min. The outlet of the chip was connected to a flowrate controller (Flowunit S, Fluigent), which allowed precise flowrate measurements and feedback control on the pressure based on the Maesflo software (Fluigent). Peek tubing (Tube Peek 1/32" × 0.25 mm, Cil Cluzeau Info Labo) was used to connect the microfluidic chip to the other elements of the experimental set up. A tube (12 cm peek tubing of 0.063 mm diameter) was positioned at the entrance of the chip to increase the hydrodynamic resistance of the device. A permanent magnet made of NdFeB 1. 47 T (N52, size 20 mm × 20 mm × 30 mm, magnetization direction through the thickness, by ChenYang technologies) was aligned with the chamber axis at a 1.5 mm distance from the chip inlet.</p><p>Operating conditions. Manual in-tube labelling ELISA. The sample (50 µL) and the detection antibodies (50 µL, 0.5 µg/mL) were incubated off-chip at room temperature for 50 min with continuous shaking. This mix (50 µL) was flowed inside the chip chamber containing functionalized magnetic beads at a 1 µL/min flowrate. The beads were then rinsed with the washing buffer for 30 min at 2 µL/min inside the chip prior to fluorescent detection. of a homogenous suspension of magnetic beads in a microfluidic chamber. The drag force applied on the beads is due to the flow of liquid in the microchannel and is balanced by a magnetic force created by a permanent magnet 40 . Beads are in free suspension in the liquid phase, a configuration that allows to avoid clogging issues. The microfluidic magnetic fluidized bed has shown interesting features regarding biomarkers analysis as it has already demonstrated its efficiency for nucleic acid analysis 47,48 , but so far it has not been applied to protein biomarker analysis in real samples. Here, we put forward the potential of this technology by combining both preconcentration in a dynamic configuration and on-beads detection. First, a high throughput extraction and preconcentration steps of the analyte are performed directly on the magnetic beads, then the detection of the target biomarker by sandwich ELISA is performed on chip and on beads in a very small volume with properties that can be tuned using the fluidized bed properties to achieve optimal performances.</p><!><p>To implement a microfluidic fluidized bed-based bioassay for IL-6 detection, we first optimized the bead grafting with capture antibodies for IL-6 capture and preconcentration. In this approach, the magnetic beads in the fluidized bed have a pivotal role as they ensure an efficient extraction of the target in a continuous flow while being used in the second step as solid support to perform the sandwich ELISA. In order to optimize the bioassay performance, we first compared two widely used grafting strategies, based on covalently-grafted capture antibody using either tosyl-activated surface or carboxylic acid modified surfaces (tosyl-activated Dynabeads® and MyOne Carboxylic M-270, respectively) 45 . These grafting strategies have been first compared in tube, using 20 µg of capture antibody per mg of beads for the tosyl-activated beads, and 4 µg of antibody per mg of beads for the MyOne Carboxylic beads as advised by the supplier, the capture antibody being here an anti-human IL6 antibody. To compare the different bead-grafting strategies, sandwich immunoassays were performed in the presence or absence of IL-6 to evaluate the specific to non-specific signal. Data shown in Table S1 demonstrated that the tosyl-activated Dynabeads® allow achieving the highest signal to noise ratio with both positive signal higher and negative signal lower than with carboxylic beads. The tosyl-activated Dynabeads™ as solid phase were thus selected as solid support for the IL-6 bioassay development.</p><p>Buffer optimization. Further optimizations were performed in tube conditions to determine the best parameters to be integrated on chip. In particular, the choice of washing buffer is very important to limit non-specific adsorption; the performances have been evaluated regarding the immunoassay specificity with different buffer compositions. To do so, washing buffers, with pH ranging from 7 to 8 with different ionic strength and ions/ counter ions nature were evaluated: NaHCO 3 (pH = 8, 100 mM), Tris EDTA (pH = 8, 100 mM), Tris HCl (pH = 8, 200 mM), and PBS (pH = 7, 150 mM). Our studies demonstrate a superiority of Tris-HCl buffer over the other ones (data not shown). In parallel, we have also investigated how the presence of BSA and Tween-20 affects the assay performances, both being well known to limit non-specific adsorption on beads as well as on PDMS. The presence of both additives indeed greatly improves the assay performances (Table S2). Finally, we selected as Vol:.( 1234567890 45 . We have thus compared both approaches in the microfluidic fluidized bed. In particular, we have evaluated if the formation of the target-detection antibody complex before its injection on chip could affect its extraction and consequently the bioassay performance. We investigated if the formation of the target-detection antibody complex prior on-chip injection could decrease the diffusion constant of the complex and potentially limits its capture in the continuous flow extraction within the fluidized bed.</p><p>For the off-chip immune complex formation, the sample, detection antibody and the enzyme were incubated together prior to their on-chip injection (Figure S1.I). The enzyme (Alkaline Phosphatase) was conjugated with the detection antibody through a streptavidin/biotin binding simultaneously with the complex IL-6/detection antibody formation in solution. Magnetic beads were functionalized as previously described (Figure S1.II). The immuno-complex was then injected in the fluidized bed to be captured on the magnetic beads (Figure S1.III). After a washing step to remove the excess of detection antibody and enzyme, the enzymatic substrate was injected within the bed to perform the detection step (Figure S1.IV).</p><p>In the case of the sequential injection, only the conjugation of the detection antibody by the enzyme was performed in tube prior to on-chip injection. Magnetic beads were functionalized as previously described (Fig. 1I). Then, as in a conventional ELISA in microtiter plate, each step was performed sequentially by injecting in the fluidized bed of each solution as follows: the sample was first injected within the fluidized bed (Fig. 1II), a washing step was performed, conjugated detection antibody was injected inside the chip (Fig. 1III), a washing step was repeated, and then the enzymatic substrate was finally injected to perform the detection (Fig. 1IV).</p><p>A series of on-chip experiments were conducted to compare these two approaches. Our results showed that a first pre-incubation seemed to slightly enhance the raw signal of detection of the antibody-enzyme complex. These results are in good agreement with previous studies 49,50 . In contrary, the nonspecific signal was significantly lower for sequential injection and the signal to noise ratio was two times higher for sequential injection compared to manual in-tube labelling (Table 1). The fluidization and continuous injection through the suspension of beads allows to reduce non-specific binding to the magnetic beads compared to in tube incubation. As our final goal www.nature.com/scientificreports/ is to inject complex matrices such as serum within the fluidized bed, the reduction of non-specific interaction needs to be prioritized. In addition, the sequential injection had the advantages of simplified automation. Thus, we selected the sequential injection mode for all subsequent experiments, for which the microfluidic fluidized bed features can be optimized to reach lower limits of detection.</p><p>Optimization of the detection step. The immunoassay format being selected, the detection antibody concentration was next optimized as a compromise between sensitivity and specificity. As previously described, the detection antibody used is an anti-human IL-6 biotinylated antibody conjugated with a streptavidin Alkaline Phosphatase enzyme. The 1X concentration corresponds to a detection antibody concentration of 0.5 µg/mL. The concentration of the antibody-enzyme complex was thus varied between 0.5 µg/mL (1 X) and 25 µg/mL (50 X). As shown in Fig. 2A, the intensity of the signal obtained in the presence of IL-6 at 5 ng/mL can be slightly increased when increasing the detection antibody concentration. However, this goes hand in hand with a significant increase of the non-specific signal and a decrease of the signal to noise ratio (Fig. 2B). The optimal concentration of detection antibody was set at 0.5 µg/mL, condition for which the higher signal to noise ratio was reached while reducing the cost per assay.</p><p>Table 1. Influence of the process of injection on the specific and non-specific signal. The experiments are performed with a sample of IL-6 at 10 ng/mL (for the specific signal) or a buffer solution mL (for the nonspecific signal) as described in the Material and Methods. parameters of the bead-based immunoassay optimized, we next leveraged the fluidized bed format to improve assay performance. The microfluidic magnetic fluidized bed design has been optimized to reach a high homogeneity of bead distribution within the microchamber 40 , but it has also been shown that the bead bed porosity can be tuned at will within the chip. A change of flowrate induces a change of the drag force applied to the beads. The balance between drag and magnetic forces is modified so that bed of beads expands as the flowrate increases.</p><!><p>The influence of the flow rate on the on-chip immunoassay has thus been investigated as a change in porosity can impact not only the analysis time (from 25 to 100 min for 50 μL of sample) but also the diffusion distance, as well as the residence time of the target biomolecule within the bed of particles.</p><p>From previous work 40 , we know that the magnetic beads in microfluidic fluidized beds tend to self-organize in cylindrical clusters of diameter d c ≈12 μm due to bead-bead interactions. In a simple 1D approximation, we can consider the bed porosity ε being defined by the distance between these clusters d s and their size d c :</p><p>For an efficient capture, the analyte needs to be able to reach, by diffusion, a magnetic bead before leaving the fluidized bed due to the flow-driven convection. If we call t d the time to reach a surface of capture by diffusion and t c the time to cross the magnetic bed by convection, we need to ensure that t d t c ≪ 1 , so that the antibodyantigen interaction can occur effectively within the residence time.</p><p>The time t d needed to travel the distance d = d s /2 allowing an analyte to reach the closer cluster by diffusion can be estimated with Einstein's relation:</p><p>8D , where D is the diffusion constant of the analyte. On the other hand, the residence time of the analyte within the bed can be approximately evaluated as t c = HL 2 tan α 2 /Q , where L is the bed's length, α the aperture angle of the chamber and H the chamber height. Hence, the ratio between both times is: Considering a diffusion constant of cytokine IL-6 D = 8.5 10 -8 cm 2 .s −151 and the aperture angle of the fluidized bed being α = 35° = 0.61 rad, we can estimate the ratio of times based on published measures of the bed length L and porosity ε 40 at flowrates of 0.5, 1 and 2 μL/min (Table 2).</p><p>While this remains an approximation, note that the ratio t d t c approaches 1 for a flowrate of 1 μL/min, already significantly above 1 for a flowrate of 2 μL/min (Table 2). Hence, we would expect our system to efficiently promote the interactions between the analyte and the surface of capture of the beads up to a maximum flowrate of ~ 1 μL/min.</p><p>We thus experimentally investigated how the flowrate of the sample injection impacts the specific signal intensity at the outlet of the chip (Fig. 2C). Our experiments showed that a shorter residence along with larger distance to the particles can significantly affect the immunoassay performances, the fluorescence intensity decreasing as the flowrate increases, in agreement with our model previously described. As a compromise between the assay sensitivity and the analysis time, the flowrate was set at 1 µL/min, allowing to reach the higher signal to noise ratio (Fig. 2D). In those conditions, the signal intensity is decreased by 20% compared to a slower flowrate but the analysis time is divided by two and the background noise is decreased by 40%. Furthermore, the correlation between the volume of the sample and the intensity of the detected signal was investigated and we were able to show a high correlation (Figure S2), showing the versatility of our device towards the volume of sample.</p><p>Finally, the choice of the fluidized bed to integrate IL-6 immunoassay was also motivated by its unique modularity to improve the immunoassay performance. As fluidization occurs when passing liquid through a packed bed of particles at a sufficient velocity to compensate magnetic forces, two regimes can be achieved with this device: below a threshold flowrate, the beads are in close contact and organized as a packed bed of particles, while above this flowrate interparticle distance increases resulting in higher porosity and improved fluid/ solid contact in a fluidized bed regime. These two regimes (packed bed and fluidized bed) can then coexist in our system as a function of the flow rate applied and have been compared here to improve the last step of the immunoassay: the enzymatic reaction. The enzymatic reaction taking place within the fluidized bed offers the possibility to consider two detection modes with potentially improved performances. Indeed, after injection of the enzymatic substrate in the bed, the fluorescent signal can be detected either continuously (in flow method) or by sequentially changing the flowrate above and below the threshold (stop-and-go method).</p><p>In the continuous in-flow detection approach, the enzymatic substrate was flowed continuously inside the chamber at 1 µL/min for 6 min (Fig. 3A). The bed is, at this flow rate, in a fluidized bed regime; the enzymatic product generated at the surface of the beads is thus continuously flowed through the bed to reach the detection area. The signal has a shape of an asymptotic curve (Fig. 3B). The value of interest is the height of the plateau, proportional to the concentration of IL-6 in the initial sample.</p><p>Evolution of the ratio t d /t c between the time of diffusion between the capture beads clusters and the residence time inside the fluidized bed due to the flowrate Q of the liquid percolating the bed. www.nature.com/scientificreports/</p><p>In the stop-and-go method, a given volume of enzymatic substrate (0.7 µL) is first flowed through the bed at 0.4 µL/min. The pressure is then decreased so that the substrate and the magnetic beads can be incubated in a packed bed regime for 10 min decreasing drastically the diffusion distances between the beads and the enzymatic substrate (Fig. 3C). After 10 min of incubation, the pressure is increased with a retro-controlled program so that the solution is flowed towards the area of detection at 1 µL/min (Fig. 3D). A fluorescence peak is thus obtained (Fig. 3E) as the quantity of product obtained during the 10-min incubation being a finite quantity. The opening of the bed after the 10 min of incubation is a critical step which could affect the shape and dimensions of the peak. By this process, a high quantity of fluorescent product can be accumulated inside the bed of beads before reaching the detection area while switching on the flow rate. With this approach, we aimed at increasing the IL-6 immunoassay sensitivity. The differences in the shapes of the recorded signals are related to the physical properties of the fluidized bed such as the porosity of bead assembly.</p><p>As shown in Table 3, the signals of three quantification methods of the fluorescent signal were compared to choose the most accurate one. A higher signal was recorded when working with the stop-and-go mode, as expected. Interestingly, the coefficient of variation was smaller when using the peak area measurement rather than the peak height whereas the mean specific signal was higher. It allowed us to reach a signal to noise ratio almost as high as the one of in-flow mode, but with a mean specific signal more than 3 times higher. Finally, despite quite similar performances in terms of signal to noise ratio, we selected the stop-and-go mode rather than the in-flow one in order to reach higher specific signal to lower the limit of detection. Our analytical model showed that an increase of the flowrate above 1 μL/min would limit the antigen-antibody interaction, not allowing improvements when working with the continuous mode. A solution to circumvent this issue lies in the addition of the incubation steps. This choice left more freedom for further optimization if needed: the sensitivity could be increased by optimizing either the injected volume of enzymatic substrate or the incubation time of the stop-and-go mode.</p><p>Evaluation of the performance of the system for IL-6 detection. To further evaluate the performances of the fluidized bed-based ELISA in terms of dynamic range and sensitivity, we established a calibration S3). A 50 µL sample was flowed through the chip at 1 µL/min then the detection antibody (0.5 µg/mL) was injected at room temperature for 50 min at the same flowrate. A quite large dynamic range was obtained with linear response from 10 pg/mL to 2 ng/mL, as shown in Fig. 4B. This dynamic range of our approach is competitive compared to those reported in the literature 52,53 or to commercial immunoassays 54 usually going from few pg/mL to ng/mL. Negative controls were performed with non-spiked buffer. We next evaluated the sensitivity of our newly developed immunoassay on the basis that significant signal is three standard deviation above the negative control. We obtained a limit of detection (LOD) at 6 pg/mL, more than two times lower than the one of the standard beads immunoassay (LOD is 15.6 pg/mL from manufacter's data).</p><p>We finally applied the on-chip method to complex samples and performed a calibration curve with IL-6 spiked in fetal bovine serum (FBS) to validate our integrated approach towards complex sample matrix analysis (Fig. 4C). This was performed on a reduced range of concentration, closer to clinical sample conditions. The results show a strong similarity with the results achieved in Tris-HCl, with a linear response in the whole dynamic range and an LOD of 60 pg/mL. Due to its tunable porosity, the microfluidic magnetic fluidized bed is well suited to work with complex matrices. However, as expected due to the high protein content of serum, it is associated with a slightly decreased sensitivity. We assume that some screening effect due to the high protein content of the serum extraction could affect IL-6 specific capture and consequently the assay sensitivity.</p><p>Altogether our results showed that the tunable properties of a magnetic and microfluidic fluidized bed allows to integrate an automated sequence of on-chip extraction and detection of Il-6. The fluidization regime can be used to limit the non-specific interactions and avoid clogging when working with complex matrices whereas the packed state could be used to enhance the detection step. The control of these two regimes (packed and fluidized states) allowed us to reach a relevant limit of detection in the tens of pg/mL, compatible with, for instance, the requirements of IL-6 detection in patient serum in sepsis [55][56][57] . Based on this first proof of concept, the modulable device may be further adapted for the detection of other cytokines. Table 3. Comparison between continuous and 'stop-and-go' methods for experiments performed with a sample of IL-6 at 10 ng/mL (for the specific signal) or a buffer solution mL (for the non-specific signal) as described in the Material and Methods.</p><!><p>Microfluidic systems have demonstrated their potential to enhance bioassay performance and integration, particularly in the case of immunoassays. However, fully integrated multi-step protocols combining analyte extraction, preconcentration and detection in a single module are still challenging, particularly when addressing high sensitivity and/or compatibility with complex matrices. We believe the microfluidic fluidized bed-based approach presented here provides a new way to tackle high-performance immunoassays in fully automated protocols. Its versatility is based on the addition of new variables: the tunable porosity and arrangement of the magnetic beads. In particular, the extraction conditions can be tuned as a function of the diffusion constant of the analyte, while the enzymatic step can further be modified to improve the assay performances. This new technology is also compatible with a large range of biomarker concentrations as well as with sample volumes ranging from one to a few hundred µL.</p><p>We demonstrate here that it allows a fast detection of the cytokine IL-6 with a large dynamic range (10 pg/ mL to 2 ng/mL), in less than 2 h, with an LOD in the picomolar range. The sensitivity achieved in this first proof of concept is applicable for instance to severe sepsis infection, where IL-6 levels can go up to a few hundreds of pg/mL in human serum samples. A LOD of 6 pg/mL for IL-6 spiked in buffer solution was achieved. This value is lower than the LOD achieved in experiments performed with the same reagents in on-bench conditions, and better overlaps with clinical ranges 19,56 . Moreover, we demonstrated its compatibility with challenging matrices of high protein content and no dilution while still ensuring a clinically-relevant sensitivity. We believe that this capability to enhance the performance of conventional assays while fully integrating complex sequential protocols make this approach a promising tool for future biomarker detection and quantification applications.</p>

Scientific Reports - Nature

Efficiency gains for thermally coupled solar hydrogen production in extreme cold

Hydrogen produced from water using solar energy constitutes a sustainable alternative to fossil fuels, but solar hydrogen is not yet economically competitive. A major question is whether the approach of coupling photovoltaics via the electricity grid to electrolysis is preferential to higher levels of device integration in 'artificial leaf' designs. Here, we scrutinise the effects of thermally coupled solar water splitting on device efficiencies and catalyst footprint for sub-freezing ambient temperatures of -20 • C. These conditions are found for a significant fraction of the year in many world regions. Using a combination of electrochemical experiments and modelling, we demonstrate that thermal coupling broadens the operating window and significantly reduces the required catalyst loading when compared to electrolysis decoupled from photovoltaics. Efficiency benefits differ qualitatively for double-and triple junction solar absorbers, which has implications for the general design of outdoor-located photoelectochemical devices. Similar to high-efficiency photovoltaics that reached technological maturity in space, application cases in polar or alpine climates could support the scale-up of solar hydrogen at the global scale.

efficiency_gains_for_thermally_coupled_solar_hydrogen_production_in_extreme_cold

<!>Author Contributions<!>Conflicts of interest<!>Device model description<!>Electrochemistry<!>58<!>Device characterisation<!>G corr

<p>Fossil fuels constitute a versatile and large fraction of our energy sources, but contribute significantly to anthropogenic warming [1]. Solar-driven water splitting (aka electrolysis) produces hydrogen, an alternative energy carrier free of greenhouse gases, sustainably and without the limitations of wind power and biomass [2][3][4]. The main obstacle preventing large-scale implementation are currently the comparatively high production costs. However, logistics for fossil fuels can also be expensive and environmentally hazardous, in particular for the year-round energy supply of remote research bases such as the Neumayer Station in Antarctica or Paranal observatory in Chile. Therefore, local hydrogen production can become both economically and environmentally favourable, but is challenging in the often cold environments [5,6].</p><p>The currently most mature approach for solar-driven hydrogen production is to supply polymer electrolyte membrane electrolysers with electricity from photovoltaic (PV) solar cells via the grid [2,[7][8][9]. The complete separation of light absorption and electrolysis does, however, come with electrical and thermal losses. Firstly, an additional DC-to-DC converter is required [7]. Secondly, internal thermalisation, i.e. de-excitation of charge-carriers to the band edges under the release of phonons, reduces the extractable energy of excited electronhole pairs. In addition, transmission losses of low-energy photons with energies below the bandgap of the light-absorbing semiconductor limit the current [3,10]. The heat generated by these two latter loss mechanisms has the potential to benefit catalysis. Therefore, a thermally tightly coupled water-splitting device -with the light absorber immersed into the electrolyte or not -represents a highly attractive concept. Such a design uses the waste heat of the absorbers to decrease the internal device electrical resistance and reduce the requirements for the catalysts, while simultaneously cooling the absorbers and hence boosting their efficiency [7,11]. Furthermore, a design that allows safe product separation without a degradation-and failure-prone membrane also reduces costs and increases operating life [12].</p><p>Recently, the beneficial effects of thermally coupled water splitting at ambient temperatures were demonstrated [13]. Solar water-splitting research in general has just recently started to consider the influence of colder ambient temperatures on device operation in the temperate zones [14]. So far, however, the impact of very low temperatures over extended times as found in high-latitudes, high-altitudes or winters in the temperate zones on operation have not been considered.</p><p>In this work, we investigate a route of expanding the thermal window that makes solar water splitting feasible down to an outdoor temperature of -20 • C. Climate data analysis shows that many world regions at high latitudes or altitudes could benefit from our considerations, prominent examples are Antarctica or the Himalaya region. We use a numerical device model to explore the influence of such low temperatures on the solar-to-hydrogen (STH) efficiency of solar water splitting devices and quantify the beneficial effects of thermal coupling and a suitable device insulation. These predictions are scrutinised under idealised laboratory conditions and the first solar water splitting device operating at -20 • C is demonstrated.</p><p>We can also show that these benefits differ qualitatively for dual-and triple-junction solar absorbers, which has significant implications for the general design of outdoor-located photoelectrochemical devices. Finally, we discuss the energy supply of high-latitude and highaltitude remote research stations as a first potentially economic competitive implementation of our considerations.</p><p>Current large-scale technologies for water splitting operate at temperatures between 50 and 1000 • C [9]. Meanwhile, laboratory studies for solar water splitting typically employ ambient temperatures of about 20 • C. For small-to medium-scale, distributed hydrogen production, however, the impact of outdoor temperatures on device operation must be considered. While the volumetric density of dissipated heat of electrolysers in the MW-range are often large enough to require cooling, this changes for smaller plants that are more effectively cooled by outdoor temperatures. In the limit of small-scale applications, such as the powering of weather stations, process temperatures will, without external heating, very closely follow ambient conditions.</p><p>Yet the mean annual temperatures of a considerable part of the world is below the freezing point of water (Fig. 1a). Low electrolyte temperatures lead to losses from higher catalysis and ion transport overpotentials, but also cause issues for (near-)neutral electrolytes, frequently used for solar water splitting [16], that do not depress the freezing point of water sufficiently.</p><p>Hydrogen production would then cease and the volumetric expansion of freezing water can damage the reactor. The energy harvesting potential for conventional solar hydrogen production can be evaluated from Fig. 1c, in which we show the annual cumulative available solar energy for days with mean temperatures above the freezing point of water. In colder regions, however, temperatures remain in a temperature envelope between -20 • C and 0 • C for a considerable fraction of the year (Fig. 1b). This would create the need for energy-intensive temperature stabilisation of the device. A distributed energy system that can operate with a maximum degree of autonomy would, however, be even more important in these regions, since they are typically more sparsely populated and fuel supply is associated with great expense and effort. Since the long-term storage of hydrogen in gas bottles at very low temperatures is not a challenge, it is a predestined energy carrier for these extreme climate conditions.</p><p>Extending the ambient operating conditions for efficient, small-scale solar water splitting to the above-defined temperature envelope is feasible through the use of electrolytes with a lowfreezing point (e.g. 30 wt% H 2 SO 4 ). Efficiency losses due to the low operating temperatures can be compensated by tight thermal coupling and device insulation as discussed below in detail. This would facilitate additional energy harvesting by solar hydrogen production in some regions considerably as depicted in Fig. 1d. Based on our considerations, solar hydrogen production could benefit in parts of China, Mongolia, the Himalayas, Russia, the Alpine region, Greenland, the Andean mountains, USA, Canada, and Antarctica. More than half of the world population is currently living in areas, where temperatures are in this envelope for at least 30 days per year.</p><p>The influence of low temperatures on the solar-to-hydrogen conversion efficiency of a solar water-splitting device, as sketched in Fig. 2a, is characterised by two contrary effects. Firstly, there is the lower catalytic performance and higher ohmic losses of the electrochemical component, and secondly, the increased solar-cell efficiency, as indicated in Fig. 2b. Which one prevails depends on a number of device parameters, such as the ohmic cell resistance and the temperature coefficient of the solar-cell open-circuit voltage (V OC ).</p><p>To understand these effects in detail and explore thermal coupling to compensate possible efficiency losses, we developed an open-source Python-based model combining solar-cell parameters, electrochemistry, and thermal fluxes. In short, the model predicts the STH efficiency based on the temperature-dependent current-voltage (IV)-characteristics of the solar cell and catalysts by computing the operating temperature in an iterative, self-consistent cycle for a quasi-steady-state condition. This means that the absorbed luminous power equals the sum of the power used to split water at thermoneutral conditions plus the power dissipated by radiation and convection (see Fig. 2a).</p><p>The following calculations are based on a device consisting of high-efficiency double-or triple-junction III-V solar cells together with Pt-and IrO x -catalysts for hydrogen/oxygen evolution reaction (HER/OER), respectively. 30 wt% H 2 SO 4 with a freezing point of −35 • C was used in the model as the electrolyte. Note that one important parameter determining the device temperature is the area ratio A housing /A solar-cell , influencing the heat dissipation from the housing by radiation and convection (see SI Fig. 1). This ratio was fixed to a practical value of 2.5 in the following considerations, representing the case of small-to medium-scale applications. A description of the full model [17] and a list of all input parameters can be found in the supplementary information. Note that a low-cost, but less-efficient alkaline device design could in principle also be realised using Si solar cells, NiFeO x (OER catalyst), NiMo (HER catalyst), and 18 wt% NaOH as an electrolyte. Similar to III-V solar cells for space applications [10], costs of the absorber are, however, probably not a major issue for niche-applications in remote world regions, where the approach competes with the supply of conventional fuels that is associated with great expense and effort. As soon as the technology is established, costs can benefit from scale-up as well as from emerging low-cost, high-efficiency approaches [18,19] that would extend the commercial use case beyond just niche. The same applies to the catalysts. For initial applications, the A catalyst /A solar-cells -ratio by YaSoFo [17] as a function of the A catalyst /A solar cells -ratio, for the decoupled, thermally coupled, and coupled as well as insulated case using a double junction (c-e) and a triple junction (f-h) solar cell. d,g, Increase in absolute STH efficiency caused by thermal coupling in comparison to the non-coupled device. e,h, Efficiency gain from insulation of the electrochemical compartment referring to the coupled configuration. could be adjusted to ensure operating potential below the the maximum power point (MPP) for a high-efficiency triple-junction solar cell. However, for the long-term goal of producing hydrogen on a terrawatt (TW) scale costs -but even more so materials availability -will play an important role. There, the use of low-performance, abundant catalysts (or low noble-catalyst loadings) is highly preferable and any additional voltage losses should be avoided as discussed below in detail. Therefore, the parameter ranges of A catalyst /A solar-cells in the model were chosen to cover operating potentials close to the MPP. Figs. 2c,f show the outdoor temperature-dependent STH efficiencies as a function of the A catalyst /A solar-cells -ratio for a thermally decoupled device based on a double-junction (V OC = 1.88 V) and triple-junction (V OC = 2.7 V) solar cell, respectively. For the double junction, the STH efficiency initially increases, but then decreases with decreasing outdoor temperature implying that the device only benefits from improved solar-cell performance for moderate temperature decreases. Consequently, considering only a narrow temperature window [20,21] could lead to the unsubstantiated generalisation of a steadily increasing or decreasing efficiency with dropping temperature. Note that this effect is very sensitive to the V OC , its temperature coefficient, and the ohmic resistance of the cell. A more negative V OC temperature coefficient and a lower V OC , for example, result in less or almost no decrease of the efficiency at very low temperature, while a higher ohmic cell resistance increases this effect (and vice versa, see SI Figs. 3 & 4). The modelling of the triple junction reveals a constant STH efficiency decrease with decreasing outdoor temperature, an effect that is more stable with respect to parameter variation. In this case, the lower performance of the electrochemical compartment always prevails. This can explained by the higher ohmic potential loss caused by the higher catalyst current densities in the triple junction device.</p><p>The absolute efficiency gain for a thermally coupled device design is illustrated in Figs. 2d,g.</p><p>As expected, thermal coupling increases the absolute STH efficiency by up to 6% at low outdoor temperatures for configurations where the operating point of the device would otherwise drop below the MPP. To further increase the efficiency, we explored the influence of thermally insulating the device housing. In the model, aluminium foil was employed to reduce radiation losses and polystyrene was used to minimise further heat dissipation. The results are shown in Figs. 2e,h as an absolute efficiency change referring to the thermally coupled design. For the double junction, the insulation has a detrimental effect on parameter regions in which the efficiency increases with decreasing outdoor temperature, while it has a positive effect in regions where the efficiency decreases with decreasing outdoor temperature (also see SI Fig. 4). For the triple junction, where the efficiency steadily decreases with decreasing outdoor temperature, the insulation is always beneficial. SI figure 3 shows, in a similar manner as Fig. 2c-h, the functional dependence with respect to the geometry-corrected distance, which is a measure for the electrode spacing, taking into account the device geometry. Again, we observe a qualitative difference for double-and triple junctions. This emphasises that these parameters span a multi-dimensional space and therefore must be carefully considered when designing a double-junction device intended to operate at low temperatures. Only considering a subset in the parameter space is probably the reason for apparently contradictory observations in the literature [14,20].</p><p>The insulation design of the electrochemical compartments could be further improved with the ongoing development of macroscopic thermal rectifiers that allow heat to transfer preferentially in one direction [22]. Such a rectifier would offer the possibility of creating and maintaining a thermal gradient between the electrochemical compartment and the solar cells, especially for short-term intermittency of the irradiance. This can then increase the efficiency boost for the device. Apart from conventional absorber materials like Si or III-V semiconductors, transition metal oxides are also feasible absorbers for photoelectrochemical water splitting [11]. Charge carriers in many of these oxides form small polarons resulting in a small drift mobility [23] implying that the carrier transport can be enhanced via thermalactivating [24]. This suggests that insulating an oxide-based device might not only increase the catalytic performance and lower the ohmic losses, but would be also beneficial for the photoabsorber. The illumination was turned on at t = 0. e, IV curves of the solar-cell array and the Pt-and IrO x -catalysts in a 2-electrode configuration of the respective configurations in thermal equilibrium, i.e. after the 3h-measurements.</p><p>To scrutinise our predictions under idealised laboratory conditions, we built a watersplitting device based on commercial triple junction GaInP/GaInAs/Ge solar cells and commercial Pt-and IrO x -catalysts. The electrodes were separated via a wedge for buoyancydriven product separation as can be seen in SI Fig. 5. Note that this membrane-less concept is a relatively novel approach and there are ongoing efforts to investigate the product crossover as a function of the cell geometry [12]. The A catalyst /A solar-cells -ratio was set to 0.34 and the total device resistance (including the multimeter) was 2.2 Ω at 21 as predicted in our model, resulting in the increase of the operating current and a decrease of the operating potential. For the insulated configuration, it can be clearly seen in Fig. 3e that the additional increase of the catalyst performance (∆V=0.18 V at 0.179 A) in comparison to the coupled configuration is not offset by the additional loss of V OC (∆V OC =0.08 V) caused by the higher device temperature.</p><p>The energy supply for research stations in high-latitude regions such as Antarctica represents an ideal test application for our considerations. There are ongoing efforts to shift the power supply away from the use of fossil fuels towards renewable energy systems [25,26], also for reasons of contamination due to spillage events. Here, hydrogen was indeed already proposed as a future energy carrier [5], and initial practical experience with an indoor, wind-powered electrolyser was gained [6]. While the overall impressions and results were positive, the complexity of the system caused some technical issues and hence relatively high maintenance efforts [6]. Here, a device that operates outdoors with a maximum degree of autonomy could be highly advantageous and represent the first economically competitive case for thermally coupled solar water splitting. Moderate light concentration could, in principle, reduce the costs for hydrogen production. However, this depends on the location.</p><p>In near-polar regions, diffuse irradiation that cannot be concentrated can prevail the direct radiation in the solar spectrum [27].</p><p>Beyond Antarctica, many research stations across the globe are situated in other remote locations at high-latitude and/or high-altitude (Fig. 1a). In almost all of the 100 stations, we considered, the mean annual temperature, T avg,y , remains below the freezing point of water for large parts of the year (Fig. 4). The developments we report here will allow to expand the thermal window that makes solar hydrogen production feasible for most stations, except for in the Antarctic interior. A realistic hydrogen fuel-cell efficiency is 65% (lower heating value) [28]. Then, powering a Raspberry Pi computing device ( For the long-term goal of producing hydrogen on a TW scale, where materials abundance for both light absorber and catlaysts will play an important role as discussed above, the increase of performance by thermal coupling and insulation offers benefits in three major areas: Firstly, it can increase the solar-to-hydrogen efficiency by shifting the operating point of a given device to higher current densities or using solar cells with lower bandgaps [13]. Furthermore, the amount of catalyst loading can be decreased or less active, yet more abundant catalyst materials become feasible. From Fig. 3e, it can be estimated that 21% and 35% of the catalyst loading or activity for the coupled and coupled/insulated case, respectively, could be saved in our test device to achieve the same operation current as in the decoupled configuration. Therefore, thermal coupling also increases the efficiency with respect to the use of catalysts in the device. Note that these numbers depend on the ohmic cell resistance, device configuration, the V OC , and its temperature coefficient as discussed above.</p><p>Finally, the reduced overpotentials from catalysis and ion transport offer the opportunity to use emerging solar cell material configurations such as III-V/Si tandem configurations, where the challenges of internal interfaces reduce the effective photovoltage [18]. These considerations are highly relevant for the design and commercialisation of highly efficient thermally coupled solar water splitting, which could use III-V/Si [19] or Perovskite/Si [30] 5 and Ref. [29]. With the expansion of the thermal window down to a threshold temperature of T 0 = 253 K (blue shading), efficient, distributed solar water splitting is feasible for most research stations with low module areas. In contrast, for a threshold temperature of T 0 = 273 K, (green shading) only some high-altitude and Arctic sites could benefit. tandem cells in a wired design or with the absorber fully immersed in the electrolyte. Some scientific and engineering challenges remain. For example, overall efficiency would benefit from a device operating at high pressures to eliminate the need for hydrogen compressors. Gaseous products need to be safely separated. The device needs to be demonstrably stable for years, not hours. Nevertheless, we lay the foundation for a thermally tightly coupled water-splitting device that can produce hydrogen under extreme climatic conditions with a maximum degree of autonomy. Our simple concept of insulating photoelectrochemical reactors with low-cost materials can boost the efficiency -both in terms of production rate and catalyst loading -of devices suffering from ohmic or catalysis losses at low temperatures.</p><p>We clearly demonstrate that the approach of highly integrated solar water splitting, where the absorber is immersed in the electrolyte [10], benefits efficiency, though it is technologically challenging. A hybrid approach is the one used in this work, where the solar cells are not immersed, but thermally coupled, providing technological maturity at the expense of thermal coupling efficiency. Our work offers a pathway for transition to a fossil-fuel-free energy supply in high-latitude and high-altitude areas, and also opens opportunities for decentralised hydrogen production in world regions with less extreme, but still cold outdoor temperatures. This can become a considerable contribution towards the decarbonisation of the energy sector at the global scale.</p><!><p>The project was developed by MM and KR, the experimental design by MK and MM.</p><p>Code for the photoelectrochemical model was implemented by MK and MM, the experiments conducted by MK, the climate data analysis done by KR. The authors jointly wrote the manuscript.</p><!><p>Electronic Supplementary Information: Efficiency gains for thermally coupled solar hydrogen production in extreme cold Moritz Kölbach, 1, 2 Kira Rehfeld, 3 and Matthias M. May 2 1 Institute for Solar Fuels, Helmholtz-Zentrum Berlin, Germany We used the latest version of the ERA5 reanalysis [1]. To assess the energy harvesting potential for solar-water splitting, we require high-resolution information on temperature and insolation. Solar radiation output in ERA5 has been favourably evaluated globally [2] and at high latitudes [3,4]. Surface temperature characteristics at high latitudes are also well captured [5,6]. Climate and solar input were analysed based on global fields of surface temperature (variable 'tas') and surface downward shortwave radiation (variable 'ssrd') at daily/less than 31 km resolution. A day (defined as 24-hour period) is considered suitable for solar-water-splitting with a device if its average (24-hour) temperature is within the temperature envelope of the device. The temperature thresholds we consider are (a) above the freezing point of water (273 K) for conventional electrolysis, (b) above 253 K, the minimal temperature we have evaluated our device for, and (c) above the freezing point of 30 wt% sulfuric acid, 238 K, which can be seen as the lowest temperature limit of the device. The added solar water-splitting potential in Jm −2 , or the energy that we estimate could have been harvested in 2019 under the expected limitations of the proposed device, is calculated as a weighted sum Q t = 365 i=1 p(t) i q i , with the weight for day, i, set to unity if its mean temperature is above the temperature threshold T 0 : p i = 1 if t i ≥ T 0 and to zero if it is not,</p><p>The harvesting potential of storable renewable energy for high-latitude and highaltitude research stations</p><p>We assess the opportunity for local production of renewable, storable energy at 100 high-latitude and high-altitude research stations and field camps. Location details for active stations were compiled based on publicly available information and publications [7].</p><p>Supplementary Table 5 provides coordinates, names, operators, and links as well as the estimated solar water-splitting energy harvesting potential for the year 2019. Temperature and radiation for the station locations was extracted from ERA5 reanalysis output using bilinear interpolation. Comparing the theoretical efficiency of a triple-junction solar cell SI 2 for AM1.5G with the efficiency for a spectrum to be expected in Antarctica [8], we find the limit to drop by 6% relative. Therefore, we estimate a relative error for the solar hydrogen harvesting potential in Fig. 4 as 10%.</p><!><p>Thermal flux for a thermally coupled device</p><p>In our model for the thermally coupled device design adapted from Min et al. [9], we assume that the temperature of the electrolyte is equal to the operating temperature of the solar cells, denoted as the device temperature (T device ). Ignoring heat transfer by educt and product flux, the device reaches its quasi-steady temperature when the absorbed luminous power (P in ) equals the sum of the fraction of the luminous power used to split water at thermoneutral conditions (f •P in ) plus the power dissipated by radiation (q rad ) and convection (q conv ):</p><p>The absorbed luminous power can be described using the following equation,</p><p>where T optical-train is the transmissivity of the optical train, A solar-cells is the area of the solar cells, and I 0 is the power density of the incident radiation. The fraction f of the absorbed luminous power that is used to split water at thermoneutral conditions can be expressed as</p><p>where j op is the temperature-dependent operation current density, C is the light concentration factor, and E th is thermoneutral voltage for water splitting of 1.48 V. Note that the influence of the temperature on E th is very small and is neglected here. [10] The power dissipated through radiation can be described as follows:</p><p>Here, σ is the Stephan-Boltzmann constant, ε solarcells is the surface emissivity, A housing is the area of the device housing, ε housing is the surface emissivity of the housing, and T out is the outdoor temperature. The power dissipated through convection can be expressed as</p><p>where h solar cells -air is the convective transfer coefficient, and U t is the overall heat transfer coefficient. The latter can be used to implement effects of insulation with a thickness of l ins and a thermal conductivity k ins around the device housing and is given by:</p><p>Thermal flux for a decoupled device For the decoupled device design, we estimate the operating temperature of the solar cell by assuming that an area of twice the solar cell area is available for heat dissipation and that the electrolyte temperature is equal to the outdoor temperature. This implies that the sun and the operating potential E op higher than the thermoneutral voltage do not heat up the electrolyte. The fraction f decoupled of the absorbed luminous power that is used to split water can then be expressed as:</p><!><p>The following part describes the temperature-dependent IV-characteristics for a 2-electrode water splitting setup based on the single OER and HER catalyst characteristics in an aqueous electrolyte. The temperature-dependent standard potential of the OER is given by:</p><p>Here, E 0,OER, T ref is the standard potential at the reference temperature T ref , and E 0,OER, T coeff is the temperature coefficient. By definition, the standard potential of the HER is zero:</p><p>The Nernst-potentials of the OER and HER can be expressed as follows:</p><p>where z is the number of electrons involved in the reaction, F is the Faraday constant, R is the universal gas constant, and pH is the pH value of the aqueous electrolyte. The thermodynamic potential of the water splitting reaction is then defined as the difference between the Nernst-potential of the HER and OER:</p><p>The OER current as a function of the applied potential E can be modelled using the anodic branch of the Butler-Volmer equation:</p><p>where α a • z is the anodic charge transfer coefficient multiplied with the number of electrons involved that can be extracted from the tafel slope, and j 0,OER is the temperaturedependent exchange current density, which can be calculated from the exchange current density j 0,OER, T ref at the reference temperature T ref and the activation energy E a,OER as follows:</p><p>Note that mass transport limitations are neglected in this model for simplicity. The HER current as a function of the applied potential can be modelled analogously:</p><p>In the model, the OER and HER current are merged to the (ohmic loss-free) 2-electrode overall water splitting current j catalysts (E, T device ). Subsequently, the ohmic loss is calculated from SI 5</p><p>the distance of the electrodes d, the geometry correction factor G corr and the temperaturedependent conductivity of the aqueous electrolyte κ electrolyte as follows:</p><p>The linear correlation of the electrolyte conductivity with temperature is given by:</p><p>Solar cell 57</p><p>Our model can perform the calculation of the temperature-dependent solar cell IVcharacteristics using the detailed balance limit or experimental parameters from the manufacturer's datasheet [11]. Here we used the latter. The temperature and intensity dependence of the short-circuit current density I sc , the open circuit potential E oc can be approximated as:</p><p>where influence of shunt and series resistances, the temperature-dependent IV-characteristics of the solar cell can be approximated by [12]:</p><p>STH efficiency</p><!><p>To obtain the operating current density j op , the model matches the IV-characteristics of the solar cell and the 2-electrode catalyst current for overall water splitting based on the SI 6</p><p>A catalyst / A solar cells -ratio:</p><p>Since the device temperature depends on the operating current density (and vice versa, see equation 3), our model iteratively solves equations 1 to 22 until convergence is reached.</p><p>Finally, the STH efficiency is calculated as follows, assuming a Faradaic efficiency of unity:</p><p>If not otherwise stated, the input parameters listed in SI Table 1,2,3 and 4 are used.</p><p>Device design</p><p>15 GaInP/GaInAs/Ge solar cells (CPV 3C44, Azur Space) with an photoactive size of 10</p><p>x 10 mm 2 were back-contacted on a 55 x 40 mm 2 polished copper sheet with a thickness of 0.35 mm using electrically conductive epoxy adhesive (Elecolit 323, Panacol) with the help of a laser-cut placing mask. The front contacts were connected in parallel by wire bonding.</p><p>The copper plate was attached to a macro-cuvette (402.000-OG, Hellma Analytics) with a thin thermally conductive double-sided adhesive foil (WLFT 404, Fischer Elektronik) to ensure good thermal coupling. The outer dimensions of the cuvette were 55 x 40 x 23.6 mm 3 with a wall thickness of 2 mm. A 5 x 55 mm 2 black polyvinyl sheet was used to mask the busbar for the front-contact resulting in a total illuminated area of 19.25 cm 2 . The positive and negative terminal of the solar cell array was wired to the OER-catalyst (IrO x on a titanium mesh with 12 g Ir/m 2 , Metakem) and HER-catalyst (platinised titanium mesh with 50 g Pt/m 2 , Metakem), respectively. Both catalysts were 3.0 x 1.0 cm 2 , which translates into a surface area of 5.1 cm 2 when accounting for the surface factor of 1.7 of the mesh.</p><p>The catalysts were positioned parallel to each other separated by 1 cm. A PTFE wedge in parallel to the catalysts at a separation of 0.75 cm to the cuvette bottom was used for product separation. The distance between the lower edge of the catalysts and the cuvette bottom was 1.9 cm. The cuvette was filled with 22 ml of 30 wt% H 2 SO 4 (pure, Carl Roth) as an electrolyte. For experiments with insulation, the cell was covered in aluminium foil (emissivity of 0.2) and inserted into a 2 cm thick foamed polystyrene housing (thermal conductivity of 0.03 Wm −1 K −1 ). Photographs of the cell are shown in SI Figure 5.</p><!><p>To perform experiments at low temperatures, the device was put into a freezer (LGUex1500, Liebherr) and illuminated through an 8 x 8 cm 2 hole in the door that was covered with a 0.5 cm thick quartz plate. As an illumination source, a WACOM Class AAA solar simulator (WXS-100S-L2H, AM1.5G, 1000 W/cm 2 ) was used. The overall power, as measured within the fridge, was set to 1000 W/m 2 using a S401C powermeter (Thorlabs). Such a single-point calibration for a multi-junction cell is, in principle, prone to errors due to the spectral mismatch between solar simulator and the AM1.5G standard spectrum. In our case of an almost perfectly current-matched triple junction, this does, however, not lead to an over-but rather an underestimation of the overall efficiency, as follows. Optimising the power ratio of the xenon and halogen lamps while keeping the total power at 1000 W/m 2 , resulted in a current density of 13.15 instead of the 15.4 as reported in the datasheet. Raising the current to the photocurrent from the datasheet of a reference cell without correcting the total power by an infrared filter, as is sometimes done in the literature [13], might artificially increase the overall efficiency due to higher thermal power available for heating the electrolyte, and is therefore avoided here. The quartz plate (without anti-reflection coating) as our optical train was inserted into the light path after light-power calibration. For the efficiency calculation, the total illuminated area of 19.25 cm 2 instead of the 15 cm 2 photoactive area was used. of the solar cell array were recorded using a Keysight 2400 source meter with a scan rate of 100 mV/s. For reference measurements in the thermally non-coupled device configuration, the solar cell array on the empty cuvette was illuminated, while the electrochemical compartment was placed in a second cuvette in the freezer located out of the direct beam path.</p><!><p>Geometry correction factor: ratio of the resistance of a restricted cell [17] with no obstacle in between the electrodes (e.g.</p><p>wedge for product separation) and the real cell resistance during operation 0.3 -</p>

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