Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2017 Supporting Information Bis(sulfonamide) transmembrane carrier allows pH-gated inversion of ion selectivity Arundhati Roy, Oindrila Biswas and Pinaki Talukdar* E-mail: [email protected] Contents Page Number I. General Methods S2 II. Physical Measurements S2 – S3 III. Synthesis S3 – S6 IV. Ion Transport Activity S6 – S15 V. Theoretical Calculations S16 – S19 VI. Crystal Structure Parameter S19 – S20 VII. NMR Spectra S21 – S30 VIII. References S31 S1 I. General Methods All reactions were carried out under the nitrogen atmosphere. All the chemicals were purchased from commercial sources and were used as received unless stated otherwise. Solvents were dried by standard methods prior to use or purchased as dry. Thin layer chromatography (TLC) was carried out with E. Merck silica gel 60-F plates and column chromatography was performed 254 over silica gel (100-200 mesh) obtained from commercial suppliers. Egg yolk phosphatidylcholine (EYPC) lipid was purchased from Avanti Polar Lipids as a solution dissolved in chloroform (25 mg/mL). HEPES buffer, monobasic sodium phosphate salt, dibasic sodium phosphate salt, HPTS dye, Safranin O dye, Triton X-100, NaOH and all inorganic salts of molecular biology grade were purchased from Sigma. Gel-permeation chromatography was performed on a column of Sephadex LH-20 gel (25×300 mm, V = 25 mL). Large unilamellar 0 vesicles (LUV) were prepared from EYPC lipid by using mini extruder, equipped with a polycarbonate membrane either of 100 nm or 200 nm pore size, obtained from Avanti Polar Lipids. II. Physical Measurements The 1H and 13C NMR spectra were recorded on 400 MHz Jeol ECS-400 (or 100 MHz for 13C) spectrometers using either residual solvent signals as an internal reference or from internal tetramethylsilane on the δ scale relative to chloroform (δ 7.26), dimethylsulphoxide (δ 2.50 ppm), acetone (δ 2.05) for 1H NMR and chloroform (δ 77.20 ppm), dimethylsulphoxide (δ 39.50 ppm), acetone (δ 29.84 and 206.26) for 13C NMR. The chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz. The following abbreviations are used: s (singlet), d (doublet) m (multiplet), td (triplet of doublet) while describing 1H NMR signals. High-resolution mass spectra (HRMS) were obtained from MicroMass ESI-TOF MS spectrometer. Fluorescence spectra were recorded by using Fluoromax-4 from Jobin Yvon Edison equipped with an injector port and a magnetic stirrer. 10 mM HEPES (with 100 mM NaCl or other salts as per necessity) buffer solutions were used for fluorescence experiment and the pH of the buffers were adjusted to 7.0 or 8.0 by NaOH and pH of the buffer solutions were measured using Helmer pH meter. 5 mM sodium phosphate buffer was prepared by mixing proper amount of monobasic and dibasic sodium phosphate solution and further pH was adjusted with extra monobasic or dibasic sodium S2 phosphate solution with NaCl or NaNO (500 mM) as per requirement and pH of the buffer 3 solution was measured using Helmer pH meter for chloride ion efflux assays by Chloride Ion Selective Electrode (ISE). Chloride efflux was monitored by Accumet Chloride Combination Ion Selective Electrode furnished with Fisher AB250 pH/Ion meter, obtained from Fischer Scientific. (FT-IR) spectra were obtained using NICOLET 6700 FT-IR spectrophotometer as KBr disc and reported in cm-1. Melting point of all compounds were measured using a VEEGO Melting point apparatus. All melting points were measured in open glass capillary and values are uncorrected. All fluorescence data were processed either by Origin 8.5 or KaleidaGraph and Chloride ISE data were processed by Origin 8.5 and finally all data were processed through ChemDraw Professional 15. III. Synthesis: Synthesis of N, N'-(1, 2-phenylene)bis(4-methylbenzenesulfonamide) (1a):S1 Scheme S1. Synthesis of compound 1a. In a 50 mL round bottom flask, o-phenylenediamine 2 (500 mg, 4.62 mmol) was dissolved in 10 mL of dry CH Cl and followed by 0.5 mL of pyridine was added and reaction mixture was 2 2 cooled to 0 oC. Subsequently, tosyl chloride 3a (1.89 g, 9.25 mmol) dissolved in 5 mL dry CH Cl was added dropwise to the reaction mixture and was stirred for 30 min at 0 oC in stirring 2 2 condition. Then the temperature was brought to room temperature and the reaction mixture was stirred for 4 h and TLC was monitored till the completion of reaction. The reaction mixture was then concentrated to remove CH Cl and pyridine and column chromatography was performed 2 2 using ethyl acetate/pet ether solvent system to obtain 1.5 g of crystalline solid, white product 1a (78%). Eluent: 22% EtOAc/PE. mp: 207 0C; IR (KBr): v/cm-1: 3317, 3220, 1917, 1805, 1593, 1498, 1403, 1327, 1152, 1084; 1H NMR (400 MHz, CDCl ): δ 2.39 (s, 6H), 6.79 (s, 2H), 6.94 ‒ 3 6.98 (m, 2H), 7.02 ‒ 7.05 (m, 2H), 7.22 (d, J = 8.0 Hz, 4H), 7.55 (td, J = 1.6 Hz, 3.6 Hz, 4H); 13C NMR (100 MHz, CDCl ): δ 22.04, 126.51, 127.79, 128.01, 130.09, 131.31, 135.98, 144.66; 3 HRMS (ESI): Calculated for C H N O S [M+H]+: 417.0942; Found: 417.0941. 20 21 2 4 2 S3 Synthesis of N, N'-(1, 2-phenylene)bis(4-nitrobenzenesulfonamide) (1b): Scheme S2. Synthesis of compound 1b. In a 50 mL round bottom flask, o-phenylenediamine 2 (200 mg, 1.85 mmol) was dissolved in 10 mL of dry CH Cl followed 0.5 mL of pyridine was added and reaction mixture was cooled to 0 2 2 oC. 4-nitrobenzenesulfonyl chloride 3b (1.27 g, 3.88 mmol) dissolved in 5 mL of dry CH Cl 2 2 was added dropwisely to the reaction mixture and was stirred for 30 min at 0 oC. Temperature was brought to room temperature and the reaction was stirred for 4 h. Then the reaction TLC was checked to confirm for completion of the reaction. The solvent of reaction mixture was concentrated to remove CH Cl and pyridine and then the residue was subjected to column 2 2 chromatography using Ethyl acetate, Methanol and Pet ether. Eluent- EtOAc/MeOH/Pet ether (70:4:26). An off-white coloured solid product 1b (760 mg, 86%) was obtained. mp: 255 oC; IR (KBr): v/cm-1: 3282, 3104, 1933, 1811, 1681, 1605, 1536, 1403, 1349, 1163, 1087; 1H NMR (400 MHz, Acetone-d ): δ 7.07 ‒ 7.12 (m, 2H), 7.15 ‒ 7.19 (m, 2H), 7.95 (td, J = 2.4 Hz, 9.2 6 Hz, 4H), 8.36 (td, J = 2.4 Hz, 9.2 Hz, 4H), 8.73 (s, 2H); 13C NMR (100 MHz, Acetone-d ): δ 6 125.27, 127.23, 128.70, 129.73, 131.77, 145.31, 151.47; HRMS (ESI): Calculated for C H N O S [M+H]+: 479.0353; Found: 479.0331. 18 15 4 8 2 Synthesis of N, N'-(1, 2-phenylene)bis(4-bromobenzenesulfonamide) (1c): Scheme S3. Synthesis of compound 1c. In a 50 mL round bottom flask o-phenylenediamine 2 (200 mg, 1.85 mmol) was dissolved in 10 mL of dry CH Cl and followed by addition of 0.5 mL of pyridine and reaction mixture was 2 2 cooled to 0 oC. Then 4-bromobenzenesulfonyl chloride 3c (0.99 g, 3.88 mmol) dissolved in 5 mL S4 of dry CH Cl was added dropwisely to the reaction mixture and was stirred for 30 min at 0 oC 2 2 .Then the temperature was brought to room temperature and the reaction was stirred for 4 h with constant stirring. Then the reaction TLC was checked to confirm for completion of the reaction. The reaction mixture was then concentrated and then the residue was subjected to column chromatography using Methanol and Chloroform. Compound 1c was obtained as crystalline, white solid product (803.2 mg, 80%). Eluent: 5% MeOH/CHCl ; mp: 224 oC ; IR (KBr): v/cm- 3 1: 3216, 1915, 1570, 1483, 1400, 1334, 1276, 1157, 1076, 1006; 1H NMR (400 MHz, DMSO- d ): δ 6.96 (m, 2H), 7.04 (m, 2H), 7.61 (d, J = 8.4 Hz, 4H), 7.76 (d, J = 8.4 Hz, H, 4H), 9.43 (s, 6 2H); 13C NMR (100 MHz, DMSO-d ): δ 123.96, 126.35, 127.13, 128.79, 129.72, 132.36, 6 138.34; HRMS (ESI): Calculated for C H Br N O S [M+H]+: 544.8840, 546.8820; Found: 18 15 2 2 4 2 544.8845, 546.8822. Synthesis of N, N'-(1, 2-phenylene)bis(4-methoxybenzenesulfonamide) (1d): Scheme S4. Synthesis of compound 1d. In a 50 mL round bottom flask o-phenylenediamine 2 (200 mg, 1.85 mmol) of was dissolved in 10 mL of dry CH Cl , followed by 0.5 mL of pyridine was added and reaction mixture was 2 2 cooled to 0 oC. 4-methoxybenzenesulfonyl chloride 1d (0.76 g, 3.70 mmol) dissolved in 5 mL of dry CH Cl was added dropwisely to the reaction mixture and was stirred for 30 min at 0 oC. 2 2 Then the temperature was brought to room temperature and the reaction was stirred for 4 h and TLC was monitored in order to confirm the completion of reaction. The reaction mixture was then concentrated and then the residue was directly subjected to column chromatography using ethyl acetate and pet ether solvent mixture. Compound 1d (730 mg, 88%) was obtained as crystalline white solid. Eluent: EtOAc /PE (40%); mp: 155 oC; IR (KBr): v/cm-1: 3240, 1909, 1589, 1501, 1397, 1326, 1258, 1154, 1086, 1017; 1H NMR (400 MHz, CDCl ): δ 3.84 (s, 6H), 3 6.81 (s, 2H), 6.86 (td, J = 3.2 Hz, 8.8 Hz, 4H), 6.95 ‒ 6.98 (m, 2H), 7.02 ‒ 7.06 (m, 2H), 7.59 (td, J = 3.2 Hz, 8.8 Hz, 4H); 13C NMR (100 MHz, CDCl ): δ 55.77, 114.30, 126.14, 127.37, 129.86, 3 S5 130.06, 131.06, 163.47; HRMS (ESI): Calculated for C H N O S [M+H]+:449.5226; Found: 20 21 2 6 2 449.5204. Synthesis of N, N'-(1, 2-phenylene)bis(4-trifluoromethyl)benzenesulfonamide)(1e): Scheme S5. Synthesis of compound 1e. In a 25 mL round bottom flask o-phenylenediamine 2 (22.09 mg, 0.20 mmol) was dissolved in 4 mL of CH Cl followed by 0.5 mL of pyridine was added and reaction mixture was cooled to 0 2 2 oC. Then 4-trifluoromethylbenzenesulfonyl chloride 3e (98 mg, 0.40 mmol) dissolved in 3 mL CH Cl was added dropwisely to the reaction mixture and was stirred for 30 min at 0 oC. Then 2 2 the temperature was brought to room temperature and the reaction was stirred for 4 h. Then the reaction TLC was checked to confirm for completion of the reaction. The reaction mixture was then concentrated and then the residue was subjected to column chromatography using ethyl acetate and pet ether. Compound 1e (102 mg, 96%) was obtained as crystalline, white solid product. Eluent: 12% EtOAc in Pet ether mp: 178 oC; IR (KBr): v/cm-1: 3263, 2925, 2856, 1602, 1502, 1403, 1329, 1179, 1057, 1010; 1H NMR (400 MHz, CDCl ): δ 6.98 ‒ 6.96 (m, 2H), 3 7.10 ‒ 7.13 (m, 4H), 7.71 (d, J = 8.4 Hz, 4H), 7.82 (d, J = 8 Hz, 4H); 13C NMR (100 MHz, CDCl ): δ 121.83, 124.54, 126.35, 126.41, 128.25, 130.42, 134.97, 141.86; HRMS (ESI): 3 Calculated for C H F N O S [M+H]+: 525.0377; Found: 525.0368. 20 15 6 2 4 2 IV. Ion Transport Activity Study HEPES Buffer, HPTS and Stock Solution Preparation: Solid HEPES and NaCl were dissolved in autoclaved water to prepare HEPES buffer (10 mM) with NaCl (100 mM), followed by dropwise addition of NaOH solution to adjust pH = 7.0. Solid HPTS dye was dissolved in above 1 mL of aforementioned buffer solution to give 1 mM solution of 10 mM HEPES containing NaCl (100 mM) of pH = 7.0. Stock solutions of all bis(sulfonamide) derivatives were prepared by dissolving in DMSO of HPLC grade. S6 Preparation of EYPC-LUVsHPTS: EYPC-LUVsHPTS ( 5.0 mM EYPC, inside: 1 mM HPTS, 10 mM HEPES, 100 mM NaCl, pH = 7.0 and outside: 10 mM HEPES, 100 mM NaCl, pH = 7.0) were prepared following reported protocol. S2, S3 Fig. S1 Representation of ion transport activity assay using EYPC vesicles, assay detail and representation of ion transport experiment using fluorescence. Ion Transport Activity Assay Protocol: 1975 L of HEPES buffer containing 10 mM HEPES, 100 mM NaCl (pH = 7.0) was taken in a clean fluorescence cuvette followed by addition of 25 L of EYPC-LUVsHPTS in the same cuvette and was placed on a magnetic stirrer equipped with the fluorescence instrument (at t = 0 s). Fluorescence intensity of pH sensitive dye HPTS, F t was monitored at  = 510 nm ( = 450 nm). After that 20 L of 0.5 M NaOH was added to em ex create pH gradient between the intra and extra vesicular system into the same cuvette at t = 20 s (Fig. S1). bis(sulfonamide) derivatives were added at t = 100 s and finally at t = 300 s, 25 L of 10% Triton X-100 was added to lyze all vesicles which resulted in destruction of pH gradient (Fig. S1) and saturation in fluorescence emission intensity was observed. The time axis was normalized according to Equation S1: t = t  100 (S1) Now the time of bis(sulfonamide) compound addition can be normalized to t = 0 s and time of Triton-X 100 addition was normalized to t = 200 s. S7 Fluorescence time courses (F) were normalized to fractional emission intensity I using t F Equation S2. % Fl Intensity (I ) = [(F - F ) / (F - F )]  100 (S2) F t 0  0 Where F = Fluorescence intensity just before the bis(sulfonamide) compound addition 0 (at t = 0 s). F = Fluorescence intensity at saturation after complete leakage (at t = 330 s). F =  t Fluorescence intensity at time t. Concentration dependent experiments were carried out by increasing concentration of bis(sulfonamide) compounds (1a ‒ 1e). Change of HPTS fluorescence emission intensity was monitored with time and addition of bis(sulfonamide) compounds resulted in destruction of pH gradient via either Na+/OH‒ symport or Na+/H+ antiport mechanism (Fig. S2). The concentration profile data were further analyzed by Hill Equation to get the Effective concentration (EC ) and Hill Coefficient (n), (Equation S3). 50 Y = Y + (Y – Y ) / [1 + (c / EC )n] (S3)  0  50 Where, Y = Fluorescence intensity just before the bis(sulfonamide) compounds (1a ‒ 1e) 0 addition (at t = 0 s). Y = Fluorescence intensity with excess bis(sulfonamide) compounds (1a ‒  1e) concentration, c = Concentration of bis(sulfonamide) compounds (1a ‒ 1e) (Fig. S3). Fig. S2 Ion transport assay of bis(sulfonamide) derivatives 1a ─ 1d using EYPC LUVsHPTS. S8 Fig. S3 Representation of Hill plots for bis(sulfonamide) compounds (1a ‒ 1e). Determination of Ion Selectivity by HPTS assay: Buffer and Stock Solution Preparation: HEPES buffers with all salts were prepared by dissolving solid HEPES (10 mM) followed by addition of appropriate salt (100 mM) in autoclaved water and adjustment of pH (pH = 7.0) was done by dropwise addition of NaOH solution. Preparation of EYPC-LUVs⊃HPTS for Cation Selectivity: EYPC-LUVsHPTS ( 5.0 mM EYPC, inside: 1 mM HPTS, 10 mM HEPES, 100 mM NaCl, pH = 7.0 and outside: 10 mM HEPES, 100 mM NaCl, pH = 7.0) were prepared following reported protocol. S3- S5 Cation Selectivity Assay: In a clean fluorescence cuvette 1975 μL of different HEPES buffer solutions (10 mM HEPES, 100 mM MCl, pH = 7.0; where, M+ = Li+, Na+, K+, Rb+ and Cs+) S9 were taken followed by addition of 25 μL of EYPC-LUVs⊃HPTS vesicle in slowly stirring condition by a magnetic stirrer equipped with the fluorescence instrument (at t = 0 s). The time course of HPTS fluorescence intensity, F was monitored at λ = 510 nm (λ = 450 nm). At t = t em ex 20 s, 20 μL of 0.5 M NaOH was added to the cuvette to make the pH gradient between the intra and extra vesicular system. The bis(sulfonamide) compounds (1c and 1e) were added at t = 100 s and at t = 300 s, 10% Triton X-100 (25 μL) was added to lyze all vesicles for complete destruction of pH gradient. For data analysis and comparison, time (X-axis) was normalized between the point of transporter addition (i.e. t = 100 s was normalized to t = 0 s) and end point of experiment (i.e. t = 300 s was normalized to t = 200 s). Fluorescence intensities (F) were normalized to fractional emission t intensity I using Equation S2. F Preparation of EYPC-LUVs⊃HPTS for Anion Selectivity: EYPC-LUVsHPTS ( 5.0 mM EYPC, inside: 1 mM HPTS, 10 mM HEPES, 100 mM NaCl, pH = 7.0 and outside: 10 mM HEPES, 100 mM NaCl, pH = 7.0) were prepared following reported protocol. S3-S5 Anion Selectivity Assay: In a clean fluorescence cuvette 1975 μL of HEPES buffer (10 mM HEPES, 100 mM NaX, at pH = 7.0; where, X− = F−, Cl−, Br− and I−) was added followed by addition of 25 μL of EYPC-LUVs⊃HPTS vesicle in slowly stirring condition by a magnetic stirrer equipped with the fluorescence instrument (at t = 0 s). HPTS fluorescence emission intensity (F) was monitored with time at λ = 510 nm (λ = 450 nm). 20 μL of 0.5 M NaOH t em ex was added to the cuvette at t = 20 s to make the pH gradient between the intra and extra vesicular system. The bis(sulfonamide) compounds (1c and 1e) were added at t = 100 s and at t = 300 s, 25 μL of 10% Triton X-100 was added to lyze all vesicles for complete destruction of pH gradient. For data analysis and comparison, time (X-axis) was normalized between the point of transporter addition (i.e. t = 100 s was normalized to t = 0 s) and end point of experiment (i.e. t = 300 s was normalized to t = 200 s) using Equation S1. Fluorescence intensities (F) were normalized to t fractional emission intensity I using Equation S2. F S10