Thiol-Activated gem-dithiols: A New Class of Controllable. Hydrogen Sulfide (H 2 S) Donors

Transcription

1 Thiol-Activated gem-dithiols: A New Class of Controllable Hydrogen Sulfide (H 2 S) Donors Yu Zhao, Jianming Kang, Chung-Min Park, Powell E. Bagdon, Bo Peng, and Ming Xian * Department of Chemistry, Washington State University, Pullman, Washington, 99164, United States S1

2 Materials and Methods: All solvents were reagent grade. Tetrahydrofuran (THF) was freshly distilled from sodium/benzophenone under argon. Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with 0.25 mm pre-coated silica gel plates. Flash chromatography was performed with silica gel 60 (particle size mm). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Proton and carbon-13 NMR spectra were recorded on a 300 MHz spectrometer. Chemical shifts are reported relative to chloroform (δ 7.26) for 1 H NMR and chloroform (δ 77.0) for 13 C NMR. Experimental Procedures and Compound Characterization Data Preparation of compound TAGDD-1 Intermediate a: Benzaldehyde (1.53 ml, 15 mmol) was dissolved in 40 ml of dichloromethane (CH 2 Cl 2 ). To this solution was added boron tribromide (20 ml of 1M CH 2 Cl 2 solution, 20 mmol). The reaction was stirred at room temperature for 1 h and then concentrated under vacuum. 3.2 g of intermediate a was obtained as yellow oil by flash chromatography. The characterization of compound a matches the reported data. 1 1 H NMR (300 MHz, CDCl 3 ) δ 7.58 (d, J = 9.0 Hz, 2H), 7.36 (m, 3H), 6.66 (s, 1H); yield: 86%. TAGDD-1: a (1.24 g, 5 mmol) was dissolved in 30 ml of DMF. To this mixture was 1 An, J.; Tang, X.; Moore, J.; Lewis, W.; Denton, R.M. Tetrahedron., 2013, 69, S2

3 added potassium thioacetate (1.71 g, 15 mmol). The resultant solution was stirred at room temperature for 1 h and then diluted with water and extracted with CH 2 Cl 2 three times. The combined organic layers were dried over anhydrous MgSO 4, filtered and concentrated. The final product TAGDD-1 was purified by flash chromatography to afford the product as light yellow solid. TAGDD-1 is a known compound. 2 m.p o C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.36 (d, J = 9.0 Hz, 2H), 7.22 (m, 3H), 6.17 (s, 1H), 2.24 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 192.8, 139.6, 128.9, 128.4, 128.0, 48.3, 30.3; FTIR (cm -1 ) 2940, 1686, 1355, 1133, 1095, 1072, 963, 690; ESI/MS m/z [M+Na] + ; calcd for C 11 H 12 NaO 2 S ; overall yield: 74% (2 steps). TAGDD-2 was obtained as white solid from 4-methylbenzaldehyde using the same procedure. m.p o C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.34 (d, J = 9.0 Hz, 2H), 7.13 (d, J = 9.0 Hz, 2H), 6.24 (s, 1H), 2.32 (s, 3H), 2.30 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 192.9, 138.3, 136.7, 129.6, 127.9, 48.2, 30.3, 21.4; FTIR (cm -1 ) 2939, 1690, 1509, 1428, 1356, 1132, 1112, 957; HRMS m/z [M+Na] + ; calcd for C 12 H 14 NaO 2 S ; overall yield: 75% (2 steps). 2 Field, L.; Hoelzel, C.B. J. Org. Chem., 1958, 23, S3

4 TAGDD-3 was obtained as white solid from 4-bromobenzaldehyde using the same procedure. m.p o C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.43 (d, J = 9.0 Hz, 2H), 7.29 (t, J = 9.0 Hz, 2H), 6.16 (s, 1H), 2.31 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 192.7, 138.8, 132.0, 129.8, 122.4, 47.6, 30.3; FTIR (cm -1 ) 2922, 1708, 1686, 1484, 1402, 1351, 1122, 1092, 1011, 951, 840; HRMS m/z [M+Na] + ; calcd for C 11 H 11 BrNaO 2 S ; overall yield: 63% (2 steps). TAGDD-4 was obtained as white solid from 4-chlorobenzaldehyde using the same procedure. m.p o C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.31 (d, J = 9.0 Hz, 2H), 7.21 (d, J = 9.0 Hz, 2H), 6.11 (s, 1H), 2.25 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 192.7, 138.2, 134.2, 129.4, 129.0, 47.5, 30.3; FTIR (cm -1 ) 2938, 1694, 1489, 1356, 1131, 1090, 1012, 956, 843; HRMS m/z [M+Na] + ; calcd for C 11 H 11 ClNaO 2 S ; overall yield: 73% (2 steps). TAGDD-5 was obtained as yellow liquid from 4-trifluoromethylbenzaldehyde using the same procedure. 1 H NMR (300 MHz, CDCl 3 ) δ 7.56 (s, 4H), 6.24 (s, 1H), 2.33 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 192.5, 143.7, 128.5, 125.9, 125.8, 47.5, 30.3; FTIR (cm -1 ) 1701, 1322, 1107, 1066, 1017, 951, 854; HRMS m/z [M+Na] + ; calcd for S4

5 C 12 H 11 F 3 NaO 2 S ; overall yield: 59% (2 steps). TAGDD-6 was obtained as white solid from 3-hydroxybenzaldehyde using the same procedure. m.p o C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.17 (t, J = 9.0 Hz, 1H), 6.96 (d, J = 9.0 Hz, 2H), 6.75 (m, 1H), 6.27 (br, 1H), 6.20 (s, 1H), 2.31 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 193.8, 156.2, 140.8, 130.3, 120.1, 115.8, 115.0, 48.2, 30.3; FTIR (cm -1 ) 3346, 2932, 1703, 1658, 1598, 1453, 1284, 1123, 958, 874; HRMS m/z [M+Na] + ; calcd for C 11 H 12 NaO 3 S ; overall yield: 51% (2 steps). TAGDD-7 was obtained as yellow liquid from 3-methylbenzaldehyde using the same procedure. 1 H NMR (300 MHz, CDCl 3 ) δ 7.22 (m, 3H), δ 7.07 (d, J = 6.0 Hz, 1H), 6.22 (s, 1H), 2.33 (s, 3H), 2.31 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 192.8, 139.4, 138.7, 129.3, 128.8, 128.6, 125.0, 48.3, 30.3, 21.6; FTIR (cm -1 ) 2949, 1687, 1352, 1130, 1093, 945; HRMS m/z [M+Na] + ; calcd for C 12 H 14 NaO 2 S ; overall yield: 75% (2 steps). S5

6 TAGDD-8 was obtained as white solid from 3-nitrobenzaldehyde using the same procedure. m.p o C; 1 H NMR (300 MHz, CDCl 3 ) δ 8.31 (s, 1H), 8.12 (d, J = 6.0 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.50 (t, J = 9.0 Hz, 1H), 6.26 (s, 1H), 2.35 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 192.4, 142.0, 134.3, 129.8, 123.3, 123.2, 47.2, 30.3; FTIR (cm - 1 ) 3073, 2952, 1683, 1533, 1347, 1102, 1084, 971; HRMS m/z [M+Na] + ; calcd for C 11 H 11 NNaO 4 S ; overall yield: 51% (2 steps). H 2 S release from TAGDD-1 in the presence of thiols in PBS buffer H 2 S generation from TAGDD-1 was initiated by adding 100 μl of donor stock solution (30 mm in THF) into 30 ml of PBS (ph 7.4, 50 mm)/thf (9:1) solution containing cysteine or GSH (500 µm). Then 1.0 ml of reaction aliquots were periodically taken and transferred to UV cuvettes containing MB cocktail (100 μl of zinc acetate (1% w/v), 200 μl of N, N-dimethyl-1,4-phenylenediamine sulfate (20 mm in 7.2 M HCl) and 200 μl of ferric chloride (30 mm in 1.2 M HCl)). The MB reaction was carried out for 15 min and the absorbance (670 nm) of the resultant solution was determined using an UV Vis spectrometer (Thermo Evolution 300). H 2 S concentration of each sample was calculated against a calibration curve of Na 2 S, which was obtained by plotting H 2 S concentration versus time. H 2 S release from all other donors was measured using this method. S6

7 Figure S1. H 2 S release from TAGDD-1 in the presence of homocysteine Product analysiss 0.25 ml of TAGDD-1 in THF (30 mm) was added to 50 ml of PBS buffer (ph 7.4, 50 mm)/ /THF (9:1, v:v) containing L-cysteine (750 µ µm). After 30 min, 1. 5 ml of the solution was taken to a 1.8 ml-autosampler vial (Thermo Scientific) via syringe and analyzed by HPLC. Compounds 7 and 2-phenylthiazolidine-4-carboxylic acid (b) were identified and quantified in comparison to standard compounds (Figure S1). HPLC gradient: 61% solvent A (0.05% TFA in water) in solvent B (0. 05% TFA in MeCN) over 10 min then 61% to 30% solvent A in solvent B over 10 min. Flow rate: 1.2 ml/min, 5-10 µl injection. Column: Thermo RP C18, mm, 5 µm (particle). SAc SAc TAGDD M + SH OH H 2 N O 750 M PBS/THF (9:1) H 2 S Standard sample injection: S7

8 TAGDD-1, t R = 14.7 min 20 PDA -254nm YZ-mech_SM4_8 20 mau mau Minutes Benzaldehyde (7), t R = 6.5 min 300 PDA -254nm YZ-mech_PheCHO_wo_Cys_ mau mau Minutes 2-phenylthiazolidine-4-carboxylic acid, t R = 2.2 min 100 PDA -254nm Mech_acid_6 S O 100 N H H O mau 50 b 50 mau Minutes Reaction solution injection: TAGDD-1 + Cysteine H 2 S 75 PDA -254nm YZ-mech_rxn-with_cys_1 75 mau No TAGDD mau Minutes Figure S2. HPLC analysis of H 2 S release from TAGDD-1 S8

9 Cell culture HeLa cells were cultured in Dulbecco s modified Eagle s medium (DMEM) high glucose medium supplemented with 10% fetal bovine serum (FBS) at 37 under a condition of 5% CO 2 atmosphere. Cell viability assay The cell counting kit (CCK)-8 (Dojindo Laboratory, Kumamoto, Japan) was applied to measure the cell viability of HeLa cells cultured in 96-well plates. After the indicated cell treatment, 100 μl of CCK-8 solution at 1:10 dilution was added to each well and cells were incubated for a further 3 h at 37. Absorbance was measured at 450 nm with a microplate reader (Tecan Infinite F200, Switzerland). The mean optical density (OD) of 4 wells in each group was used to calculate cell viability as follows: (experiments were performed in triplicate) Cytotoxicity of TAGDDs % Cell viability 100 HeLa cells were incubated with TAGDDs at varied concentrations (5-25 µm) for 1 h and then washed by PBS. CCK-8 assay was applied to measure cell viability as described above. S9

10 Figure S3. Effects of representative TAGDDs on cell viability.. HeLa cells were treated with different concentrations of TAGDDs (5 25 μm) for 1 h. The cell counter kit (CCK)-8 assay was performed to measure cell viability. Data were shown as the mean ± SD (n = 4). H 2 S release in HeLa cells HeLa cells were inoculated in a 24-well plate and cultured overnight. The cells were co- incubated with 25 µm of TAGDD-1 at 37 C for 300 min and washed by PBS to remove extracellular TAGDD-1. Cells were then co-incubated with WSP-4 (50 μm) in PBS (containing 100 µm of CTAB) at 37 C for 30 min. Cell imaging was carried out after washing the cells three times with PBS (ph 7.4). All of the microscopy images were taken on a fluorescence microscope with excitation at 490 nm (green channel). S10

11 Figure S4. Fluorescence spectra of: a) 25 μm TAGDD1 only; b) 10 μm WSP4 only; c) 10 μmm WSP μm TAGDD1; d) 10 μm WSP μm TAGDD μm Na 2 S. The reactions were carried out for 5 min at room temperature in PBS buffer (10 mm, ph 7.4) with 1 mm CTAB. Data were acquired with excitation at 521 nm. S11

12 1 H NMR (300 MHz, CDCl 3 ) spectrum of TAGDD C NMR (75 MHz, CDCl 3 ) spectrum of TAGDD-1 S12

13 1 H NMR (300 MHz, CDCl 3 ) spectrum of TAGDD C NMR (75 MHz, CDCl 3 ) spectrum of TAGDD-2 S13

14 1 H NMR (300 MHz, CDCl 3 ) spectrum of TAGDD C NMR (75 MHz, CDCl 3 ) spectrum of TAGDD-3 S14

15 1 H NMR (300 MHz, CDCl 3 ) spectrum of TAGDD C NMR (75 MHz, CDCl 3 ) spectrum of TAGDD-4 S15

16 1 H NMR (300 MHz, CDCl 3 ) spectrum of TAGDD C NMR (75 MHz, CDCl 3 ) spectrum of TAGDD-5 S16

17 1 H NMR (300 MHz, CDCl 3 ) spectrum of TAGDD C NMR (75 MHz, CDCl 3 ) spectrum of TAGDD-6 S17

18 1 H NMR (300 MHz, CDCl 3 ) spectrum of TAGDD C NMR (75 MHz, CDCl 3 ) spectrum of TAGDD-7 S18

19 1 H NMR (300 MHz, CDCl 3 ) spectrum of TAGDD C NMR (75 MHz, CDCl 3 ) spectrum of TAGDD-8 S19