Supporting Information Section:

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1 Supporting Information Section: Effects of Low to Intermediate Water Concentrations on Proton Coupled Electron Transfer (PCET) Reactions of Flavins in Aprotic Solvents and a Comparison with the PCET Reactions of Quinones Serena L. J. Tan, Maria L. Novianti and Richard D. Webster* Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore * webster@ntu.edu.sg; Telephone: ; Fax: S1

2 Contents Page S3 1. Synthesis Experiments 1.1. General Experimental (ethyl(m-tolyl)amino)-3-methylpyrimidine-2,4(1H,3H)-dione Page S (ethyl(m-tolyl)amino)pyrimidine-2,4(1H,3H)-dione Page S ethyl-3,6-dimethyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridine 5-oxide Page S ethyl-6-methyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridine 5-oxide Page S ethyl-3,6-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (Flavin 2) Page S ethyl-6-methylbenzo[g]pteridine-2,4(3H,10H)-dione (Flavin 1) Page S9 Figure S1. Variable scan rate cyclic voltammograms of 1 mm flavin 1 in acetonitrile with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C. Page S10 Figure S2. Variable scan rate cyclic voltammograms of 1 mm flavin 2 in acetonitrile with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C. Page S11 Figure S3. Cyclic voltammogram of approximately 1 mm flavin 1 in acetonitrile containing 3 mm water with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C at a scan rate of 0.1 V s -1. Page S12 Figure S4. Cyclic voltammograms of 1 mm flavin 1 in DMSO with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C, before reductive electrolysis (black line) and after oxidation back to starting material (red dashed line). Page S13 Figure S5. Cyclic voltammograms of 1 mm flavin 2 in DMSO with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C, before reductive electrolysis (black line) and after oxidation back to starting material (red dashed line). Page S14 Figure S6. Cyclic voltammograms of 1 mm riboflavin in DMSO with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C, before reductive electrolysis (black line) and after oxidation back to starting material (red dashed line). Page S15 Figure S7. Variable scan rate cyclic voltammograms of 1 mm riboflavin in DMSO with 0.2 M n-bu 4 NPF 6, from 0.1 V s 1 (black line) to 20 V s 1 (red line), after 2 e bulk reduction, recorded at a 1 mm Pt electrode at 22 (±2) o C. S2

3 1. Synthesis Experiments 1.1. General Experimental 1 H NMR (400 MHz) spectra were recorded on Bruker AVANCE 400 instrument in CDCl 3 and DMSO. Spectra were calibrated using the residual 1 H chemical shift in CDCl 3 (7.26 ppm) and DMSO (2.50 and 3.30 ppm), which were used as internal reference standards. 13 C NMR (100 MHz) spectra were recorded on Bruker AVANCE 400 instrument in CDCl 3 and DMSO. Spectra were calibrated using CDCl 3 (77.0 ppm) and DMSO (39.5 ppm) for 13 C NMR spectra. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = double of doublets, dt = double triplet, m = multiplet. IR spectra were recorded on a Shimazu IR Prestige-21 FT-IR Spectrometer. Highresolution mass spectra were obtained with a JEOL MS-700P mass spectrometer and a Finnigan MAT 95 XP mass spectrometer (Thermo Electron Corporation) and Q-Tof Premier (ethyl(m-tolyl)amino)-3-methylpyrimidine-2,4(1H,3H)-dione To a round-bottom flask containing a large magnetic stirrer bar, was added 6-chloro-3-methyluracil (4.80 g, 30.0 mmol) and N-ethyl-m-toluidine (8.10 g, 60.0 mmol). The solvent-free reaction mixture was heated under N 2 for 24 h at 175 C, and subsequently cooled to room temperature, forming a thick gum. A mixture of ethanol/ hexane (50 ml) was added to the reaction flask, and stirred till the gum has dissolved. The white precipitate formed was filtered and washed with hexane. The crude product was purified by recrystallization (EtOH/EA) to give the product (5.19 g, 67%) as white solid. White solid; IR (neat): 3395, 3017, 2399, 2361, 1697, 1628, 1497, 1450, 1381, 1350, 1219, 1034, 926 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 1.22 (2H, t, J = 7.1 Hz), 1.71 (1H, s), 2.39 (3H, s), 3.24 (3H, s), 3.62 (2H, q, J = 7.1 Hz), 5.00 (1H, d, J = 2.7 Hz), (2H, m), (1H, m), (2H, m); 13 C NMR (100 MHz, CDCl 3 ) δ 12.1, 21.31, 26.7, 47.0, 76.4, 125.2, 128.8, 129.9, 130.5, 139.4, 141.1, 150.4, 151.2, 164.3; HRMS ESI (m/z): found, , calcd for C 14 H 18 N 3 O 2 : [M+H] +, ; m.p C. S3

4 (ethyl(m-tolyl)amino)pyrimidine-2,4(1H,3H)-dione To a round-bottom flask containing a large magnetic stirrer bar, was added 6-chlorouracil (3.38 g, 23.1 mmol) and N-ethyl-m-toluidine (6.25 g, 46.2 mmol). The solvent-free reaction mixture was heated under N 2 for 24 h at 175 C, and subsequently cooled to room temperature, forming a thick gum. A mixture of ethanol/ hexane (50 ml) was added to the reaction flask, and stirred until the gum has dissolved. The white precipitate formed was filtered and washed with hexane/ethanol. The crude product was purified by recrystallization (EA/EtOH) to give the product (3.72 g, 66%) as white solid. White solid; IR (neat) 3132, 2361, 1670, 1628, 1574, 1281, 1227, 903, 779, 710 cm -1 ; 1 H NMR (400 MHz, DMSO) δ 1.06 (3H, t, J = 7.0 Hz), 2.33 (3H, s), 3.68 (2H, q, J = 6.9 Hz), 4.15 (1H, d, J = 1.2 Hz), (2H, m), (1H, m), (1H, m), (1H, s), (1H, s); 13 C NMR (100 MHz, DMSO) δ 13.3, 21.3, 46.2, 78.1, 125.1, 128.5, 128.6, 130.1, 139.9, 142.5, 151.8, 154.7, 164.2; HRMS ESI (m/z): found, , calcd for C 13 H 16 N 3 O 2 : [M+H] +, ; m.p C. S4

5 ethyl-3,6-dimethyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridine 5-oxide To a round-bottom flask containing acetic acid (80 ml), was added 6-(ethyl(m-tolyl)amino)-3- methylpyrimidine-2,4(1h,3h)-dione (7.07 g, 27.3 mmol) and stirred till completely dissolved. Sodium nitrite (7.53 g, mmol) was added portion wise, and the resulting mixture was stirred at room temperature for 2 h. The reaction mixture was then filtered and the residue was washed with cold ethanol. The crude product was purified by recrystallization (EtOH) to give the product (7.76 g, 100%) as yellow solid. Yellow solid; IR (neat) 2361, 1690, 1643, 1535, 1273, 1242, 1188, 1150, 1042, 972, 826, 725 cm -1 ; 1 H NMR (400 MHz, DMSO) δ 1.32 (3H, t, J = 7.0 Hz), 2.56 (3H, s), 3.2 (3H, s), 4.61 (2H, q, J = 6.9 Hz), 7.43 (1H, d, J = 8.4 Hz), 7.87 (1H, s), 8.24 (1H, d, J = 8.4 Hz); 13 C NMR (100 MHz, DMSO) δ 12.6, 22.1, 28.0, 117.0, 121.1, 125.0, 127.7, 133.3, 134.0, 147.5, 151.6, 154.8, 156.6; HRMS ESI (m/z): found, , calcd for C 14 H 15 N 4 O 3 : [M+H] +, ; m.p C. S5

6 ethyl-6-methyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridine 5-oxide To a round-bottom flask containing acetic acid (80 ml), was added 6-(ethyl(m-tolyl)amino)pyrimidine- 2,4(1H,3H)-dione (5.92 g, 24.1 mmol) and stirred till completely dissolved. Sodium nitrite (6.66 g, 96.6 mmol) was added portion wise, and the resulting mixture was stirred at room temperature for 2 h. The reaction mixture was then filtered and the residue was washed with cold ethanol. The crude product was purified by recrystallization (EtOH) to give the product (6.60 g, 100%) as yellow solid. Yellow solid; IR (neat) 3441, 2723, 2361, 1689, 1597, 1543, 1281, 1242, 1188, 1096, 887, 818, 779, 725 cm -1 ; 1 H NMR (400 MHz, DMSO) δ 1.32 (3H, t, J = 7.0 Hz), 1.90 (1H, s), 2.56 (2H, s), 4.58 (2H, q, J = 6.8 Hz), 7.42 (1H, d, J = 8.8 Hz), 7.83 (2H, s), 8.21 (1H, d, J = 8.8 Hz); 13 C NMR (100 MHz, DMSO) δ; HRMS ESI (m/z): found, , calcd for C 13 H 13 N 4 O 3 : [M+H] +, ; m.p C. S6

7 ethyl-3,6-dimethylbenzo[g]pteridine-2,4(3H,10H)-dione (Flavin 2) To a round-bottom flask containing H 2 O/EtOH (60 ml) in a 1:1 mixture, was added 10-ethyl-3,6- dimethyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridine 5-oxide (2.0 g, 7.0 mmol), and cooled in an ice bath. Sodium dithionite (2.88 g, 14.0 mmol) was added to the solution and stirred in open atmosphere until the dirty orange solution turns bright yellow (~12 h). The bright yellow solids were filtered and washed with cold ethanol. The crude product was purified by recrystallization (CH 2 Cl 2 / EtOH 1:1) to give the product (1.90 g, 100%) as bright yellow solid. Yellow solid; IR (neat) 3425, 3017, 2399, 2361, 2330, 2099, 1651, 1558, 1420, 1273, 1211, 1042, 926 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 1.51 (3H, t, J = 7.2 Hz), 2.66 (3H, s), 3.53 (3H, s), 4.78 (2H, q, J = 7.1 Hz), 7.44 (1H, d, J = 2.8 Hz), 8.22 (1H, d, J = 8.4 Hz); 13 C NMR (100 MHz, CDCl 3 ) δ 12.3, 23.0, 28.8, 40.1, 114.6, 128.3, 132.4, 133.2, 134.6, 135.8, 148.0, 148.6, 156.1, 160.0; HRMS ESI (m/z): found, , calcd for C 14 H 15 N 4 O 2 : [M+H] +, ; m.p C. S7

8 ethyl-6-methylbenzo[g]pteridine-2,4(3H,10H)-dione (Flavin 1) To a round-bottom flask containing H 2 O/EtOH (60 ml) in a 1:1 mixture, was added 10-ethyl-6-methyl- 2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridine 5-oxide (2.0 g, 7.4 mmol), and cooled using an ice bath. Sodium dithionite (3.04 g, 14.8 mmol) was added to the solution and stirred in open atmosphere for 2 days. The dark green solution was extracted with dichloromethane (20 x 100 ml). The organic layer was dried with MgSO 4 and evaporated. The crude mixture was purified by recrystallization (EtOH/ DCM) to give the product (1.20 g, 40%) as yellow solid. Yellow solid; IR (neat) 3426, 2723, 2677, 2361, 1713, 1659, 1543, 1296, 1258, 1211, 1180, 1126, 1089, 1026, 972, 894, 825, 771, 725 cm -1 ; 1 H NMR (400 MHz, DMSO) δ 1.33 (3H, t, J = 7.0 Hz), 2.60 (3H, s), 4.63 (2H, q, J = 7.1 Hz), 7.49 (1H, d, J = 8.4 Hz), 7.83 (1H, s), 8.03 (1H, d, J = 8.4 Hz), (1H, s); 13 C NMR (100 MHz, DMSO) δ 12.3, 23.0, 28.8, 40.1, 114.6, 128.3, 132.4, 133.2, 134.6, 135.8, 148.0, 148.6, 156.1, 160.0; HRMS ESI (m/z): found, , calcd for C 13 H 13 N 4 O 2 : [M+H] +, ; m.p C. S8

9 0.1 V s V s V s -1 1 V s -1 2 V s -1 5 V s V s V s -1 Figure S1. Variable scan rate cyclic voltammograms of 1 mm flavin 1 in acetonitrile with 0.2 M n- Bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C. The current data were scaled by multiplying by 0.5. S9

10 0.1 V s V s V s -1 1 V s -1 2 V s -1 5 V s V s V s -1 Figure S2. Variable scan rate cyclic voltammograms of 1 mm flavin 2 in acetonitrile with 0.2 M n- Bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C. The current data were scaled by multiplying by 0.5. S10

11 Figure S3. Cyclic voltammogram of approximately 1 mm flavin 1 in acetonitrile containing 3 mm water with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C at a scan rate of 0.1 V s -1. S11

12 Figure S4. Cyclic voltammograms of 1 mm flavin 1 in DMSO with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C, before reductive electrolysis (black line) and after oxidation back to starting material (red dashed line). S12

13 Figure S5. Cyclic voltammograms of 1 mm flavin 2 in DMSO with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C, before reductive electrolysis (black line) and after oxidation back to starting material (red dashed line). S13

14 Figure S6. Cyclic voltammograms of 1 mm riboflavin in DMSO with 0.2 M n-bu 4 NPF 6, recorded at a 1 mm Pt electrode at 22 (±2) o C, before reductive electrolysis (black line) and after oxidation back to starting material (red dashed line). S14

15 Figure S7. Variable scan rate cyclic voltammograms of 1 mm riboflavin in DMSO with 0.2 M n- Bu 4 NPF 6, from 0.1 V s 1 (black line) to 20 V s 1 (red line), after 2 e bulk reduction, recorded at a 1 mm Pt electrode at 22 (±2) o C. S15