5.6 Chemical synthesis
General
1H and 13C NMR spectra were recorded in CDCl3-d and D2O using a Bruker AVANCE 400 Plus Nanobay instrument (400 and 101 MHz) and a Bruker AVANCE 500 instrument fitted with a cryoprobe (500 and 126 MHz), respectively. Chemical shifts (δ) are given in ppm and referenced to tetramethylsilane (0.00 ppm) or the internal solvent signal used as an internal standard. Assignments in the NMR spectra were made by first-order analysis of the spectra and were supported by 1H−1H COSY and 1H−13C HMQC correlation results. Matrix-assisted laser desorption ionization time-of-flight high-resolution mass spectrometry (MALDI-TOF HRMS) spectra were recorded on a Jeol JMS-S3000 using 2,5-dihydroxylbenzoic acid as the matrix. Unless otherwise stated, all commercially available solvents and reagents were purchased from FUJIFILM Wako Pure Chemical Corporation and Tokyo Chemical Industry Company Limited without further purification.
2-Bromoethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (2)
To a solution of compound 1 (3.00 g, 6.09 mmol) and 2-bromoethanol (743 µL, 10.4 mmol) in dichloromethane (28.4 mL) was added molecular seives 3Å powder (3.00 g), and cooled at −40°C. To the mixture was added boron trifluoride-ethyl ether complex (229 µL, 1.82 mmol) diluted in dichloromethane (2.1 mL) and stirred at −40°C for 30 min. The solution was diluted with dichloromethane and filtered through celite. The filtrate was washed with aqueous sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel chromatography with 8:1 to 4:1 (v/v) hexane-ethyl acetate to give 2 (1.84 g, 66%): 1H NMR (CDCl3, 400 MHz) δ 5.22 (t, 1 H, J2,3 =9.5 Hz, H-3), 5.09 (t, 1 H, J3,4 =9.5 Hz, H-4), 5.02 (dd, 1 H, Hz, H-2), 4.58 (d, 1 H, J1,2 = 8.0 Hz, H-1), 4.58 (dd, 1 H, J = 4.8 Hz,, J6a,6b = 12.3 Hz, H-6a), 4.20-4.13 (m, 2 H, H-6b, -OCH2-), 3.85-3.79 (m, 1 H, -OCH2-), 3.74-3.69(m, 1 H, H-5), 3.49-3.45 (m, 2 H, -CH2Br), 2.10 (s, 3 H, Ac), 2.08 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.02 (s, 3 H, Ac); 13C{1H} NMR δ (CDCl3, 101 MHz): 170.6, 170.2, 169.4, 169.4, 101.0, 72.6, 71.9, 71.0, 69.8, 68.3, 61.8, 29.8, 20.7, 20.6, 20.5.
6-Bromohexyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (3)
To a solution of compound 1 (3.00 g, 6.09 mmol) and 6-bromoethanol (2.49 mL, 10.3 mmol) in dichloromethane (28.4 mL) was added molecular sieves 4 Å powder (3.00 g) and cooled at −40°C. To the mixture was added boron trifluoride-ethyl ether complex (229 µL, 1.82 mmol) diluted in dichloromethane (2.1 mL) and stirred at −40°C for 30 min, at −20°C for 30 min and then at 0°C for 30 min. The solution was diluted with dichloromethane and filtered through celite. The filtrate was washed with aqueous sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel chromatography with 10:1 to 3:1 (v/v) hexane-ethyl acetate to give 3 (2.06 g, 66%): 1H NMR (CDCl3, 400 MHz) δ 5.21 (t, 1 H, J2,3 =9.5 Hz, H-3), 5.09 (t, 1 H, J3,4 =9.7 Hz, H-4), 4.99 (dd, 1 H, Hz, H-2), 4.49 (d, 1 H, J1,2 = 8.0 Hz, H-1), 4.27 (dd, 1 H, J = 4.7 Hz,, J6a,6b = 12.3 Hz, H-6a), 4.14 (dd, 1 H, J5,6a = 2.4 Hz, H-6b), 3.91-3.85 (m, 1 H, -OCH2-), 3.72-3.67 (m, 1 H, H-5), 3.51-3.46 (m, 1 H, -OCH2-), 3.41 (t, 2 H, J = 6.8 Hz, -CH2Br), 2.11 (s, 3 H, Ac), 2.05 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 1.89-1.82 (m, 2 H, -CH2-), 1.62-1.55 (m, 2 H, -CH2-), 1.46-1.32 (m, 4 H, -CH2-); 13C{1H} NMR (CDCl3,101 MHz) δ 170.7, 170.3, 169.4, 169.2, 100.8, 72.8, 71.7, 71.3, 69.9, 68.4, 62.0, 33.7, 32.6, 29.2, 27.7, 25.0, 20.7, 20.6, 20.6, 20.6.
2-S-Thioacetylethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (4)
To a solution of compound 2 (500 mg, 1.10 mmol) and thioacetic acid (117 µL, 1.64 mmol) in N,N-dimethylformamide (3.67 mL) was added potassium carbonate (228 mg, 1.65 mmol) and stirred at room temperature for 12 h. The solution was diluted with ethyl acetate, washed with aqueous sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel chromatography with 4:1 (v/v) hexane-ethyl acetate to give 4 (492 mg, 99%): 1H NMR (CDCl3, 400 MHz) δ 5.21 (t, 1 H, J2,3 =9.5 Hz, H-3), 5.09 (t, 1 H, J3,4 =9.7 Hz, H-4), 4.99 (dd, 1 H, Hz, H-2), 4.53 (d, 1 H, J1,2 = 8.0 Hz, H-1), 4.26 (dd, 1 H, J = 4.8 Hz,, J6a,6b = 12.3 Hz, H-6a), 4.14 (dd, 1 H, J = 2.4 Hz,, J6a,6b = 12.3 Hz, H-6b), 4.01-3.95 (m, 1 H, -OCH2-), 3.73-3.68 (m, 1 H, H-5), 3.65-3.59 (m, 1 H, H-5, -OCH2-), 3.26-3.01 (m, 2 H, -CH2S), 2.34 (s, 3 H, SAc), 2.09 (s, 3 H, Ac), 2.08 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.01 (s, 3 H, Ac); 13C{1H} NMR (CDCl3, 101 MHz) δ 195.2, 170.6, 170.2, 169.4, 169.3, 100.8, 72.7, 71.9, 71.1, 68.6, 68.3, 61.8, 30.5, 28.8, 20.7, 20.7, 20.6, 20.5.
6-S-Thioacetylhexyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (5)
To a solution of compound 3 (700 mg, 1.37 mmol) and thioacetic acid (146 µL, 1.79 mmol) in N,N-dimethylformamide (4.57 mL) was added potassium carbonate (284 mg, 2.05 mmol) and stirred at room temperature for 17.5 h. The solution was diluted with ethyl acetate, washed with water and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by silica gel chromatography with 8:1 to 4:1 (v/v) hexane-ethyl acetate to give 5 (561 mg, 83%): 1H NMR (CDCl3, 400 MHz) δ 5.20 (t, 1 H, J2,3 =9.5 Hz, H-3), 5.09 (t, 1 H, J3,4 =9.7 Hz, H-4), 4.98 (dd, 1 H, Hz, H-2), 4.49 (d, 1 H, J1,2 = 8.0 Hz, H-1), 4.27 (dd, 1 H, J = 4.7 Hz,, J6a,6b = 12.3 Hz, H-6a), 4.14 (dd, 1 H, J = 2.4 Hz,, J6a,6b = 12.3 Hz, H-6b), 3.90-3.84 (m, 1 H, -OCH2-), 3.71-3.67 (m, 1 H, H-5), 3.50-3.44 (m, 1 H, H-5, -OCH2-), 2.85 (t, 2 H, J = 7.3 Hz -CH2S), 2.33 (s, 3 H, SAc), 2.09 (s, 3 H, Ac), 2.04 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 1.59-1.52 (m, 4 H, -CH2-), 1.37-1.32 (m, 4 H, -CH2-); 13C{1H} NMR (CDCl3, 101 MHz) δ 196.0, 170.7, 170.3, 169.4, 169.3, 100.8, 72.9, 71.8, 71.3, 69.0, 68.5, 62.0, 30.6, 29.4, 29.2, 29.0, 28.4, 25.3, 20.8, 20.7, 20.6, 20.6.
2-S-Ethyl-β-D-glucopyranose S-linked maleimide (6) [34]
A solution of 4 (50.0 mg, 111 µmol) in methanol (950 µL) was added to 28% NaOMe-MeOH (50 µL) and then stirred at room temperature for 1 h. Diaion™ SK1B was added to the solution for quenching and filtration and evaporation. The residue was dissolved in methanol (2.22 mL), Et3N (22 µL) and maleimide (53.9 mg, 555 µmol) were added, and the mixture was stirred at room temperature for 1 h. The solution was concentrated, and the residue was purified by silica gel chromatography with 50:1 to 5:1 (v/v) dichloromethane-methanol to give 6 (30.3 mg, 81%): 1H NMR (D2O, 400 MHz) δ 4.30 (d, 1 H, J1,2 =8.0 Hz, H-1), 3.98-3.90 (m, 2 H, -CH2SCH-, -OCH2-), 3.75-3.72 (m, 2 H, H-6, -OCH2-), 3.53 (dd, 1 H, J5,6a= 6.0 Hz,, J6a,6b= 12.1 Hz, H-6), 3.31-3.25 (m, 2 H, H-3, H-5), 3.21-3.07 (m, 3 H, H-2, H-4, -SCHCH2-), 2.90-2.86 (m, 1 H, -CH2S-), 2.80-2.75 (m, 1 H, -CH2S-), 2.63-2.58 (m, 1 H, -SCHCH2-); 13C{1H} NMR (CDCl3, 126 MHz) δ 180.9, 179.7, 102.3, 75.8, 75.5, 72.9, 69.4, 69.0, 60.6, 41.5, 41.4, 37.2, 30.3; MALDI-TOF HRMS m/z: [M+Na]+: Calcd for C12H19N1O8S1+Na+: 360.0724; Found: 360.0676.
6-S-Hexyl-β-D-glucopyranose S-linked maleimide (7) [34]
A solution of 5 (50.0 mg, 101 µmol) in methanol (950 µL) was added to 28% NaOMe-MeOH (50 µL) and then stirred at room temperature for 2.5 h. Diaion™ SK1B was added to the solution for quenching, filtration, and evaporation. The residue was dissolved in methanol (2.02 mL), Et3N (20 µL) and maleimide (49.0 mg, 505 µmol) were added, and the mixture was stirred at room temperature for 1 h. The solution was concentrated, and the residue was purified by reverse-phase C18 silica gel chromatography with 1:0 to 4:1 (v/v) H2O-CH3CN to give 6 (27.5 mg, 72%): 1H NMR (D2O, 500 MHz) δ 4.32 (d, 1 H, J1,2 =8.0 Hz, H-1), 3.98-3.93-3.90 (m, 1 H, -CH2SCH-), 3.82-3.78 (m, 2 H, H-6a, -OCH2-), 3.61-3.52 (m, 2 H, H-6b, -OCH2-), 3.38-3.30 (m, 2 H, H-3, H-5), 3.27-3.10 (m, 3 H, H-2, H-4, -SCHCH2-), 2.67-2.57 (m, 3 H, -SCHCH2-, -CH2S-), 1.52-1.47 (m, 4 H, -CH2-), 1.32-1.24 (m, 4 H, -CH2-); 13C{1H} NMR (CDCl3, 126 MHz) δ 181.2, 179.9, 102.0, 75.8, 75.7, 73.0, 70.4, 69.5, 60.6, 41.4, 37.2, 30.0, 28.4, 28.1, 27.4, 24.4; MALDI-TOF HRMS m/z: [M+Na]+: Calcd for C16H27N1O8S1+Na+: 416.1350; Found: 415.1309.
The study is reported in accordance with ARRIVE guidelines. All mouse experiments were performed in accordance with the ethical guidelines for animal care of National Center for Geriatrics and Gerontology, and the experimental protocols were approved by the Animal Care Committee of National Center for Geriatrics and Gerontology [17,18, 28-32].