33% |
Stage #1: 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one; 7-(2,2'-bithien-5-yl)-3-(2-deoxy-3-O-acetyl-beta-D-ribofuranosyl)imidazo[4,5-b]pyridine; bis(tri-n-butylammonium) pyrophosphate In 1,4-dioxane; pyridine for 0.166667h;
Stage #2: With tributyl-amine; bis(tri-n-butylammonium) pyrophosphate In N,N-dimethyl-formamide at 20℃; for 0.166667h;
Stage #3: With ammonia; iodine; sodium hydrogensulfite In 1,4-dioxane; pyridine; water; N,N-dimethyl-formamide for 4.75h; |
5.2
7-(2,2'-Bithien-5-yl)-3-(2-deoxy-3-O-acetyl-β-D-ribofuranosyl)imidazo[4,5-b]pyridine (44 mg, 0.1 mmol) was azeotropically dried with pyridine and was then dissolved in pyridine (100 µL) and dioxane (100 µL). A solution of 1 M 2-chloro-1,3,2-benzodioxaphosphorin-4-one in dioxane (110 µL, 0.11 mmol) was added thereto, followed by stirring for 10 min. To this solution, a solution of tributylamine (100 µL) and a solution of bis(tributylammonium)pyrophosphate in 0.5 M DMF (300 µL, 0.15 mmol) was added, followed by stirring at room temperature for 10 min. A solution of 1% iodine in pyridine/water (98:2, v/v, 2.0 mL) was added thereto, followed by stirring for 15 min, and an aqueous 5% sodium hydrogen sulfite solution (150 µL) was added thereto. Water (5.0 mL) was further added thereto, followed by stirring for 30 min. Then, 28% aqueous ammonia (20 mL) was added thereto, followed by stirring at room temperature for 4 hr. The reaction solution was concentrated under reduced pressure, and 7-(2,2'-bithien-5-yl)-3-(2-deoxy-β-D-ribofuranosyl)imidazo[4,5-b]pyridine 5'-triphosphate (33 µmol, 33%) was obtained through purification by DEAE Sephadex (A-25) column chromatography (elution with a solution of 50 mM to 1.0 M TEAB) and C18-HPLC (elution with 0% to 50% acetonitrile in 100 mM TEAA). 1H NMR (300 MHz, D2O) δ 8.49 (s, 1H), 8.08 (d, 1H, J = 5.4 Hz), 7.58 (d, 1H, J = 4.0 Hz), 7.33-7.30 (m, 2H), 7.06 (dd, 1H, J = 1.1,4.7 Hz), 6.99 (dd, 1H, J = 3.7, 5.1 Hz), 6.91 (d, 1H, J = 3.9 Hz), 6.29 (t, 1H, J = 6.9 Hz), 4.68 (m, 1H, overlapping with D2O), 4.18 (m, 1H), 4.10-4.02 (m, 2H), 3.05 (q, 22H, J = 7.3 Hz), 2.68 (m, 1H), 2.41 (m, 1H), 1.14 (t, 34H, J = 7.3 Hz). 31P NMR (121 MHz, D2O) δ-9.71 (d, 1P, J = 19.8 Hz), -10.72 (d, 1P, J = 19.8 Hz), - 22.54 (t, 1P, J = 20.0 Hz). UV-vis spectral data in 10 mM sodium phosphate buffer (pH 7.0): λmas = 264 nm (ε 9900), 368 nm (ε 31400). ESI-MS (C19H20N3O12S2P3); calculated value: 637.96 (M-H)-, observed value: 637.87 (M-H)-. |