Synthese
2-(((((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)oxophosphoryl)oxy)-N,N,N-trimethylethanaminium was used as a raw material to synthesize 2-((((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)oxophosphoryl)oxy)-N,N,N-trimethylammonium ethyl. -amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)oxidizedphosphoryl)oxy)-N,N,N-trimethylethane-1-ammonium sodium salt (1:1) in the following general procedure: CMP (100 mg, 0.31 mmol) was prepared in DMF (2 mL) and tributylamine (150 μL , 0.62 mmol) in a free acid-stirred suspension was heated at 50 °C for 10 min and subsequently cooled to room temperature. Upon addition of CDI (250 mg, 1.55 mmol), the suspension changed to a clarified solution within minutes. The reaction mixture was stirred anhydrically at room temperature for 30 min, followed by treatment with anhydrous methanol (100 μL, 2.48 mmol) to hydrolyze the excess of CDI.After 30 min, anhydrous zinc chloride (59 mg, 0.43 mmol) was added, followed by choline phosphate solution (0.77 M anhydrous DMF solution, 2 mL, 1.55 mmol). The reaction mixture was stirred at room temperature for 24 hours. After evaporation of the solvent in vacuo, CDP-Cho 5 was purified by anion-exchange chromatography.The residue was dissolved in water and purified by ion-exchange column chromatography on a Sephadex DEAE A-25 resin using a linear gradient elution of 0-0.5 M ammonium bicarbonate, followed by chromatographic separation on an RP-18. Triethylammonium counterbalance ions were exchanged for sodium ions by passing the nucleotide solution through a Dowex 50WX8 column. Freeze-drying gave 1-(β-D-ribofuranosyl)cytosine-5'-diphosphorylcholine (sodium salt) as a white solid (115 mg, 74% yield). hplc: tR = 8.8 min (λmax 276 nm); 1H NMR (400 MHz, D2O) δ 7.86 (d, 1H, H6, J6-5 = 7.6 Hz), 6.04 ( d, 1H, H5, J5-6 = 7.6 Hz), 5.92 (d, 1H, H10, J10-20 = 4.0 Hz), 4.31 (sl, 2H, CH2-O), 4.28-4.08 (m, 5H, H2O H30H40H50 and H50b), 3.60 (t, 2H, CH2-N, J = 4.2 Hz), 3.14 ( s, 9H, CH3); 13C NMR (101 MHz, D2O) δ 166.2 (s, C4), 157.7 (s, C2), 141.4 (s, C6), 96.6 (s, C5), 89.3 (s, C10), 82.6 (d, C40, JC-P = 8.9 Hz), 74.1 (s, C20), 69.3 (s C30), 65.9 (s, CH2N), 64.7 (d, C50, JC-P = 5.1 Hz), 59.9 (s, CH2O), 53.9 (s, CH3); 31P NMR (162 MHz, D2O) δ 11.35 (d, 2Jab = 21.4 Hz), 12.14 (d, 2Jab = 21.4 Hz); HRMS (ESI) m/z: [M + H]+ C14H26N4O11NaP2 calculated value 511.0971, measured value 511.0973.
