46% |
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5-Fluoro-deoxycytidine (60 mg, 0.245 mmol) was dried under reduce pressure at 80 C for 2 h. After cooling down, F5dC was suspended in trimethyl phosphate (0.6 ml) in argon purged vial and POCl3 (27 muL, 0.294 mmol) was added. The mixture was then stirred at 0 C for 2.5 h, an ice-cooled solution of (NHBu3)2H2P2O7 (600 mg, 1.09 mmol) and Bu3N (0.22 ml, 0.92 mmol) in dry DMF (2.2 ml) was added and the mixture was stirred at0 C for another 1 h. Then the reaction was quenched by addition of 2 M aqueous TEAB (0.5 ml) and the solvents were evaporated in vacuo and the residue was co-distilled with water several times. The product was purified by using semi-preparative reversed-phase HPLC (C18) with TEAB in H2O (0.1 M) to TEAB in H2O/MeOH (1:1, 0.1 M) linear gradient as eluent. Several co-distillations with water and conversion into the sodium salt form (Dowex 50WX8 in Na+ cycle) followed by freeze-drying from water gave white powder of F5dCTP (54.7 mg, 46%). F5dCTP: 1H NMR (400 MHz, D2O, ref(dioxane) = 3.75 ppm) delta 8.05 (d, J = 6.4 Hz, 1H, H-6), 6.26 (td, J = 6.6, 1.8 Hz, 1H, H-1'), 4.61 (dt, J = 6.6, 3.5 Hz, 1H, H-3'), 4.20 (t, J = 4.5 Hz, 2H, H-5'), 2.42 (ddd, J = 14.1, 6.2, 3.9 Hz, 1H, H-4'), 2.29 (dt, J = 13.7, 6.6 Hz, 2H, H-2'). 31P NMR (162 MHz, D2O) delta -10.52 (d, J = 19.7 Hz), -11.43 (d, J = 20.0 Hz), -23.02 (t, J = 19.7 Hz). MS (ESI-): m/z (%): 404.0 (100) [M-PO3+2H]- 506.0 (35) [M+Na+2H]-; HR/MS: calcd (m/z) for C9H12O14N2FNaP3 506.93886; found 506.93908 [M+Na+2H]-. |