반응 #164143
ord-378d117f99294673a959d188d10309fd
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후처리
- 1기타evaporating solvent under reduced pressure and purification by preparative reverse phase HPLC
실험 절차
To a solution of 5 (30 mg, 0.0738 mmol) in P(O)(OCH3)3 (0.5 mL) was treated with POCl3 (70 μL, 0.73 mmol) at 0° C., followed by stirring at r.t for 2-3 h and then evaporating solvent under reduced pressure and purification by preparative reverse phase HPLC to afford the corresponding mono phosphate 6. 31P NMR (CD3OD, 121.4 MHz) δ 0.79 (s); 19F NMR (CDCl3), 282.2 MHz) δ −77.37 (s, CF3). To a dried solution of 6 in DMF (1 mL) were added 1,1′-carbonyldiimidazole (CDI, 33 mg, 0.21 mmol), followed by stirring at r.t. for 5 h and then quenching with MeOH (8 μL, 0.21 mmol). A solution of tetrabutylammonium pyrophosphate (TBAPP, 143.0 mg, 0.33 mmol) and tributyl amine (0.15 mL, 0.66 mmol) in DMF (1.5 mL) was added to the above solution at r.t. under Ar, followed by stirring overnight and then quenched with 2.0 M TEAB. After evaporating solvent, the organic residue was purified by preparative reverse phase HPLC with 100 mM TEAA/AcCN (0 to 50% gradient) to give the corresponding triphosphate 7. 1H NMR (D2O, 300 MHz) δ 7.74 (s, 1H, H6), 5.95 (dd, 1H, H1′, J=3.3 and 6.3 Hz), 4.29-4.20 (m, 1H, H4′), 4.18-4.07 (m, 3H, CH2 and H5′), 4.02-3.92 (m, 1H, H5′), 3.58 (t, 2H, CH2, J=5.1 Hz), 3.38 (t, 2H, CH2, J=5.1 Hz), 3.20 (s, 2H, CH2), 2.35-2.22 (m, 1H, H2), 2.10-1.92 (m, 3H, H2, and 2H3′). 31P NMR (D2O, 121.4 MHz) δ −10.52 (d, J=19.2 Hz), −10.95 (d, J=20.3 Hz), −22.96 (t, J=20.3 Hz); 19F NMR (D2O, 282.2 MHz) δ −76.37 (s, CF3). Removal of trifluoro acetate (TFA) protecting group was accomplished by treatment with NH4OH (33%, 500 μL) at 60° C. for 20 min and then at r.t for 1 h to give the corresponding triphosphate 8 (17 mM, 500 μL).