反应详情

To a stirred solution of the triphosphate (8.4) (5 mg, 0.05 mmol) in 0.2M Na2CO3-NaHCO3 buffer (800 μpH 8.5) was added an anhydrous DMF solution (600 μl) of the N-hydroxysuccinimidyl ester of 5-carboxyfluorescein (10 mg, 0.02 mmol) at room temperature and stirring continued overnight. After evaporating the reaction mixture under reduced pressure, the yellow coloured residue obtained was dissolved in a minimum amount of 1:1 aqueous methanol, loaded on a glass column (40 cm×2 cm) packed up to 20 cm height with 35-70 microns silica gel 60 (EM-Separations, cat no. 9389-5). The excess dye was eluted using 1:1 chloroform/methanol to neat methanol and the desired nucleotide-fluorescein conjugate was eluted using 6:3:1 i-PrOH:NH4OH:H2O to obtain (8.4) as a yellow solid after pooling and evaporation. Compound (8.5) was further purified by HPLC on a 15 microns Delta Pak C18 column (1.9 cm×30 cm) under the gradient conditions of 0-50% buffer A (0.1 M TEAB, pH 7.1) and buffer B (25% acetonitrile in 0.1M TEAB, pH 7.0) at 15 ml per minute in 30 minutes. The desired compound (8.5) fractions were pooled, evaporated and lyophilised to get pure (8.5) as a yellow solid (quantitative yield).

1. customAfter evaporating the reaction mixture under reduced pressure
2. customthe yellow coloured residue obtained
3. washThe excess dye was eluted
4. washthe desired nucleotide-fluorescein conjugate was eluted