Gas-liquid chromatography of nucleosides. Effect of silylating reagents and solvents

The aims of this investigation were to study the completeness of silylation of nucleosides with three different reagents, bis(trimethylsilyl)trifluoroacetamide (BSTFA), bis(trimethylsilyl)acetamide (BSA) and trimethylsilylimidazole (TMSI), and to investigate the effect of different solvents (acetonitrile, pyridine, dimethylformamide, chloroform, methylene chloride, hexane, benzene, and toluene) on quantitation of derivatization. Closed-tube silylations of the nucleosides were performed with BSTFA, BSA, and TMSI, and for the most complete silylation, the optimal time, temperature, and molar excess of reagent were: for BSTFA, 150 degrees-15 min and 225 molar excess; for TMSI, 60 degrees-3 h and 1000 molar excess; and for BSA, 120 degrees-2 h and 250 molar excess. Also, silylations of seven major and minor nucleosides were carried out using a 1000 molar excess of BSTFA, BSA, and TMSI at 25 degrees with 5 min sonication, and at optimal silylation conditions as described above for the three reagents. The silylating strengths were determined by the increase in RWR (= weight response of nucleoside/weight response of pyrene) values, and are summarized for the amino group containing nucleosides silylated at room temperature as BSTFA greater than TMSI greater than BSA, and for silylation under optimal conditions as BSTFA greater than BSA greater than TMSI. The efficiency of silylation for the hydroxyl group-containing nucleosides silylated at room temperature was BSTFA greater than TMSI greater than BSA, and for silylation under optimal conditions BSTFA greater than TMSI = BSA...