The mechanism of the phosphoramidite synthesis of polynucleotides

Abstract

The mechanism of the coupling step in polynucleotide synthesis using 5′-4,4′-dimethoxytritylthymidine-3′-β-cyanoethyl-N,N-diisopropylphosphoramidite as the phosphitylating agent and catalysed by the salt of saccharin and N-methylimidazole in acetonitrile has been studied by ³¹P NMR. Pre- and post-equilibria between the activator salt and released diisopropylamine have been examined by ¹H NMR and ITC, which show that the salt between saccharin and diisopropylamine will be present in acetonitrile. Activation of the phosphoramidite by the salt of saccharin and N-methylimidazole involves nucleophilic catalysis and the formation of a reactive saccharin adduct bonded through its carbonyl oxygen to phosphorus. The rate constants for the reaction of the 4-methoxyphenol with 5′-4,4′-dimethoxytritylthymidine-3′-β-cyanoethyl-N,N-diisopropylphosphoramidite in the presence of saccharin–N-methylimidazole salt show a non-linear dependence on phenol concentration, becoming independent at high phenol concentrations, compatible with a change in rate limiting step from the alcoholysis step to the activation step.