The mechanism of the coupling step in polynucleotide synthesis using 5'-4,4'-dimethoxytritylthymidine-3'-beta-cyanoethyl-N,N-diisopropylphosphoramidite as the phosphitylating agent and catalysed by the salt of saccharin and N-methylimidazole in acetonitrile has been studied by (31)P NMR. Pre- and post-equilibria between the activator salt and released diisopropylamine have been examined by (1)H 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'-beta-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.