Phosphorothioate anti-sense oligonucleotides: the kinetics and mechanism of the sulfurisation of phosphites by phenylacetyl disulfide (PADS)

In the pharmaceutical industry the sulfurisation of nucleotide-phosphites to produce more biologically stable thiophosphates is often achieved using `aged' solutions of phenylacetyl disulfide (PADS) which consist of a mixture of polysulfides that are more efficient sulfur transfer reagents. However, both `fresh' and `aged' solutions of PADS are capable of the sulfurisation of phosphites. The rates of both processes in acetonitrile are first order in sulfurising agent, phosphite and a pyridine base, although with `aged' PADS the rate becomes independent of base at high concentrations. The Bronsted beta values for sulfurisation using `fresh' and `aged' PADS with substituted pyridines are 0.43 and 0.26, respectively. With `fresh' PADS the Bronsted beta(nuc) = 0.51 for substituted trialkyl phosphites is consistent with a mechanism involving nucleophilic attack of the phosphite on the PADS disulfide bond to reversibly generate a phosphonium intermediate, the rate-limiting breakdown of which occurs by a base catalysed elimination process, confirmed by replacing the ionisable hydrogens in PADS with methyl groups. The comparable polysulfide phosphonium ion intermediate seen with `aged' PADS presents a more facile pathway for product formation involving S-S bond fission as opposed to C-S bond fission.