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 Brönsted ß values for sulfurisation using 'fresh' and 'aged' PADS with substituted pyridines are 0.43 and 0.26, respectively. With 'fresh' PADS the Brönsted ßnuc = 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.

Phosphorothioate anti-sense oligonucleotides: the kinetics and mechanism of the generation of the sulfurising agent from phenylacetyl disulfide (PADS).

The synthesis of phosphorothioate oligonucleotides is often accomplished in the pharmaceutical industry by the sulfurisation of the nucleotide-phosphite using phenylacetyl disulfide (PADS) which has an optimal combination of properties. This is best achieved by an initial 'ageing' of PADS for 48 h in acetonitrile with 3-picoline to generate polysulfides. The initial base-catalysed degradation of PADS occurs by an E1cB-type elimination to generate a ketene and acyldisulfide anion. Proton abstraction to reversibly generate a carbanion is demonstrated by H/D exchange, the rate of which is greatly increased by electron-withdrawing substituents in the aromatic ring of PADS. The ketene can be trapped intramolecularly by an o-allyl group. The disulfide anion generated subsequently attacks unreacted PADS on sulfur to give polysulfides, the active sulfurising agent. The rate of degradation of PADS is decreased by less basic substituted pyridines and is only first order in PADS indicating that the rate-limiting step is formation of the disulfide anion from the carbanion.