Phosphorothioate Oligonucleotides with Low Phosphate Diester Content:  Greater than 99.9% Sulfurization Efficiency with “Aged” Solutions of Phenylacetyl Disulfide (PADS)

In this report, we describe the design of an efficient and robust sulfurization step in the production process of phosphorothioate (PS) oligonucleotides by solid-phase synthesis. Average stepwise sulfurization efficiencies >99.9% were obtained with “aged” solutions of phenylacetyl disulfide (PADS) in the solvent system acetonitrile/3-picoline (0.2 M, 1:1, v/v). Preparation of the reagent at least 1 day prior to use yields PS-oligonucleotides with low-level phosphate (PO) diester content. The use of freshly prepared PADS solutions gave oligonucleotide products with increased PO-diester content (stepwise sulfurization efficiency 99.5−99.7%). Short aging periods combined with short sulfurization contact times lead to formation of oligonucleotide 4,4‘-dimethoxytrityl-C-phosphonate derivatives. Investigation of reagent stability and sulfurization efficiency revealed significant changes in solution composition over time caused by dissociation of the PADS molecule. The “in situ” formation of a reactive intermediate, presumably a polysulfide, appears to be crucial for optimal synthesis results.