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

• Published in: Organic process research & development 2004-11, Vol.8 (6), p.852-858
• Main Authors: Krotz, Achim H, Gorman, Dennis, Mataruse, Paul, Foster, Craig, Godbout, James D, Coffin, Christopher C, Scozzari, Anthony N
• Format: Article
• Language: English
• ISSN: 1083-6160; 1520-586X
• DOI: 10.1021/op040208v

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.