Development of 2‘-O-Methoxyethyl Phosphorothioate Oligonucleotides as Antisense Drugs under Stereochemical Control

A systematic investigation of the stereoreproducibility of 2'-O-methoxyethylribonucleoside phosphoramidite-based phosphorothioate oligonucleotide synthesis reveals that 1H-tetrazole-catalyzed phosphoramidite coupling followed by sulfurization with phenylacetyl disulfide (PADS) is under inherent stereochemical control. Analyses of single phosphorothioate linkages in oligonucleotides as well as in dimers by 31P NMR spectroscopy reveal reproducible stereochemical control in deoxy/2'-O-methoxyethylribo- and 2'-O-methoxyethylribo/deoxydinucleotide phosphorothioates and in 2'-O-methoxyethylribo/2'-O-methoxyethylribonucleotide phosphorothioate linkages. Particularly interesting is the fact that the R,S ratio of the phosphorothioate linkage is reproducible (within limits of experimental error) between syntheses and is independent of diastereomeric composition of phosphoramidites, synthesis scale, solid support, reactor, concentration during coupling or synthesis conditions. Activators, however, play a major role in determining the diastereomeric ratio of 2'-O-methoxyethyl RNA phosphorothioate internucleotide linkages.