Oligodeoxyribonucleoside phosphorothioates (PS-ODNs) have been, and are still, extensively studied as potential therapeutic agents against various types of cancer and infectious diseases in humans. Given that these oligonucleotide analogues are P-chiral, each of the internucleotidic phosphorothioate linkages adopts either a Rp or a Sp configuration. Chemically synthesized PS-ODNs are therefore composed of a population of stereoisomers. Most studies addressing the biological activity, pharmacokinetic properties, and toxicology of PS-ODNs have been performed with oligonucleotides having undefined chirality at phosphorus. Stereopure Rp-oligodeoxyribonucleoside phosphorothioates have been prepared enzymatically and exhibited a lower stability to nucleases endogenous to human serum than the parent PS-ODNs with undefined P-chirality. Conversely, chemically synthesized stereopure Sp-oligodeoxyribonucleoside phosphorothioates have demonstrated superior stability to these nucleases than P-diastereomeric PS-ODNs. Thus, to further investigate the biological, pharmacokinetic, and toxicologic properties of P-stereopure PS-ODNs improved chemical methods are required to synthesize these biomolecules and increase their availability for clinical studies. We have discovered that deoxyribonucleoside cyclic N-acylphosphoramidites are efficient monomers for the stereospecific synthesis of PS-ODNs, and these are easily prepared from commercial precursors. Chromatographic resolution of these diastereomeric phosphoramidites to P-stereopure cyclic N-acylphosphoramidites on silica gel is facile because the mobility of each diastereomer is quite different from that of its epimer. Base-assisted condensation of the 5'-OH function of a nucleoside or a nucleotide covalently linked to a solid support with deoxyribonucleoside cyclic N-acylphosphoramidites led to rapid and efficient formation of an internucleoside phosphite triester linkage. The phosphite triester function can be either oxidized to a phosphate triester(PO)by treatment with tert-butyl hydroperoxide or sulfurized by a sulfur transfer reagent to a P-stereodefined thiophosphate phosphotriester(PS)function to permit, for example, stereocontrolled synthesis of chimeric PO/PS-ODNs. In this regard, the stereospecific synthesis of the PS-ODNs [Rp,Rp]- and [Sp,Sp]-d(CpsCpsC) and [Rp,Sp,Rp]-d(CpsCpsCpsC) have been successfully accomplished. The method is currently being optimized to enable the incorporation of the four different nucleobases into DNA chains of at least 20 bases long. Interestingly, deoxyribonucleoside cyclic N-acylphosphoramidites can, unlike conventional deoxyribonucleoside phosphoramidites, effectively produce oligonucleotides under relatively wet conditions. This property is particularly indicated for parallel synthesis of oligonucleotides on arrayable surfaces using robotic equipment under atmospheric conditions. These arrays can be invaluable for high-throughput screening of genetic mutations characterizing specific diseases.
