DNA oligonucleotides containing stereodefined phosphorothioate linkages in selected positions.
This unit describes a method for the synthesis of DNA chimeric PO/PS-oligonucleotides with a stereodefined phosphorothioate bond in the selected position. Diastereomerically pure 5'-O-DMTr-N-protected-deoxyribonucleoside-3'-O-(2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane)s obtained according to the previously described protocol (UNIT 4.17) are transformed via a stereospecific 1,3,2-oxathiaphospholane-ring opening condensation into the corresponding dinucleoside phosphorothioates. Such dimers cannot be introduced into an oligonucleotide chain via the phosphoramidite approach since the unprotected P-S(-) bond is easily oxidized during routine I(2)/Py/water oxidation of the phosphite function. In the methodology described here, the reversible alkylation of the PS function is applied. Subsequently, the 3'-phosphoramidites of such PS-protected dimers prepared in situ are used for routine synthesis of chimeric PO/PS-oligonucleotides according to the phosphoramidite method. The presence of the alkylated PS-function requires modified conditions for oligonucleotide deprotection and cleavage from the solid support. Detailed procedures for the synthesis of PS-dimers and their incorporation into an oligonucleotide chain, as well as deprotection/purification steps are presented.
Nawrot
Barbara
B
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland.
Rebowska
Beata
B