SOLID SUPPORT SYNTHESIS OF ALL-RP-OLIGO(RIBONUCLEOSIDE PHOSPHOROTHIOATE)S

The first method for solid support synthesis of all-R(P)-oligo(ribonuc leoside phosphorothioate)s is presented as well as attempts to increas e the stereoselectivity of the key step in this approach, The syntheti c strategy consists of (i) a solid support synthesis procedure, using ethyl)-2'-O-tert-butyldimethylsilyl-ribonucleoside 3'-H-phosphonates, that due to stereoselectivity in the condensation step, gives oligomer s with mostly S-P-H-phosphonate diesters (72-89% under standard condit ions), (ii) stereospecific sulfurization with S-8 in pyridine to produ ce oligo(ribonucleoside phophorothioate)s enriched with internucleosid ic linkages of R(P) configuration, (iii) treatment of the deprotected oligonucleotides with the enzyme Nuclease P1 from Penicillium citrinum , that specifically catalyses cleavage of S-P-phosphorothioate diester linkages, which leaves a mixture of oligomers having all internucleos idic linkages as R(P)-phosphorothioates, and finally (iv) isolation an d HPLC purification of the full length all-R(P) oligomer, Mixed sequen ces containing the four common nucleosidic residues up to the chain le ngth of a heptamer were synthesized, Change of N-4-protection on the c ytidine building block from propionyl to N-methylpyrrolidin-2-ylidene gave a slightly improved diastereoselectivity in H-phosphonate diester formation, Increased selectivity up to 99+% was obtained with the gua nosine building block when the amount of pyridine in the coupling step was reduced.