SYNTHESIS OF 3'-THIORIBONUCLEOSIDES AND THEIR INCORPORATION INTO OLIGORIBONUCLEOTIDES VIA PHOSPHORAMIDITE CHEMISTRY

Oligoribonucleotides containing 3'-S-phosphorothiolate linkages are va luable probes in nucleic acid biochemistry, but their accessibility ha s been limited because 3'-thioribonucleoside phosphoramidites have not been available. We synthesized 3'-thioribonucleoside derivatives (C, G, and U) via glycosylations of nucleoside bases with 3-S-thiobenzoyl- 5-O-toluoyl-1,2-O-diacetylfuranose 5, which was obtained from l-3-trif luoromethane-sulfonyl-alpha-D-xylofuranose 2 by S(N)2 displacement wit h sodium thiobenzoate. Additionally, a 3'-thioinosine derivative was p repared from inosine via direct modification of the ribose, analogous to the previously reported synthesis of 3'-thioadenosine, except that the intermediate 2',3'-epoxide 9 was first protected as the 5'-O-tert- butyldiphenylsilyl ether prior to subsequent synthetic steps. This hyd rophobic silyl group facilitated extraction and isolation of synthetic intermediates. After removal of the protecting groups, the 8'-thionuc leosides (C, G, U, and I) were treated with 2,2'-dipyridyl disulfide t o protect the free thiol group as a disulfide. The 3'-thionucleosides were converted to the corresponding phosphorothioamidites using proced ures analogous to those for standard phosphoramidites. The amino group s of 3'-thiocytidine and 3'-thioguanosine were protected as benzoyl an d isobutyryl amides, respectively, and the 5'- and 5'-hydroxyl groups of each nucleoside were protected as dimethoxytrityl and tert-butyldim ethylsilyl ethers, respectively. The 3'-thiol group was deprotected by reduction with DTT and phosphitylated to afford analytically pure 3'- S-phosphorothioamidites 15, which were incorporated into oligoribonucl eotides by solid-phase synthesis. Chemical assays and mass spectrometr y of the synthetic RNA showed that ribose-3'-S-phosphorothiolate linka ges were installed correctly and efficiently into RNA oligonucleotides using phosphoramidite chemistry.