2′-C-branched ribonucleosides:: Synthesis of the phosphoramidite derivatives of 2′-C-β-methylcytidine and their incorporation into oligonucleotides

We describe a strategy for the incorporation of a 2'-C-branched ribonucleoside, 2'-C-beta-methylcytidine, into oligonucleotides via solid-phase synthesis using phosphoramidite derivatives. 4-N-Benzoyl-2'-C-beta-methylcytidine (2b) was synthesized by coupling persilylated 4-N-benzoylcytosine with 1,2,3,5-tetra-O-benzoyl-2-C-beta-methyl-alpha-(and beta)-D-ribofuranose (1) in the presence of SnCl4 in acetonitrile, followed by selective deprotection with NaOH in pyridine/methanol. The 3'- and 5'-hydroxyl groups were blocked as a cyclic di-tert-butylsilanediyl ether 3 by treatment with di-tert-butyldichlorosilane/AgNO3 in DMF. The 2'-hydroxyl group was then protected as a tert-butyldimethylsilyl ether 4a by treatment with tert-butylmagnesium chloride followed by addition of tert-butyldimethylsilyl trifluoromethanesulfonate in THF. As an alternative to 2'-silyl protection, the corresponding 2'-O-tetrahydropyranyl ether 4b was prepared by treatment of 3 with 4,5-dihydro-2H-pyran in the presence of a catalytic amount of 10-camphorsulfonic acid in methylene chloride. The di-tert-butylsilanediyl groups of 4a and 4b were removed by treatment with pyridinium poly(hydrogen fluoride) to afford 5a and 5b, respectively. Protection of the 5'-hydroxyl group as a dimethoxytrityl ether and phosphitylation of the S'-hydroxyl group by the standard procedure gave the phosphoramidite derivatives 7a and 7b. Both 7a and 7b could be used to incorporate 2'-C-beta-methylcytidine into oligonucleotides efficiently via standard solid-phase synthesis, but the tetrahydropyranyl group of 7b was more readily removed from oligonucleotides than the tert-butyldimethylsilyl group of 7a. Oligonucleotides containing 2'-C-beta-methylcytidine undergo base-catalyzed degradation analogous to natural RNA.