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

We describe a strategy for the incorporation of a 2'-C-branched ribonucleos ide, 2'-C-beta-methylcytidine, into oligonucleotides via solid-phase synthe sis 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-buty ldichlorosilane/AgNO3 in DMF. The 2'-hydroxyl group was then protected as a tert-butyldimethylsilyl ether 4a by treatment with tert-butylmagnesium chl oride followed by addition of tert-butyldimethylsilyl trifluoromethanesulfo nate 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-dihyd ro-2H-pyran in the presence of a catalytic amount of 10-camphorsulfonic aci d in methylene chloride. The di-tert-butylsilanediyl groups of 4a and 4b we re removed by treatment with pyridinium poly(hydrogen fluoride) to afford 5 a and 5b, respectively. Protection of the 5'-hydroxyl group as a dimethoxyt rityl ether and phosphitylation of the S'-hydroxyl group by the standard pr ocedure gave the phosphoramidite derivatives 7a and 7b. Both 7a and 7b coul d be used to incorporate 2'-C-beta-methylcytidine into oligonucleotides eff iciently via standard solid-phase synthesis, but the tetrahydropyranyl grou p of 7b was more readily removed from oligonucleotides than the tert-butyld imethylsilyl group of 7a. Oligonucleotides containing 2'-C-beta-methylcytid ine undergo base-catalyzed degradation analogous to natural RNA.