SYNTHESIS OF 2'-MODIFIED NUCLEOTIDES AND THEIR INCORPORATION INTO HAMMERHEAD RIBOZYMES

Several 2'-modified ribonucleoside phosphoramidites have been prepared for structure-activity studies of the hammerhead ribozyme, The aim of these studies was to design and synthesize catalytically active and n uclease-resistant ribozymes, Synthetic schemes for stereoselective syn thesis of the R isomer of 2'-deoxy-2'-C-allyl uridine and cytidine pho sphoramidites, based on the Keck allylation procedure, were developed, Protection of the 2'-amino group in 2'-deoxy-2'-aminouridine was opti mized and a method for the convenient preparation of '-O-dimethoxytrit yl-2'-deoxy-2'-phthalimidouridine 3'-O-(2-cyanoethyl-N,N-diisopropylph osphoramidite) was developed, During the attempted preparation of the 2'-O-t-butyldimethylsilyl-3'-O-phosphoramidite of arabinouridine a rev ersed regioselectivity in the silylation reaction, compared with the p ublished procedure, was observed, as well as the unexpected formation of the 2,2'-anhydronucleoside. A possible mechanism for this cyclizati on is proposed, The synthesis of 2'-deoxy-2'-methylene and 2'-deoxy-2' -difluoromethylene uridine phosphoramidites is described, Based on a ' 5-ribose' model for essential 2'-hydroxyls in the hammerhead ribozyme these 2'-modified monomers were incorporated at positions U4 and/or U7 of the catalytic core, A number of these ribozymes had almost wild-ty pe catalytic activity and improved stability in human serum, compared with an all-RNA molecule.