Synthesis of 2′-modified nucleotides and their incorporation into hammerhead ribozymes

Abstract

Several 2′-modifled ribonucleoside phosphoramldites have been prepared for structure-activity studies of the hammerhead ribozyme. The aim of these studies was to design and synthesize catalytically active and nuclease-resistant ribozymes. Synthetic schemes for stereosetective synthesis of the R isomer of 2′-deoxy-2′-C-ally1 uridine and cytidine phosphoramidites, based on the Keck allylatlon procedure, were developed. Protection of the 2′-amino group in 2V-deoxy-2′-aminourldine was optimized and a method for the convenient preparation of 5′/O-dimethoxytrltyl- 2′-deoxy-2′-phthalimidourldlne 3′-0(2-cyanoethyl- A/,W-dlisopropylphosphoramidrte) was developed. During the attempted preparation of the 2′-O-f-butyldimethylsilyl- 3′-O-phosphoramldite of arabinouridine a reversed regioselectivity in the sllylatlon reaction,compared with the published procedure, was observed, as well as the unexpected formation of the 2,2′-anhydronucleoslde. A possible mechanism for this cyclization 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′-modrfied monomers were incorporated at positions U4 and/or U7 of the catalytic core. A number of these ribozymes had almost wild-type catalytic activity and improved stability in human serum, compared with an all-RNA molecule. © 1995 Oxford University Press.