Chemical synthesis of biologically active oligoribonucleotides using β-cyanoethyl protected ribonucleoside phosphoramidites

Abstract

The preparation of fully protected diisopropylamino-.beta.-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity > 99.95% is described. It is demonstrated that the combination of standard DNA protecting groups, 5''-O-DMT, N-Bz (Ade and Cyt), N-iBu (Gua), .beta.-cyanoethyl for phosphate, in conjunction with TBDMS for 2''-hydroxyl protection, constitutes a reliable method for the preparation of fully active RNA. Average stepwise coupling yields in excess of 99% were achieved with these synthons on standard DNA synthesizers. Two steps completely deprotect the oligoribonucleotide and workup is reduced to a fifteen minute procedure. Futher, it is shown that the deprotected oligoribonucleotides are free from 5''-2'' linkages. This methodology was applied to the chemical synthesis of a 24-mer microhelix, a 35-mer minihelix and two halves of a catalytic ''Hammerhead Ribozyme''. These oligoribonucleotides were directly compared to two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.