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

Nucleic Acids Res. 1990 Sep 25;18(18):5433-41. doi: 10.1093/nar/18.18.5433.

Abstract

The preparation of fully protected diisopropylamino-beta-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity greater than 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. Further, 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 in two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Electrophoresis, Polyacrylamide Gel
  • Molecular Sequence Data
  • Molecular Structure
  • Nitriles / chemical synthesis*
  • Nitriles / chemistry
  • Oligoribonucleotides / chemical synthesis*
  • Oligoribonucleotides / chemistry
  • Oligoribonucleotides / isolation & purification
  • RNA, Catalytic / chemical synthesis
  • Trityl Compounds / chemical synthesis*
  • Trityl Compounds / chemistry

Substances

  • Nitriles
  • Oligoribonucleotides
  • RNA, Catalytic
  • Triphenylmethyl Compounds