Unit

UNIT 3.22 2′-Hydroxy Protection of Ribonucleosides as 2-Cyano-2,2-Dimethylethanimine-N-Oxymethyl Ethers in Solid-Phase Synthesis of RNA Sequences

  1. Jacek Cieślak,
  2. Cristina Ausín,
  3. Andrzej Grajkowski,
  4. Serge L. Beaucage

Published Online: 8 OCT 2013

DOI: 10.1002/0471142700.nc0322s54

Current Protocols in Nucleic Acid Chemistry

Current Protocols in Nucleic Acid Chemistry

How to Cite

Cieślak, J., Ausín, C., Grajkowski, A. and Beaucage, S. L. 2013. 2′-Hydroxy Protection of Ribonucleosides as 2-Cyano-2,2-Dimethylethanimine-N-Oxymethyl Ethers in Solid-Phase Synthesis of RNA Sequences. Current Protocols in Nucleic Acid Chemistry. 54:3.22:3.22.1–3.22.28.

Author Information

  1. Food and Drug Administration, Bethesda, Maryland

Publication History

  1. Published Online: 8 OCT 2013

Abstract

The reaction of 2′-O-aminooxymethylribonucleosides with 2-cyano-2-methyl propanal leads to the formation of stable and yet reversible 2′-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl)ribonucleosides in post-purification yields of 54% to 82%. Phenoxyacetylation of the exocyclic amino functions of these ribonucleosides proceeds in yields of 74% to 89%, and subsequent 5′-O-dimethoxytritylation and 3′-O-phosphitylation of the corresponding N-phenoxyacetylated ribonucleosides provide the fully protected ribonucleoside phosphoramidite monomers in isolated yields of 69% to 88%. These ribonucleoside phosphoramidites are employed in solid-phase synthesis of three chimeric RNA sequences, each differing in purine/pyrimidine content. The stepwise coupling efficiency of the ribonucleoside phosphoramidites (as 0.15 M solutions in acetonitrile) averages 99% over a coupling time of 180 s when 5-benzylthio-1H-tetrazole is used as an activator. Upon completion of RNA chain assembly, removal of the nucleobase- and phosphate-protecting groups and release of sequences from the solid support are carried out under standard basic conditions. Finally, the 2′-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl) protective groups are cleaved from the RNA sequences by treatment with 0.5 M tetra-n-butylammonium fluoride in dry DMSO for 24 to 48 hr at 55°C without releasing RNA-alkylating side-products. Characterization of the fully deprotected RNA sequences by PAGE, enzymatic hydrolysis, and MALDI-TOF mass spectrometry confirms the identity and high quality of these sequences. Curr. Protoc. Nucleic Acid Chem. 54:3.22.1-3.22.28. © 2013 by John Wiley & Sons, Inc.

Keywords:

  • 2′-O-aminooxymethyl ribonucleosides;
  • 2′-O-(2-cyano-2,2-dimethyl-ethanimine-N-oxymethyl)ribonucleosides;
  • solid-phase RNA synthesis;
  • fluoride-mediated 2′-deprotection;
  • chimeric RNA sequences