title: 2'-Hydroxy protection of ribonucleosides as 2-cyano-2,2-dimethylethanimine-N-oxymethyl ethers in solid-phase synthesis of RNA sequences.
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.
authors: Food and Drug Administration, Bethesda, Maryland.
authors: Jacek
authors: J
authors: Cieślak
authors: Food and Drug Administration, Bethesda, Maryland.
authors: Cristina
authors: C
authors: Ausín
authors: Food and Drug Administration, Bethesda, Maryland.
authors: Andrzej
authors: A
authors: Grajkowski
authors: Food and Drug Administration, Bethesda, Maryland.
authors: Serge L
authors: SL
authors: Beaucage
mesh_terms: D003485:Cyanates / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004987:Ethers / Q000737:chemistry; D007097:Imines / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012313:RNA / Q000138:chemical synthesis*; D012263:Ribonucleosides / Q000737:chemistry*; D060327:Solid-Phase Synthesis Techniques; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003485:Cyanates; D004987:Ethers; D007097:Imines; D009943:Organophosphorus Compounds; D000644:Quaternary Ammonium Compounds; D012263:Ribonucleosides; C434331:phosphoramidite; D012313:RNA; C009405:tetrabutylammonium
keywords: 2′-O-(2-cyano-2,2-dimethyl-ethanimine-N-oxymethyl)ribonucleosides; 2′-O-aminooxymethyl ribonucleosides; chimeric RNA sequences; fluoride-mediated 2′-deprotection; solid-phase RNA synthesis