Jacek Cieślak, Cristina Ausín, Andrzej Grajkowski, Serge L. Beaucage
{"title":"2′-Hydroxy Protection of Ribonucleosides as 2-Cyano-2,2-Dimethylethanimine-N-Oxymethyl Ethers in Solid-Phase Synthesis of RNA Sequences","authors":"Jacek Cieślak, Cristina Ausín, Andrzej Grajkowski, Serge L. Beaucage","doi":"10.1002/0471142700.nc0322s54","DOIUrl":"10.1002/0471142700.nc0322s54","url":null,"abstract":"<p>The reaction of 2′-<i>O</i>-aminooxymethylribonucleosides with 2-cyano-2-methyl propanal leads to the formation of stable and yet reversible 2′-<i>O</i>-(2-cyano-2,2-dimethylethanimine-<i>N</i>-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′-<i>O</i>-dimethoxytritylation and 3′-<i>O</i>-phosphitylation of the corresponding <i>N</i>-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-1<i>H</i>-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′-<i>O</i>-(2-cyano-2,2-dimethylethanimine-<i>N</i>-oxymethyl) protective groups are cleaved from the RNA sequences by treatment with 0.5 M tetra-<i>n</i>-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. <i>Curr. Protoc. Nucleic Acid Chem</i>. 54:3.22.1-3.22.28. © 2013 by John Wiley & Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471142700.nc0322s54","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1