Polymer support oligonucleotide synthesis XVIII^1.2): use of<i>β</i>-cyanoethyi-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product

TLDR

The authors synthesized 5′‑O‑protected deoxynucleoside‑3′‑O‑β‑cyanoethyl phosphoramidites from β‑cyanoethyl monochlorophosphoramidites and performed one‑step cleavage, N‑deacylation, and β‑cyanoethyl deprotection on polymer support using concentrated aqueous ammonia, with resulting oligodeoxynucleotides characterized by Maxam‑Gilbert sequencing. The β‑cyanoethyl phosphoramidites proved effective for automated oligonucleotide synthesis on controlled pore glass, offering stable monomers, easy preparation of dichloro‑β‑cyanoethoxy‑phosphine without PCl3 contamination, and a preference for N,N‑diisopropylamine derivatives.

Abstract

Various 5′-0-N-protected deoxynucleoside-3′-0-β-cyanoethyl-N,N-dialkylamino-/N-morpholinophosphoramidites were prepared from β-cyanoethyl monochlorophosphoramidites of N,N-dimethylamine, N,N-diisopropylamine and N-morpholine. These active deoxynucleoside phosphites have successfully been used for oligodeoxy-nucleotide synthessis on controlled pore glass as polymer support and are very suitable for automated DNA-synthesis due to their stability in solution. The intermediate dichloro-β-cyanoethoxy-phosphine can easily be prepared free from any PCl3 contamination. The active monomers obtained from β-cyanoethyl monochloro N,N-diisopropylaminophosphoramidites are favoured. Cleavage of the oligonucleotide chain from the polymer support, N-deacylation and deprotection of β-cyanoethyl group from the phosphate triester moiety can be performed in one step with concentrated aqueous ammonia. Mixed oligodeoxynucleotides are characterized by the sequencing method of Maxam and Gilbert.