Alkyl phosphotriester modified oligodeoxyribonucleotides. V. Synthesis and absolute configuration of Rp and Sp diastereomers of an ethyl phosphotriester (Et) modified EcoRI recognition sequence, d[GGAA(Et)TTCC]. A synthetic approach to regio- and stereospecific ethylation-interference studies

Abstract

Protected deoxynucleoside 3'-O-ethyl-N,N-diisopropylphosphoramidite reagents were prepared for use in the automated synthesis of ethyl phosphotriester (Et) modified oligonucleotides. The title diastereomers were separated by reversed-phase HPLC, and chirality at phosphorus was assigned by an improved configurational correlation scheme that was verified by NMR spectroscopic studies (accompanying paper, Part VI). This generally applicable correlation scheme involved enzymatic digestions of each diastereomer to give the corresponding diastereomer of d[A(Et)T]; phosphite triester sulfurization to obtain diastereomeric O-ethyl phosphorothioates, d[AS(Et)T], which were separated by HPLC for stereoretentive oxidation with H2O2 to give d[A(Et)T], and stereoretentive de-ethylation with PhSH-Et3N to give diastereomeric phosphorothioates, d[AST], whose configurations at phosphorus had been assigned previously. Neither the Rp-Rp nor Sp-Sp duplex, (d[GGAA(Et)TTCC])2, was cleaved by EcoRI endonuclease under conditions that led to cleavage of both the unmodified duplex, [d(GGAATTCC)]2, and the mixture of diastereomeric phosphorothioate-modified duplexes, [d(GGAASTTCC)]2. Cleavage of the latter substrates was Sp-selective.