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

摘要

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 (1.) enzymatic digestions of each diastereomer to give the corresponding diastereomer of d[A(Et)T]; (2.) phosphite triester sulfurization to obtain diastereomer O-ethyl phosphorothioates, d[A_S(Et)T], which were separated by HPLC for (3.) stereoretentive oxidation with H₂O₂ to give d[A(Et)T], and (4.) stereoretentive de-ethylation with PhSH-Et₃N to give diastereomeric phosphorothioates, d[A_ST], whose configurations at phosphorus had been assigned previously. Neither the R_p?R_p nor S_p?S_p 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(GGAA_STTCC)]₂. Cleavage of the latter substrates was S_p-selective.