Deoxyribonucleoside cyclic N-acylphosphoramidites as a new class of monomers for the stereocontrolled synthesis of oligothymidylyl- and oligodeoxycytidylyl- phosphorothioates

A simple and straightforward synthesis of the pyrimidine 2'-deoxyribonucleoside cyclic N-acylphosphoramidites R-P-1 and S-P-1 is described. Specifically, (+/-)-2-amino-1-phenylethanol 2 was chemoselectively N-acylated to 4 by treatment with ethyl fluoroacetate 3 followed by reaction with hexaethylphosphorus triamide to afford the cyclic N-acylphosphoramidite 5 as a mixture of diastereomeric rotamers (5a and 5b). Condensation of N-4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2-deoxycytidine 8 with 5 in the presence of 1H-tetrazole gave, after silica gel chromatography, pure R-P-1 and S-P-1. P-31 NMR studies indicated that when Rp-l or Sp-l is reacted with 3'-O-acetylthymidine and N,N,N',N'-tetramethylguanidine in CD3CN, the dinucleoside phosphotriester S-P-9 or R-P-9 is formed in near quantitative yield with total P-stereospecificity (delta(P) 144.2 or 143.9 ppm, respectively). Sulfurization of Sp-9 or R-P-9 generated the P-stereodefined dinucleoside phosphorothioate R-P-11 or S-P-11 (delta(P) 71.0 or 71.2 ppm, respectively). The 2'-deoxycytidine cyclic N-acylphosphoramidite derivatives R-P-1 and S-P-1 were subsequently applied to the solid-phase synthesis of [R-P,R-P]- and [S-P,S-P] -trideoxycytidilyl diphosphorothioate d(CPSCPSC), and [R-P,S-P,R-P]-tetradeoxycytidilyl triphosphorothioate d(CPSCPSCPSC) Following deprotection, reversed-phase (RP) HPLC analysis of these oligonucleotide analogues showed a single peak for each oligomer. By comparison, RP-HPLC analysis of purified P-diastereomeric d(CPSCPSC) and d(CPSCPSCPSC) prepared from standard 2-cyanoethyl deoxyribonucleoside phosphoramidites exhibited 4 and 8 peaks, respectively, each peak corresponding to a specific P-diastereomer (see Figure 3A). The thymidine cyclic N-acylphosphoramidite derivatives R-P-14 and S-P-14 were also prepared, purified, and used successfully in the solid-phase synthesis of [R-P](11)-d[(T-PS)(11)T]. Thus, the application of deoxyribonucleoside cyclic N-acyl phosphoramidites to P-stereocontrolled synthesis of oligodeoxyribonucleoside phosphorothioates may offer a compelling alternative to the methods currently used for such syntheses. [References: 51]

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