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 1 H- tetrazole gave, after silica gel chromatography, pure R P -1 and S P -1 . 31 P NMR studies indicated that when R P -1 or S P -1 is reacted with 3‘- O -acetylthymidine and N, N, N ‘, N ‘ -tetramethylguanidine in CD 3 CN, the dinucleoside phosphotriester S P -9 or R P -9 is formed in near quantitative yield with total P -stereospecificity (δ P 144.2 or 143.9 ppm, respectively). Sulfurization of S P -9 or R P -9 generated the P -stereodefined dinucleoside phosphorothioate R P -11 or S P -11 (δ 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(C PS C PS C), and [ R P, S P, R P ]-tetradeoxycytidilyl triphosphorothioate d(C PS C PS C PS C). 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(C PS C PS C) and d(C PS C PS C PS C) 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.