A simple and straightforward synthesis of the pyrimidine 2′-deoxyribonucleoside cyclic Nacylphosphoramidites 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 1Htetrazole 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.
The 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl group for phosphate protection in the synthesis of oligodeoxyribonucleotides has been developed to completely prevent nucleobase alkylation by acrylonitrile that could potentially occur upon deprotection of the traditional 2-cyanoethyl phosphate protecting group. The properties of this new phosphate protecting group were evaluated using the model phosphotriester 9. The mechanism of phosphate deprotection was studied by treating 9 with concentrated NH4OH. NMR analysis of the deprotection reaction demonstrated that cleavage of the N-trifluoroacetyl group is rate-limiting. The resulting phosphotriester intermediate 13 was also shown to undergo rapid cyclodeesterification to produce O,O-diethyl phosphate 15 and N-methylpyrrolidine 16 (Scheme ). Given the facile removal of the 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl phosphate protecting group under mild basic conditions, its utilization in oligonucleotide synthesis began with the preparation of the deoxyribonucleoside phosphoramidites 4a−d (Scheme ). The coupling efficiency of 4a−d and conventional 2-cyanoethyl deoxyribonucleoside phosphoramidites 24a−d was then compared in the solid-phase synthesis of the 20-mer d(ATCCGTAGCTAAGGTCATGC). As previously observed in the deprotection of 9, the 4-[N-methyl,N-(2,2,2-trifluoroacetyl)amino]butyl phosphate protecting groups were easily and completely removed from the oligonucleotide by using either concentrated NH4OH or pressurized ammonia gas. Analysis of the deprotected oligomer by polyacrylamide gel electrophoresis (Figure ) indicated that the phosphoramidites 4a−d are as efficient as the 2-cyanoethyl phosphoramidites 24a−d in the synthesis of the 20-mer. Furthermore, following digestion of the crude 20-mer by snake venom phosphodiesterase and bacterial alkaline phosphatase, HPLC analysis showed complete hydrolysis to individual nucleosides and no detectable nucleobase modification.
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