AN IMPROVED SYNTHESIS OF OLIGODEOXYRIBONUCLEOSIDE PHOSPHOROTHIOATES

研制了一种新型硫化剂， 通过标准品制备寡脱氧核苷硫代磷酸酯 “亚磷酰胺”方法。这些寡核苷酸类似物已经被 据报道抑制细胞培养物中的基因表达。 几种多硫化物作为潜在的硫转移试剂进行了测试。的 这些，硫代磺酸盐3 H-1，2-苯并噻唑-3-酮1，1-二氧化物， 特别有效。 0.05 M试剂乙腈溶液， 将二核苷单亚磷酸酯三酯转化成相应的 在30秒内以接近定量的产率进行硫代磷酸酯化。该试剂 导致快速，有效（99%的逐步产率）和可靠的合成 硫代磷酸酯寡聚体（15-28聚体），其仅携带或携带 预定数目的P（S）键合而没有可检测的核苷 改性此外，具有以下结构的低聚物的P-31 NMR分析是优选的： 仅P（S）键（24-mer）揭示了存在-“仅小的 P（O）键的百分比（小于1%）。已经进一步 证明了P（O）键的存在（约. 24)内 硫代磷酸低聚物没有改变其对 myc基因产物在外周血单个核细胞中的表达 相对于不含P（O）键的相同低聚物。 上述硫代磺酸盐在各种有机溶剂中的溶解度在 除了其在乙腈溶液中的长期稳定性外 （至少1个月）而不损失效率， 许多实验室都能获得硫代磷酸寡核苷酸，因此， 为这项工作的重要性增添了新的内容。

A novel sulfurizing reagent was developed to facilitate the automated preparation of oligodeoxyribonucleoside phosphorothioates via the standard "phosphoroamidite" approach. These oligonucleotide analogues have been reported to inhibit gene expression in cell cultures. Several polysulfides were tested as potential sulfur-transfer reagents. Of these, the thiosulfonate 3H-1,2-benzothiole-3-one 1,1-dioxide was particularly efficient. A 0.05M solution of the reagent in acetonitrile, converted a dinucleoside monophosphite triester into the corresponding phosphorothioate within 30 sec in near quantitative yields. This reagent led to rapid, efficient (stepwise yields of 99%) and reliable synthesis of phosphorothioate oligomers (15-28 mers) carrying either exclusively or a predetermined number of P(S) linkages without detectable nucleosidic modification. Moreover, the P-31 NMR analysis of an oligomer having exclusively P(S) linkages (24-mer) revealed the presence of-'only a small percentage of P(O) linkages (less than 1%). It has been further demonstrated that the presence of P(O) linkages (ca. 24) within a phorphorothioate oligomer did not alter its effectiveness against the expression of the myc gene product in peripheral blood mononuclear cells relative to the same oligomer that is free of P(O) linkages. The solubility of the above thiosulfonate in various organic solvents in addition to its stability in acetonitrile solution for a prolonged period of time (at least 1 month) without losing efficiency provide a facile access to phosphorothioate oligonucleotides to many laboratories and, thus, add a new dimension to the significance of this work.