Development of synthetic method of DNA oligomer by phosphite procedure using dimer as condensation unit

对自动合成仪合成寡核苷酸的方法进行了改进，用二聚体代替单体作为缩合单元。以胸苷为原料，经二甲氧基三苯甲基化，再用邻氯苯基磷酰三唑磷酰化，以较好的收率合成了5 ′-O-二甲氧基三苯甲基胸苷3 ′-（邻氯苯基）磷酸酯。在MSNT作为缩合剂的存在下，通过将未保护的胸苷缩合为二甲氧基三苯甲基胸苷3 '-（邻氯苯基）磷酸来制备二核苷一磷酸。缩合产物中含有3 ′-3 ′键的副产物，经硅胶柱层析除去。然后，将具有3 '-5'键的主要产物用甲基-N，N-二异丙基亚磷酰胺进行亚磷酸化，产率为85%。在自动化合成仪上连接胸苷二聚体单元溶液，合成十五胸苷酸。重复二聚体的缩合7次，并通过氨水处理裂解结合在聚合物载体上的寡核苷酸。然后，通过反相HPLC和随后的阴离子交换HPLC纯化所合成的寡核苷酸。当使用1 μ mol胸腺嘧啶连接的聚合物载体时，产率为39个A260单位（30%）。这一结果是极好的，因此二聚体单元代替单体单元的缩合是有用的，并且使用自动合成仪的合成方法应改进。

Improvement of synthetic method of oligonucleotides using automated synthesizer has been performed by using dimer unit as condensation unit in place of monomer unit.5'-O-Dimethoxytritylthymidine 3'-(o-chlorophenyl)phosphate was synthesized from thymidine by dimethoxytritylation and subsequent phosphorylation using o-chlorophenyl phosphoroditriazolide in a good yield. Preparation of dinucleoside monphosphate was carried out by condensation of unprotected thymidine to dimethoxytritylthymidine 3'-(o-chlorophenyl)phosphate in presence of MSNT as condensing agent. The products of the condensation contained byproduct which had 3'-3' linkage and removed by silica gel column chromatography. Then, major product having 3'-5' linkage was phosphitylated with methyl-N,N-diisopropylphosphoramidite in 85% yield. Thus, the dimer unit for the blockwise synthesis of oligonucleotides was prepared conveniently in good yield.The synthesis of pentadecathymidylate was performed by automated synthesizer which was attached with thymidine-dimer unit solution. The condensation of dimer was repeated 7 times and oligonucleotide bound in polymer support was cleaved by aqueous ammonia treatment. Then, oligonucleotide synthesized was purified by reversed phase HPLC and susequent anion exchange HPLC. The yield was 39 A260 units (30%) When 1 umol of thymidine-linked polymer support was used. This result is excellent, so that the condensation of dimer unit in place of monomer unit is useful and synthetic method using automated synthesizer should be improved.