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，用二聚体单元代替单体单元作为缩合单元。以胸腺嘧啶核苷为原料，经二甲氧基三酰化和邻氯苯基磷三氮唑的磷酸化反应，以较高的产率合成了5‘-O-二甲氧基三丁基胸苷3’-(邻氯苯基)磷酸。以MSNT为缩合剂，将未保护的胸腺嘧啶核苷与二甲氧基三甲基胸腺嘧啶核苷3‘-(邻氯苯基)磷酸缩合，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，用甲基-N，N-二异丙基亚磷酰胺对主要产物3‘-5’键进行亚磷酸化，产率为85%。因此，可以方便地制备用于嵌段合成寡核苷酸的二聚体单元，并以较高的产率合成十五硫代肌酸。二聚体的缩合反应重复7次，结合在聚合物载体上的寡核苷酸用氨水处理裂解。合成的寡核苷酸经反相高效液相色谱柱纯化和阴离子交换柱层析分离纯化。当使用1umol的胸腺嘧啶核苷连接的聚合物载体时，产率为39A260单位(30%)。结果表明，用二聚体单元代替单体单元的缩合反应是可行的，应改进自动合成器的合成方法。

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.