EXPLORATION OF ULTIMATE NEW SYNTHETIC METHOD OF DNA/RNA BY USE OF "PROTON-BLOCK APPROACH"-DIRECTED TOWARD ESTABLISHMENT OF INOVATIVE METHOD FOR THE SYNTHESIS OF NUCLEIC ACIDS WITHOUT BASE PROTECTION-

以前，在N-未保护的亚磷酰胺方法中聚合物载体上的O-选择性磷酸化不能进行，因为dA和dC的氨基对三价磷（III）型亚磷酸化试剂具有高反应性。在本文中，我们开发了一种新的偶联策略，命名为“活化亚磷酸酯法”，其中的亚磷酸三酯中间体，如化合物1介导的亚磷酰化反应。在TpA和TpC衍生物的液相合成中，使用HOBt的缩合显示几乎100%的O-选择性，并且它们可以以高产率分离。随后，我们研究了通过使用各种1-羟基苯并三唑和苯酚型促进剂在聚合物载体上合成二聚体（ApT、CpT和GpT）和三聚体（TpApT、TpCpT和TpGpT）的磷酸化的O-选择性。将HOBt作为促进剂应用于固相合成，得到了大于99.7%的优异的O-选择性。我们解释了这种O-选择性 关于我们 基于反应性中间体与亲核试剂如核苷的氨基或羟基之间的FMO相互作用。研究还发现，在使用HOBt型促进剂的缩合反应中，随着促进剂酸性的增加，O-选择性降低。详细的研究我们目前的方法的机制披露，在此缩合，亚磷酸酯苯并三唑基酯中间体通过重排转化成相应的五价磷物种，发现仍然是反应性的羟基功能。据估计，这种五价物质对总磷酸化的贡献在缩合中至多为4 - 10%。此外，我们不仅通过人工操作，而且通过DNA合成仪成功合成了较长的寡核苷酸。我们的新方法的效用被证明是由一个碱不稳定的修饰的寡脱氧核苷酸具有4-N-乙酰脱氧胞苷残基的成功合成。少

Previously, Ο-selective phosphorylation on polymer supports in the N-unprotected phosphoramdite method could not be carried out since the amino groups of dA and dC have high reactivity toward tervalent phosphorus(III)-type phosphitylating reagents. In this paper, we developed a new coupling strategy named 'activated phosphite method' in which the phosphitylation reaction is mediated by phosphite triester intermediates such as compound 1. In the solution-phase synthesis of TpA and TpC derivatives, the condensation by use of HOBt showed almost 100% Ο-selectivity and, they could be isolated in high yields. Subsequently, we examined the Ο-selectivity of the phosphorylation by use of various 1-hydroxybenzotriazole-and phenol-type promoters in the synthesis of dimers (ApT, CpT, and GpT) and trimers (TpApT, TpCpT, and TpGpT) on polymer supports. Application of HOBt as the promoter to the solid-phase synthesis resulted in excellent Ο-selectivity of more than 99.7%. We explained this Ο-selectiv … More ity based on the FMO interactions between the reactive intermediates and the nucleophiles such as the amino or hydroxyl groups of nucleosides. It was also found that the Ο-selectivity decreased in the condensation by use of HOBt-type promoters with an increase of the acidity of the promoter. The detailed studies on the mechanism of our present approach disclosed that, during this condensation, a phosphite benzotriazolyl ester intermediate is converted via rearrangement into the corresponding pentavalent phosphorus species which was found to be still reactive to hydroxy functions. It was estimated that contribution of this pentavalent species to the overall phosphorylation is at most 4 -10% in the condensation. Furthermore, we succeeded in the synthesis of longer oligonucleotides not only by a manual operation but also by a DNA synthesizer. The utility of our new method was demonstrated by the successful synthesis of an alkali-labile modified oligodeoxyribonucleotide having 4-N-acetyl deoxycytidine residues. Less