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，由于da和dC的氨基对三价磷(III)型磷酰化试剂有很高的反应活性，所以在N-无保护磷酸盐方法中，聚合物载体上的Ο选择性磷酸化不能进行。在本论文中，我们提出了一种新的偶联策略--活性亚磷酸酯法。在化合物1等亚磷酸三酯中间体的催化下进行的亚磷酸化反应。在TPA和TPC衍生物的液相合成中，使用HOBt进行的缩合反应显示出几乎100%的Ο选择性，并且它们可以高产率地分离出来。随后，我们考察了在聚合物载体上合成二聚体(APT、CPT和GPT)和三聚体(TpApT、TpCpT和TpGpT)时，使用不同的1-羟基苯并三氮唑和苯酚类型的促进剂对磷酸化的Ο选择性。将该催化剂应用于固相合成，Ο选择性可达99.7%以上。我们解释了这一选择-Ο…更多的是基于活性中间体和亲核试剂(如核苷的氨基或羟基)之间的FMO相互作用。研究还发现，在HOBt型促进剂的作用下，缩合反应中Ο选择性随着助剂酸度的增加而降低。对本方法机理的详细研究表明，在缩合过程中，亚磷酸苯并三唑酯中间体通过重排转化为相应的五价磷物种，发现该物种仍对羟基功能起反应作用。据估计，在缩合反应中，这种五价物种对整个磷酸化的贡献至多为4-10%。此外，我们不仅通过人工操作而且通过DNA合成器成功地合成了较长的寡核苷酸。我们的新方法的实用性被成功地合成了含有4-N-乙酰脱氧胞苷残基的不耐碱的修饰的寡核苷酸。较少

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