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-

在此之前，由于dA和dC的氨基对三价磷（III）型磷酸化试剂具有很高的反应性，因此无法在无n保护的磷酸方法中进行Ο-selective在聚合物载体上的磷酸化。在本文中，我们开发了一种新的偶联策略，称为“活化亚磷酸酯法”，其中磷酸化反应是由亚磷酸酯三酯中间体如化合物1介导的。在固相合成TpA和TpC衍生物的过程中，利用HOBt的缩合率几乎达到100% Ο-selectivity，并且可以得到高收率的分离产物。随后，我们检测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，使用各种1-羟基苯并三唑和酚型启动子的磷酸化Ο-selectivity。将HOBt作为促进剂应用于固相合成，获得了优异的Ο-selectivity收率，达到99.7%以上。我们根据反应中间体与亲核试剂（如核苷的氨基或羟基）之间的FMO相互作用解释了这一点Ο-selectiv…More ity。还发现，随着促进剂酸度的增加，使用hobt型促进剂的缩合过程中Ο-selectivity降低。对该方法机理的详细研究表明，在此缩合过程中，亚磷酸酯苯并三唑酯中间体通过重排转化为相应的五价磷，该五价磷仍然具有羟基活性。据估计，在缩合反应中，这种五价物质对整个磷酸化的贡献最多为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更长的寡核苷酸。成功合成了具有4- n -乙酰脱氧胞苷残基的碱不稳定修饰寡脱氧核糖核苷酸，证明了新方法的实用性。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