Chemical Research on Pathological Role of Nitric Oxide

探讨炎症中NO过量产生的促癌机制具有重要意义。在此项目之前，我们证明了NO可以高产地将dGuo转化为脱氧氧苷（dOxo），并将dCyd转化为dCyd-重氮酸盐（一种反应中间体）。在这个项目中，我们揭示了以下几点。(1)确定了dGuo-重氮酸盐作为dGuo转化为dOxo的中间体，并建立了完整的反应机理。基于该机理，我们发现原来认为是NO破坏碱的氧化脱氨作用并不适用于dGuo，而是由NO攻击NH_3引发开环，然后形成重氮酸盐，水分子加入到所产生的阳离子中，然后闭合环。(2)已确定dOxo和重氮酸盐之间的紧密连接反应，表明NO促进癌症和细胞死亡。研究还发现，抑制基因p53的CpG位点的主要成分5-甲基胞苷（5-methycytidine）被转化为^<5me> dcyd -重氮酸盐。(3)建立了毛细管电泳和高效液相色谱结合荧光标记的dOxo检测方法。HPLC法检测限为2 ~ 3 fmol，估计HNO_2环境下大肠杆菌中dOxo的生成量小于检测限。(4)为了探究dOxo形成引起的DNA结构变化及其误读机制，我们尝试制备了酰胺磷单体。由于5'-OH的反应活性较低，DMTr-dOxo的产率为30%。(5)我们在已知的修复酶中探索了识别dOxo的修复酶，发现AlkA和Endo VIII具有非常轻微的缺失活性。(6)通过2D-NMR研究了人类易受NO攻击的端粒中重复的DNA寡聚物dCCCTAA，发现了3个同分异构体的四链结构，提示如此简单的序列如此复杂的结构多样性可能在控制细胞死亡和促癌中发挥作用。少

Exploration of cancer promotion mechanism caused by NO overproduced in inflammation is of great importance. Before this project, we demonstrated that NO converts dGuo to deoxyoxanosine (dOxo) with high yield and dCyd to dCyd-diazoate, a reaction intermediate. In this project, we have revealed the followings.(1) We have identified dGuo-diazoate as an intermediate for the dGuo conversion to dOxo and established the complete reaction mechanism. Based on the mechanism, oxydative deamination which has been thought to be responsible for the base damages by NO, has been found to be not true for dGuo, and ring-opening initiated by NO attack to NH_3 followed by the diazoate formation, water molecule addition to the resulting cations, and ring-closure has been found to be involved.(2) Closs-linking reactions between dOxo and diazoates have been identified, indicative of promotion to cancer and cellular death by NO. Also it has been determined that 5-methycytidine, major component in the CpG site … More of inhibitory gene p53 which is known as a hot spot for cancer promotion, is converted to ^<5me>dCyd-diazoate.(3) Detection methods for dOxo have been developed based on capillary electrophoresis and HPLC combined with fluorescent labeling. Using the HPLC method with the detection limit of 2-3 fmol, the amount of dOxo generated in E. coli exposed to HNO_2 was estimated to be less than the detection limit.(4) To explore structural change of DNA caused by dOxo formation and the following misreading mechanism, we tried to prepare phosphoroamidite monomer. Because of the low reactivity of 5'-OH, the yield of DMTr-dOxo was 30%.(5) We explored repair enzymes recognizing dOxo among known repair enzymes and found that AlkA and Endo VIII show a very slight deletion activity.(6) A DNA oligomer, dCCCTAA which is a sequence repeated in human telomere susceptible to NO attack, has been investigated by 2D-NMR and the three isomeric four-stranded structures have been found, implying that such a complex structural diversity of such a simple sequence may play a role controlling cell death and cancer promotion. Less