Chemical Research on Pathological Role of Nitric Oxide

探讨炎症过程中NO过度产生所致的促癌机制具有重要意义。在本项目之前，我们证明了NO高产率地将dGuo转化为脱氧氧鸟苷(Doxo)，并将dCyd转化为反应中间体dCyd-重氮酸酯。在本课题中，我们主要做了以下几方面的工作：(1)确定了dGuo-重氮酸酯作为dGuo转化为Doxo的中间体，并建立了完整的反应机理。根据这一机制，被认为是NO造成碱基损伤的氧化脱氨作用对dGuo是不成立的，并发现NO攻击NH_3后形成重氮酸盐而引发的开环，所产生的阳离子中的水分子加成，以及环的关闭。(2)Doxo与重氮酸盐之间的Clos键反应已被确定，表明NO促进癌症和细胞死亡。此外，还确定了CpG位点…的主要成分5-甲基胞苷作为促癌热点的抑癌基因P53更多地被转化为重氮环磷酸盐。(3)建立了基于毛细管电泳法和高效液相色谱结合荧光标记法的多柔比星检测方法。利用高效液相色谱方法，检测下限为2-3fmol，估计在HNO_2作用下，大肠杆菌产生Doxo的量小于检测限。(4)为了探索Doxo形成引起的DNA结构变化及后续的误读机制，我们尝试制备了亚磷酰胺单体。由于5‘-OH的低活性，DMTR-Doxo的产率为30%。(5)我们探索了已知修复酶中识别Doxo的修复酶，发现Alka和Endo VIII具有非常轻微的缺失活性。(6)DNA寡聚体dCCCTAA是在人类端粒中重复的序列，容易受到攻击，已发现三个异构体四链结构，这意味着如此复杂的结构多样性如此简单的序列可能在控制细胞死亡和癌症促进中发挥作用。较少

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