Chemical Research on Pathological Role of Nitric Oxide

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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