It is the goal of our research to provide knowledge about disease processes that are important for genetic toxicology and carcinogenesis caused by endogenous nitrosation. Significant progress toward this goal can be achieved by the investigation of the chemical mechanisms of nucleobase deamination, by the identification of the reactive intermediates that are responsible for DNA damage and mutation, and by studies of their reactions. It is the principal hypothesis of this proposal that the key intermediates in the nitrosative deaminations of the three nucleobases, guanine, cytosine and adenine, may undergo pyrimidine ring-opening reactions. It is our aim to provide experimental and computational evidence in support of this mechanistic hypothesis. This new understanding of the mechanisms will not only provide a consistent explanation for all known products of nucleobase deaminations, but it will help guide the search for new toxins and new modes of DNA modification including new types of interstrand cross-links. Only a full knowledge of the deamination mechanisms can allow us to know all of the possible products of the deaminations. This, in turn, can lead to a better understanding of many disease processes within the body. Our proposal contains the following specific aims: 1. To test our hypothesis that pyrimidine ring-opening occurs during the nitrosative deamination of the nucleobases by way of 17O- and 18O-labeling experiments in aqueous media. 2. Independent syntheses of selected postulated intermediates will be carried out and their chemistry will be explored in aqueous media to test our hypothesis that pyrimidine ring-opening occurs during the nitrosative deamination of the nucleobases and to test the chemical competence of the postulated intermediates to form the observed products. 3. Adduct and cross-link formation in synthetic oligonucleotides will be studied under conditions that closely mimic gastric nitrosation to test our hypothesis that the products formed by pyrimidine ring-opening during the nitrosative deamination of the nucleobases may lead to novel adduct formation and the formation of interstrand cross-links. 4. High level ab initio molecular orbital calculations will be employed to characterize the structures and the thermodynamic and the kinetic stabilities of all of the postulated intermediates, and to determine the most likely course of their reactions. 5. The computational studies will include studies of the bimolecular reaction mechanisms for dediazoniation of the nucleobase diazonium ions.
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