Nitrosative guanosine deamination : pyrimidine ring opening implications of effects in homogeneous solution as well as anisotropic environments
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] In recent years a great deal of interest has been focused on the exposure to nitrous acid (HNO2), nitric oxide (NO) and other nitrosating species because they are mutagenic agents. These mutagenic agents are known to cause two types of chemical reactions with DNA bases, deamination and interstrand cross linking involving the nitrosation of exocyclic amino group of guanine, adenine and cytosine. The ingestion or inhalation of nitrosating reagents (NO[subscript x][superscript -] ) contained in foods, or the environments have been known to cause in vivo nitrosation. The significance of this knowledge greatly increased with the discovery of endogenous NO synthesis by different nitric oxide synthase enzymes, and the emergence of NO releasing drugs and NO-synthase inhibitors. So, nitrosative DNA base deamination represents an important mechanism of endogenous DNA oxidation and contributes towards various diseases via cytotoxicity and mutagenesis. DNA base deamination involves the replacement of the exocyclic amino group of a nucleobase by a hydroxyl group on treatment with nitrogen oxides. Nitrogen oxides enter the body as nitrates and nitrites where they are subsequently metabolized to nitrous acid. Deamination of DNA bases can occur spontaneously, converting the nucleobases guanine to xanthine, cytosine to uracil, and adenine to hypoxanthine resulting in highly mutagenic lesions. The cellular level of these lesions increased substantially when cells are exposed to this DNA damaging agent, either from dietary or endogenous sources. This dissertation mainly focuses on the mechanism of guanosine deamination in homogenous as well as ss-DNA and anisotropic environment of ds-DNA by [superscript 17]O- and [superscript 18]O-labeling studies. On the basis of chemistry of primary amine, it was believed that guanosine deamination involves the nitrosation of the exocyclic amine resulting in the formation of guanine diazonium ion, while keeping the pyrimidine ring intact. Yet, this diazonium ion has never been isolated or observed experimentally. Our theoretical studies of isolated guaninediazonium ion established that its unimolecular dediazoniation is accompanied by concomitant amide bond cleavage and leads to the pyrimidine ring-opened intermediate 5-cyanoimino-4-oxomethylene-4,5- dihydroimidazole via our proposed pathway. Addition and recyclization chemistry of the intermediate could explain the formations of all known products of nitrosative guanosine deamination, xanthosine, oxanosine and the cross-links. To explore all possible mechanisms, and to establish unequivocally that the pyrimidine ring-opening event occurs along the reaction path that leads to the formation of oxanosine, we performed series of reactions. We studied the nitrosative deamination of guanosine in (17O)water and implications are discussed in Chapter 1. We also studied nitrosative deamination of guanosine in ([superscript18]O)water, and of [6-[superscript18]O]- guanosine in normal water. For that we synthesized [6-[superscript 18]O]-guanosine, which is discussed in Chapter 3. While we wanted to synthesize [7-[superscript18]O]-oxanosine by employing the enzyme adenosine deaminase, surprisingly we found a new compound: 1-[beta]-(D-ribofuranosyl)-5-ureido-1Himidazole-4-carboxylic acid from oxanosine while it was treated with ADA. We identified and characterized the compound and discussed implications of oxanosine for the quest for a toxicological marker for nitrosation activity in Chapter 2. We also studied anisotropic environmental effect in nitrosative guanosine deamination and provided the results in Chapter 4. Our comparative analysis of nitrosation in dG, ss- and ds-DNA environment proved that the xanthosine (dX and after depurination, X) is the main product in all experiments because in ds-DNA, cytosine catalysis at near-neutral pH provides for a preference for water addition to the cyanoimine moiety as compared to the ketene moiety of 1. Whereas, at lower pH the cytosine catalysis mechanisms can account for an increase in dO formation. Again in very acidic condition N7 protonation lead to depurination and followed by water attack in cyanoimine moiety lead to X. By ESI(+)MS, we provided direct experimental evidence for the existence of the pyrimidine ring-opened cations we had proposed on the basis of theoretical studies as intermediates in nitrosative nucleobase deamination in Chapter 5. Chapters 6 and 7 are about computational study of formation of aziridinium ion from a tricyclic marine alkaloid Fasicularin, containing a thiocyanate moiety and a hexyl side chain; and its analogue 1-methyl-3-thiocyanato-piperidine (TCP). The equilibrium structures of fasicularin parent system, 1m and TCP, and formation of fasicularin derived aziridinium ions from both are investigated using B3LYP methods. The low energy barrier of the formation of aziridinium ion from boat-conformation of fasicularin in the presence of the continuum solvent model proves fasicularin as the first natural product, which has the ability to form DNA-alkylating aziridinium ion by displacing thiocyanate moiety. The thesis ends with the Chapter 8, where we studied oxanine formation mechanism in gas phase as well as aqueous phase.
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