The action of many non-hydrolytic DNA cleaving agents is believed to be based on reactive phenyl radicals that abstract hydrogen atoms from deoxyribose in DNA. Various issues relating to this process, including the drug's binding site on DNA, the mechanism of radical formation, its accessibility to various DNA sites, and the fate of the resulting DNA radicals, are being addressed by scientists to advance the rational design of better antitumor and antiviral drugs. However, the key part of the process, the reaction of the radical intermediate with DNA, remains unexplored. Although information on the factors that control this reaction is critically needed for drug design, nearly nothing is known about the radical intermediates formed from drugs because of severe experimental difficulties in studying such highly reactive species. Our goal is to learn how to control the reactivity and intrinsic selectivity of a radical warhead by structural changes. We have developed a novel experimental approach based on mass spectrometry to study biologically relevant radical reactions. The method involves attaching a charged group to a radical of interest for manipulation in an FT-ICR mass spectrometer wherein small neutral biomolecules are introduced by laser-induced acoustic desorption. This proposal presents a continuation of a four-year project that involved 1) purchase and set-up of the necessary instrumentation, 2) demonstration of the feasibility of the approach, 3) examination of known systems to show that the method yields data relevant to neutral radicals and solution conditions, and 4) collection of kinetic reactivity data to provide several novel structure/reactivity relationships for advancement of the design of better non-hydrolytic DNA cleavers. The most important areas of the currently proposed work include (1) development of structure/reactivity relationships for more complex systems, (2) exploration of the selectivity of different radicals toward different sites in oligonucleotides, (3) examination of the propensity of the radicals to attack peptides to model protein damage, and (4) based on the insights gained in the above studies, development of a reactivity paradigm that provides general predictive power. (5) This paradigm will be used to design target-specific radical warheads and prodrugs. Professors Rick Borch (Medicinal Chemistry and Molecular Pharmacology, Purdue), a specialist in cancer drug design and mechanisms,and Mark Lipton (Organic Chemistry, Purdue), an expert in synthesis of biradical producing prodrugs, will guide the research and help in finding the best way to incorporate knowledge into the design and testing of novel drugs.
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