In this proposal we plan to continue our long-term objectives of elucidating the precise mechanism of DNA fragmentation by metal-drug complexes. Specifically, we will focus on the chemistry of bleomycin-induced DNA degradation. Our recently developed approach to this question involves the synthesis of DNA models which are specifically tritiated at various positions on the deoxyribose ring of a defined nucleotide. Analysis of the fate of the tritium upon degradation of the DNA model by bleomycin, ferrous ion, and oxygen has enabled us to deduce a mechanistic sequence which accounts for the monmeric products formed during the reaction. Our findings are consistent with an initial hydrogen abstraction by bleomycin of the 4'-hydrogen, followed by formation of a 4'-peroxyl or hydroxyl species which lead to base propenal or free base, respectively. The mechanism for free base formation is based on the chemical labilization of the 3'- and 5'-protons in response to a 4'-keto group generated exclusively by this process. During the tenure of this grant, we plan to investigate: 1) the nature of the large selection effect against tritium in the 4'-hydrogen abstraction; 2) the isolation and identification of the carbohydrate moiety formed during free base release; 3) the effects of different metals and reaction conditions on the fragmentation event; 4) the mechanism of O2 incorporation into the products of bleomycin-induced degradation of DNA; and 5) the effects of bleomycin analogs and synthetic """"""""mimics"""""""" on these mechanisms. Our approach provides a unique opportunity to thoroughly study the precise mechanistic sequence of this chemotherapeutically important DNA-drug reaction.
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