Mechanistic studies of the natural DNA-cleaving agents neocarzinostatin chromophore, calicheamicin [gamma]1, and dynemicin A

Citation

Cohen, Scott B. (1995) Mechanistic studies of the natural DNA-cleaving agents neocarzinostatin chromophore, calicheamicin [gamma]1, and dynemicin A. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ZJH6-AC74. https://resolver.caltech.edu/CaltechETD:etd-09182007-090157

Abstract

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. Experiments establishing the intermediacy of the cumulene derived from neocarzinostatin chromophore are described. It is shown that [...]95% of DNA cleavage arises via the cumulene. The sequence specificity and efficiency of DNA cleavage by externally generated cumulene are identical to that by the cumulene formed in situ, supporting the proposal that the cumulene determines the sequence specificity of DNA cleavage. It is shown that DNA and a water-soluble cyclohexadiene derivative are equally effective in trapping of the biradical intermediate at concentrations of 5 mM and 1 M, respectively, supporting the idea that the biradical must be generated as a DNA-bound species to induce DNA cleavage. The reaction of calicheamicin [...] with glutathione has been studied in the presence of DNA and is shown to produce all four products arising from S-S bond exchange. The calicheamicin-glutathione disulfide is formed as the major product of this reaction, and is shown to be 2-3 orders of magnitude less reactive toward glutathione than is calicheamicin [...]. The rate of DNA cleavage by calicheamicin [...] is essentially independent of the concentration of DNA, while the rate of DNA cleavage by the calicheamicin-glutathione disulfide is inversely proportional to the concentration of DNA. The data support the hypothesis that calicheamicin [...] undergoes thiol activation as a DNA-bound species, while the calicheamicin-glutathione disulfide is activated free in solution. Binding constants of dynemicin A and synthetic analogs to DNA show that the two E-ring hydroxyls of the anthraquinone contribute approximately 2.7 kcal/mol binding energy, and that neutralization of the negatively-charged carboxylate stabilizes the drug DNA binding complex by ~3 kcal/mol. Dynemicin A and the synthetic analogs display an inverse rate dependence on the concentration of DNA, supporting the proposal that these drugs must dissociate from DNA prior to chemical activation.

Thesis Files