Type of Document Dissertation Author Breiner, Boris Author's Email Address email@example.com URN etd-11132006-133123 Title Enediynes: C1 - C5 Cyclization and Application to DNA Photocleavage Degree Doctor of Philosophy Department Chemistry and Biochemistry, Department of Advisory Committee
Advisor Name Title Igor V. Alabugin Committee Chair Jack Saltiel Committee Member Kenneth A. Goldsby Committee Member Michael Blaber Committee Member Keywords
- DNA Cleavage
Date of Defense 2006-11-07 Availability unrestricted AbstractThe naturally occurring enediynes are compounds with astonishingly high biological activity, based upon their ability to generate DNA-damaging reactive species. In an effort to find a photochemical way for enediyne activation, the Alabugin lab found a new reaction of enediynes, namely the C1 – C5 cyclization of tetrafluoropyridinyl (TFP)-substituted enediynes to substituted indenes. This study consists of two parts: In the first one, the mechanism of the C1 – C5 cyclization is investigated, while in the second one, lysine-conjugates of TFP-substituted enediynes (along with related fulvenes and acetylenes) are used in DNA photocleavage.
The mechanism of the C1 – C5 cyclization was investigated using photophysical, photochemical, and computational methods and found to be based on subsequent electron-transfer reactions. The initial electron transfer from a suitable donor to the enediyne creates the enediyne radical-anion. An enediyne dianion is formed in the next step, most probably through disproportionation of two radical-anions. The dianion cyclizes and gets protonated twice to form a fulvene intermediate. This fulvene intermediate exchanges an electron with yet another enediyne radical-anion to form the fulvene radical-anion. After protonation and hydrogen abstraction, the final indene product is formed.
The application of lysine-conjugates to DNA photocleavage was investigated using a variety of biochemical methods. The activity of the compounds was determined using plasmid relaxation assays, while the selectivity was tested using radioactively labeled DNA oligomers. The activity of DNA photocleavage was found to be pH-dependent, which is a highly desirable property, considering the acidic conditions commonly found in cancer cells. The reason for this dependence is a combination of tighter binding, favorable photophysics, and improved solubility at lower pH. The selectivity of the cleavage reactions was found to be the result of a compromise between the guanosine (G) selectivity of oxidative DNA damage, and the tendency of ammonium groups to bind at AT-tracts. Surprisingly, this is also true for control compounds that were expected to react in a radical-based fashion. During the investigation, the radioactive label (part of a phosphate group) was found to be the preferred site of attack and cleavage by the lysine-conjugates. This required the implementation of an alternative labeling technique, which improved both the robustness of the label and the sensitivity of the assay.
Recognition of the phosphate group was the basis for a new method to induce DNA damage in a sequence specific fashion. By annealing a target strand with DNA oligomers of varying length, with either terminal phosphate or hydroxyl groups, it was possible to construct a variety of simulated DNA damage sites. The lysine-conjugates recognize these damage sites and the resulting photocleavage is clearly enhanced at the desired location.
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