Synthesis and characterization of putative triplet sensitizers in the dark pathway to cyclobutane pyrimidine dimers of DNA
Date of Award
Doctor of Philosophy (PhD)
The cyclobutane pyrimidine dimer (CPD) is the major photoproduct resulting from direct absorption of UVB radiation in sunlight by DNA that results in C to T mutations found in human skin leading to skin cancer. CPDs can also be produced through the action of photosensitizers that can absorb longer wavelength UVA that is not absorbed efficiently by DNA and transfer triplet energy to the DNA. Most recently, CPDs was discovered to be formed in the dark following UV irradiation of melanocytes. This dark pathway was proposed to result from an indirect pathway to CPDs that involves triplet energy transfer from an as yet unknown chemi-excited molecule. It was suggested that when oxidized by peroxynitrite, melanin precursors might function as precursors to dioxetanes that decompose to excited triplet states capable of sensitizing CPD formation in DNA. To investigate this possibility, we synthesized and characterized the oxidation products of indole derivatives related to the proposed dioxetanes dertived from melanin that were proposed to be involved in the dark reaction. We first studied the ability of indole oxidation products to generate CPDs in DNA via photosensitization and concluded that melanin precursors are an unlikely source of the chemisensitizers. Then we investigated CPD formation induced by peroxynitrite in the presence of various classes of molecules and reductants to test our hypothesis that chemisensitization does not involve dioxetanes, but rather occurs via an excited state of a reduction product of peroxynitrite. The oxidized indole products of indole (IOP), tryptamine (TOP), hydroxy-indole (HOP) and dimethoxy-indole (DOP) were prepared by standard organic synthesis. The triplet state energies and lifetimes of these indole oxidation products were obtained by analysis of time dependent phosphorescence measurements and compared with norfloxacin (NFX) which has the lowest energy triplet energy known to triplet excite DNA. The results showed that while all four compounds efficiently form triplet states, only IOP and TOP have triplet energies that are higher than (NFX). The ability of the compounds to photosensitize CPD formation in DNA was then determined using a T4 endonuclease V (T4-pdg) assay. The result showed that only IOP and TOP can photosensitize DNA to form CPD, which is consistent with having triplet energies hgher than NFX. Therefore, the products of dioxetanes of oxygen-substituted indoles are unlikely to be involved in the “dark” pathway to CPDs in melanocytes. To investigate the possibility that the organic molecules causing chemi-sensitization were not functioning as substrates for peroxynitrite, but rather as reductants, preoxynitrite was incubated with various organic compounds and inorganic reducing reagents at pH 8.8. CPD formation was observed to occur with the organic compounds 5-hydroxyindole-2-carboxylic acid (5OH2CA), 5-hydroxyindole (HI), phenol, 2,3-dihydroxybenzoic acid (DHBA), 3,4-dihydroxy-DL-phenylalanine (DL-DOPA), and tryptophan (Try), and the inorganic reagents Na2S2O3, NaBH4 and NH2OH. Given that inorganic reducing agents were able to induce CPD formation in the presence of peroxynitrite, as well or better than the organic compounds and that organic compounds with high oxidation potential were ineffective, we conclude that chemisensitization takes place through reduction of peroxynitrite. Therefore, we propose that the dark pathway takes place via a triplet excited state produced by reduction peroxynitrite that excites CPD formation in DNA.
Chair and Committee
John-Stephen Taylor, Kevin Moeller, Michael L. Gross, Kimberly Parker,
Wang, Yanjing, "Synthesis and characterization of putative triplet sensitizers in the dark pathway to cyclobutane pyrimidine dimers of DNA" (2021). Arts & Sciences Electronic Theses and Dissertations. 2470.