The Characterization of Excited States and Reactive Intermediates in the Photosensibilization of Alkylating Quinones to Produce Dna Covalent Adducts
Díaz Espinosa, Yisaira
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Cancer is the second most common cause of death in the US. About 1,600 people per day are expected to die of cancer in 2014. Cancer have no cure. Many treatments have been developed to treat this group of diseases, characterized by the uncontrolled growth of abnormal cells. One of the most promising anticancer therapies, discovered in the early 1900s and still under investigation, is photodynamic therapy (PDT). PDT is a cancer therapy in which a tumor-localizing drug, called a photosensitizer (PS), combines with molecular oxygen and visible light to produce selective tumor necrosis. In the presence of oxygen, the PS photosensitizes the production of singlet oxygen and superoxide. In the Type II pathway, singlet oxygen is proposed to be the most important species in the process of killing tumor cells. However, 90 % of human cancers are solid tumors that present a lower overall level of oxygen than normal tissues and/or extremely hypoxic areas. Because of the need of the presence of oxygen, direct killing of these hypoxic cells by singlet oxygen is very limited in this pathway. The photo-reduction and photo-oxidation of substrates, occurring in the Type I pathway, have also been proposed as phototoxic events in PDT, especially, in oxygen deficient environments. These photosensitizers could also photoreduce molecules having nearly equal or more positive redox potentials than oxygen in anoxic/hypoxic cells. Quinones have a reduction potential similar to oxygen and can be actived by photosensibilization and used as a bioreductive agent in the PDT. Quinones are important compounds used in antitumor therapy and to treat other human illnesses. The common feature of these compounds is that they are easily reduced to produce reactive intermediates and products. In addition, upon reduction under hypoxic conditions alkylating quinones covalently bind DNA. Recently, Alegría et al. demonstrated that aziridinyl-benzoquinones (BQ) can be reduced by photosensitization, under anoxia, followed by DNA covalent binding. However, the mechanism and specific interactions in these processes have not been established. Due to the lack of information on the mechanism of the reaction, studies on the photochemical reactions of the BQ in the presence of a photosensitizer dye and/or DNA nucleosides are needed. The characterization of the intermediate species generated is very important for proposing a possible mechanism for this reaction. A photophysical and photochemical study of pheophoride-a (PHEO) in the presence of diaziridinyl-benzoquinones (BQ) and/or DNA nucleosides is presented in this thesis. Our ultimate goal is to provide insight on the mechanism whereby the BQ is photosensibilized and to form covalent adducts with DNA. To this end, he photophysical properties of the PHEO has been studied and characterized. The ground-state interaction of the dyes with the BQ and DNA was studied using the UV-VIS absorption technique. Reactivity of the singlet excited state of the dyes in presence of the BQ and DNA was studied by steady-state and time-resolved fluorescence methods. Transient intermediates involved in the triplet excited state photo-reaction were identified and characterized by a laser technique. The PHEO ground state complexes formation with the BQ was studied in EtOH and PBS/7.4. The association constant, the number of BQ molecules interacting with PHEO, and the fluorescence quantum yield of the complex was determined. The complexes showed higher association constants in buffer solution than in ethanol. The PHEO-CARBOQ and PHEO-AZQ complexes showed higher fluorescence quantum yield in EtOH than in buffer solution, which is also higher than of the PHEO monomer. Quenching of the PHEO fluorescence by DNA nucleosides and double-stranded oligonucleotides was also observed and the bimolecular quenching rate constants were determined. The quenching rate constants increased as the oxidation potential of the DNA nucleoside increases. Larger quenching constants were obtained in the presence of the CARBOQ and pBQ, suggesting that these quinones enhances DNA photo-oxidation, presumably by inhibiting the back-electron transfer reaction form the photoreduced PHEO to the oxidized base. The enhanced DNA-base photosensitized oxidation by PHEO in presence of CARBOQ was related to the large extent by which the quinone covalently binds to DNA. The triplet-excited state of PHEO was quenched by the quinones in both solutions, PBS/7.4 and EtOH. The radical anions of each quinone were characterized. The quenching rate constants were obtained for the electron transfer reaction between PHEO and the quinone. The values of the quenching rate constants were higher in PBS/7.4 than in EtOH and were related to the reduction potential of the quinones. The formation of the radical anion of the quinone demonstrated that the ABQ can be activated by photosensibilization. This specie forms covalent bonds with DNA, which could result in cell death. These results support the hypothesis that ABQ could be used as bioreductive drug in PDT Type I pathway.