Estudo eletroquímico e eletroanalítico da fluoresceína e dos antineoplásicos raltitrexato e procarbazina usando técnicas voltamétricas e biossensores de DNA

Abstract

An electrochemical and electroanalytical study of the biomarker Fluorescein (FLSC) and the antineoplastic drugs Raltitrexed (RTX) and Procarbazine (PCZ) was carried out using voltammetric techniques and electrochemical DNA biosensors with the purpose of investigating the oxidation mechanisms of these compounds and the reactivity/toxicity of RTX and PCZ with the dsDNA molecule. Potentialities of redox properties of these compounds for electroanalytical applications were also investigated. The electrochemical and electroanalytical studies of FLSC, PCZ and RTX were performed at GCE, for a wide range of electrolytes with different pH values, using cyclic, square wave and differential pulse voltammetry. The results showed that FLSC, RTX and PCZ are electroactive and undergo irreversible electroxidation, in which FLSC and RTX are eletroxidized under diffusion controled and pH-dependent processes, while PCZ is irreversibly eletroxidized under an adsorption controled and pH-dependent process. in two consecutive steps with the formation of an electroactive product. Both oxidation steps involve the transfer of one electron and one proton, corresponding to the oxidation of the phenolic groups with formation of ortho-quinone derivatives, which are reversibly reduced to form catechol derivatives and/or polymer products. One electron and one proton are removed from the phenolic group at the C6' position in the first oxidation step and at the C3' position in the second step. A FLSC oxidation mechanism is proposed. The diffusion coefficient of FLSC was estimated in pH = 7.0 phosphate buffer (DFLSC = 9.77  10-5 cm2 s-1). A differential pulse voltammetric method for quantification of FLSC in physiological medium was proposed, with a limit of detection of 0.64 μmol L-1 and quantification of 2.13 μmol L-1. The oxidation of RTX at GCE occurs in two consecutive irreversible steps of charge transfer. The first oxidation step is pH independent and occurs with the withdrawal of one electron from the tertiary amine at the N10 position. The second step is pH dependent and involves the withdrawal of one electron and one proton from the carbon at the C9 position. It was demonstrated from the studies with the DNA biosensors the existence of an interaction via electrostatic attraction and/or intercalation between the RTX and the dsDNA molecules. On the other hand, no significant lesion on the dsDNA molecule, such as double helix breakage or oxidative damage, was detected. The oxidation of PCZ occurs in four consecutives charge transfer steps. The first, second and third steps are pH-dependent and occur with the transfer of one electron and one proton, leading to the formation of an electroactive product in acid media, while the last one is pH-independent, involving the transfer of one electron. A reaction mechanism for the electrochemical oxidation of PCZ is proposed, where, in the first and second step, the oxidation occurs on the hydrazine group, followed by isomerization and, after hydrolysis, conversion to benzaldehyde-procarbazine. The third and fourth steps are associated with the oxidation of the benzaldehyde-procarbazine with the production of the Nisopropylterephthalic acid. The chemical degradation of PCZ was investigated in different media (acid, physiological and alkaline) for different periods. A change in the anode behavior of PCZ in physiological media was attributed to the chemical degradation of PCZ with homogeneous formation of electroactive degradation product (s). The in-situ interaction of DNA with PCZ and PCZ-Cu2+ were investigated using electrochemical DNA biosensors. Damage to the dsDNA molecule was clearly detected after biosensor incubations in PCZ-Cu2+ solutions, which were associated with changes in the structure of the double helix and oxidative damage in the purine bases. The knowledge of the oxidation mechanisms of FLSC, RTX and PCZ as well as the electroanalytical methods developed for the determination of these substances and the RTX-DNA and PCZ-DNA interaction studies presented here correlate with each other and enrich the literature with information for the interpretation of the molecular biochemistry of these species.