Estudo da reatividade ao DNA de derivados ferrocênicos, utilizando técnicas eletroquímicas e espectroscópicos

Abstract

DNA-reactive agents are associated with several types of interaction with DNA, including intercalation, non-covalent binding to the grooves, covalent bonding / crosslinking, DNA breakage and the incorporation of nucleosides producing alterations in its structure. When these changes are irreversible, they might be transmitted, causing genotoxicity. DNA is an important therapeutic target, despite its lower selectivity. In recent years, organometallic molecules have been described as promising compounds in the therapy against cancer and other diseases. Given the attractive pharmacological profile identified for ferrocene derivatives, this work aims to use biosensors of dsDNA and ssDNA in solution, for a pharmacoelectrochemical study of the interaction with CT-DNA, also using UV-vis/fluorescence titration of tamoxifen and benzo-hydrazide-based ferrocenic derivatives, investigating their DNA-reactivity profile. In the present work, dsDNA biosensor, ssDNA in solution, quartz crystal microbalance (EQCM) and electrochemical evaluation were used; UV-Vis spectroscopy at a fixed DNA and increasing concentration of the analytes, in Tris-HCl buffer pH 7.2, with 10 and 30% ethanol. The inhibition of ethidium bromide (BE)-DNA complex fluorescence was assessed at increasing concentrations of the compounds, at the same conditions as used for UV-Vis studies. The retardation of electrophoretic mobility (REM) was performed on DNA pUC 19 at compounds’ concentration of 100 mol L-1. The electrochemical results showed a disturbance of the native structure of dsDNA and exposure of Guanine and Adenine bases, in one case, and only adenine, in the other case, upon interaction with P41 and P365, respectively. Ferrocene itself did not show interaction. A direct interaction with ssDNA was observed only with P41; by EQCM, with evidence of the mass increase of p41 in the presence of the SAM of DNA. UV-Vis spectroscopy allowed the calculation of Kf values based on DNA uptake, which were higher for P15, with Kf = 1 x 105 L mol-1 and P41 Kf = 2.4 x 104 L mol-1. The constants calculated by fluorescence were also obtained and compared with the data of Doxorrubicine, well-recognised as a intercalating drug. Fc-25 showed higher Kf values, all showed substantial accommodations binding stoichiometry 1:1. Through REM, a difference in the presence of H3, comparable to cisplatin, was noticed. The other compounds did not alter the mobility. The combination of techniques and the results obtained suggest that the type of interaction between the compounds and CT-DNA is by intercalation except for H3, which possibly interacts directly with the bases, by hydrogen bonding.