ELECTROCHEMICAL OXIDATION OF SOME BIOLOGICALLY SIGNIFICANT PURINES AT SOLID ELECTRODES

Abstract

The electron transfer processes that occur in biological systems are appreciably more complex than in vitro systems. These processes consist a series of complex steps in which an electron transfer is usually accompanied by a proton transfer. Molecular oxygen is usually involved in such reactions and requires a carrier before initiating the process and infact, each step in the mechanism is enzyme catalyzed. Electrochemical oxidation has been found to provide rather unique insights into the mechanism of biological redox reactions, as the electrochemical and biological redox reactions have several superficial similarities. The electrochemical studies of biologically significant molecules represent one of the most useful applications of electrochemistry. In recent years it has been demonstrated that electrochemical studies when coupled with other instrumental techniques such as XH NMR, mass spectrometry and molecular spectroscopy can probe useful insights into the mechanism of complex enzyme catalyzed reactions. In view of the importance of electrochemical investigations, the redox behaviour of some biologically important molecules such as alkyl purines and their derivatives are elucidated in the present dissertation. Studies were carried out using variety of electroanalytical techniques along with other sophisticated spectroscopic and analytical techniques. For simplicity and clarity the results of the investigation are organized in the dissertation as follows : (») The FIRST CHAPTER of the thesis is General Introduction and highlights the results of the significant work reported in the literature in this area. Dialkylpurines are claimed to possess pharamacological and physiological activity such as cardiodepressive, hypotensive, antidiuretic and antilipolytic as they are competitive antagonist at A1 and A2 adenosine receptor subtypes. In view of the importance of dialkylpurines, the SECOND CHAPTER of the dissertation is devoted to the electrochemical and enzymatic oxidation of two dialkyl purines viz., 1,3-Dimethyluric acid (I) and 7,9-Dimethyluric acid (1). These compounds were selected to study the effect of methyl groups on the redox mechanism of purines. In the first case (I) methyl groups are in the pyrimidine ring of purine and in the second case (1) in the imidazole ring. The effect of electrode material on the redox reaction of these compounds has also been studied by using Pyrolytic graphite, Glassy carbon and Platinum electrode. 0 CH HN n cAAn H | CH3 (I) (1) The oxidation of these compounds occurs in a single well defined peak in linear and cyclic sweep voltammetry at all the electrodes used. The peak potential of oxidation peak Ia in case of 1,3-Dimethyluric acid was pH dependent. The Ep vs. pH plot exhibited a break at around pH 5.6 which (iii) N 0 corresponded to the pKa of compound (I). The nature of the electrode reaction was established as EC in which charge transfer is followed by irreversible chemical steps. Spectral changes during electrooxidation were monitored and the kinetics of the decomposition of UV-absorbing intermediate generated during electrooxidation was followed by change in absorbance with time at selected wavelengths. The products of oxidation were separated by Gel permeation chromatography, HPLC etc. and were characterized by m.p., lH NMR, Mass and IR spectrometry and a tentative mechanism for the oxidation of 1,3-Dimethyluric acid has also been suggested. Similar studies were carried out for 7,9-Dimethyluric acid and the products of electrooxidation were characterized and found as alloxan and 1,3-dimethylurca at pH 3.0 and 1,3-dimethylallantoin and 1,3-dimethyl- 5-hydroxyhydantoin-5-carboxamide at pH 7.0. The effect of methyl group on ease of oxidation has also been evaluated. The peroxidase catalyzed oxidation of both these compounds has also been studied using Horseradish peroxidase type VIII enzyme and catalyzed by hydrogen peroxide. The spectral changes during enzymatic oxidation and kinetics of decomposition of UV-absorbing intermediate generated were carried out. The decay of the intermediate was found to follow first order kinetics. A comparison of the electrochemical and enzymatic oxidation studies indicates that both the oxidations proceeds by identical mechanism. The THIRD CHAPTER of the thesis deals with the electrochemical oxidation of two methylated xanthines viz., 1,3-Dimethylxanthine (1) (iv) and 7-methylxanthine (I). ° o <rH3 CHj-N'V \ CH3 H J, (1) (I) Studies were carried out in phosphate buffers and in both the compounds a well defined peak Ia was observed when the sweep was initiated in the positive direction. The electrode process was found to involve excessive adsorption of the reactant at the surface of PGE. As electrochemical oxidation of xanthine has been found to proceed via uric acid, it was considered interesting to check whether the electrooxidation of these two xanthines proceeds via their corresponding uric acid. For this purpose, cyclic voltammograms of 1,3-Dimethyluric acid and 7-Methyluric acid were also recorded under identical conditions. It was found that a well defined peak IIa was observed in the second sweep at exactly same potential as observed in the case of compounds (1) and (I) with the same dE /dpH. Tentative mechanisms for the oxidation are also suggested. In biological systems purines usually occur as nucleosides or nucleotides and it is generally observed that the redox behaviour of nucleotides and nucleosides is much more complicated than their parent bases. Hence, it was considered interesting to investigate the electrochemical behaviour of a purine nucleoside, 7-Methylxanthosine (I) and is the subject (v) CH3 O I ' X J* O n I H HOH,C ^O ^ OH OH (I) matter of FOURTH CHAPTER of the dissertation. The oxidation of 7-methylxanthosine (I) occurred in a single well defined oxidation peak Ia in the entire pH range of 2.3 to 10.7. In the reverse sweep a reduction peak IIIC was noticed. When the direction of sweep was again changed one more anodic peak IIa was observed at less positive potentials. The UV-Vis spectral changes during electrooxidation were monitored and a UV-absorbing intermediate was found to generate in the wavelength region 230 to 290 nm. The kinetics of the decay of the intermediate was followed spectrophotometrically. The products of the electrode reaction were also separated and analysed. On the basis of these studies, it is concluded that ribose group strongly influences the adsorption of this molecule through production of additional adsorption sites in the molecule. Thus, on the basis of these studies it can be concluded that electrochemical oxidation coupled with molecular spectroscopy provides important information about the hidden aspects of biological reactions.