OXIDATION CHEMISTRY OF SOME BIOLOGICALLY IMPORTANT PURINES AND THEIR NUCLEOSIDES

Abstract

For the last two decades, the electrochemical behaviour of biologically important organic compounds and their precursors has been investigated extensively. Reactions in biological systems are usually complex and biologically active site is probably deep within the molecular frame work, hence it is very difficult to get an idea about these reactions. The best way of obtaining the information, closer to the actual situation of biological phenomenon on a certain compound, would be by examining the electrochemical behaviour of that compound at the electrode-solution interface. It is now a well established fact that such studies provide much better picture of the chemical aspects of the enzyme-catalysed redox reactions of these compounds. In recent years, it has been demonstrated that the electrochemical studies when coupled with other instrumental methods such as molecular spectroscopy, H NMR, mass spectrometry, etc. have the potentialities to probe and elucidate the mechanistic aspects of such complex enzyme-catalysed redox reactions. In view of the importance of electrochemical investigations, the redox behaviour of some biologically important organic compounds, viz., N-methylated derivatives of purines and nucleoside of guanine, are elucidated in the present dissertation using various electrochemical techniques in combination with other sophisticated spectroscopic and analytical techniques. The results of the electrochemical investigations on N-methylated purines and guanine nucleoside are organised as follows : The first chapter is the general introduction and presents a review of the relevant work reported in the literature. It also presents some salient features of the techniques used in the dissertation. Uric acid is the primary end product of purine metabolism and is a constituent of many body fluids. In view of the importance of uric acid in biological processes, the second chapter of the thesis has been devoted (i) to the extensive electrochemical investigations carried out on three Nmethylated uric acids, viz., 9-methyluric acid; 1,7-dimethyluric acid and 1,3,7- trimethyluric acid. The electrochemical oxidation of these N-methylated uric acids was studied at different solid electrodes to monitor the effect of electrode material on the redox reactions. The electrodes selected for the studies were pyrolytic graphite electrode (PGE), glassy carbon electrode (GCE) and platinum electrode (Pt). Linear sweep voltammetry of all the three methylated uric acids exhibited a well defined, pH-dependent anodic peak (Ia) at each of the electrodes used. The peak potential shifted to a less positive potential with an increase in pH in each case. The shape of the peak obtained was slightly different at different electrodes. The peak was broader at platinum electrode in comparison to that at GCE and PGE while it was spiky at PGE. The Ep versus pH plots at all the three electrodes exhibited a break at pH -4.5, -5.8 and - 6.0 in 9-methyl- ; 1,7-dimethyl- and 1,3,7-trimethyluric acid respectively which corresponds to the pKa values of these compounds. In cyclic voltammetry at a sweep rate of 100 mV s"1 a single anodic peak (Ia) was observed at all the three electrodes. However, in the reverse sweep different behaviour was noticed. In 9-methyluric acid at PGE and GCE the reduction peaks Ic and IIC were observed whereas no reduction peak was obtained at platinum electrode. In 1,7-dimethyluric acid at PGE two reduction peaks Ic and IIC were obtained, whereas at GCE only peak Ic was obtained. No cathodic peak was obtained at platinum electrode. In 1,3,7-trimethyluric acid at PGE three reduction peaks (Ic, IIC and IIIC) were noticed whereas at GCE and platinum only one reduction peak (Ic) was observed. The nature of the electrode reaction was established as EC (electrochemical step followed by chemical step) in all the three methylated derivatives. The UV-spectral changes and the kinetics of the decay of the UV-absorbing intermediate generated during the reaction were found essentially same at each of the three electrodes in various methylated uric acids. The products of the electrode reaction were (ii) separated by using gel-permeation chromatography. Products were characterized by mp, H NMR, mass, etc. and based on the experimental results obtained, tentative mechanisms for the oxidation of these compounds have been proposed. The effect of methyl group on the oxidation of uric acid has also been evaluated. The methyl group beside producing an electron releasing effect also restricts the number of resonating structures in the case of methylated uric acids. It has been suggested on the basis of our studies that methylation of nitrogen atom at pyrimidine ring affects the oxidation mechanism resulting in products different from that obtained in uric acid. The enzymic oxidation of 9-methyl- ; 1,7-dimethyl- and 1,3,7-trimethyluric acid has also been studied using horseradish peroxidase Type VIII and catalysed by hydrogen peroxide. The spectral changes during enzymic oxidation and the decay of the UV-absorbing intermediate generated have been studied. Catalase was used for quenching the enzymic oxidation for studying the decay of the UV-absorbing intermediate. A comparison of the electrochemical behaviour of these N-methylated uric acids with their enzymic oxidation, has also been presented. Purines usually occur in biological system as nucleosides or nucleotides. The behaviour of the nucleosides are much more complicated than their parent bases. It was therefore, considered interesting to investigate the electrochemical behaviour of guanosine. As 2e, 2H+ oxidation of guanosine readily gives 8-hydroxyguanosine, therefore, electrochemical studies on guanosine as well as 8-hydroxyguanosine were carried out at PGE and GCE using various electroanalytical techniques and the results obtained are presented in the third chapter. It was found that an overall 4e, 4H+ electrooxidation of guanosine occurred in two 2e, 2H+ reactions. On the basis of the electrochemical, spectroelectrochemical and product analysis of guanosine and 8-hydroxyguanosine at both PGE and GCE, it has been suggested that oxidation (iii) of guanosine proceeds via the formation of 8-hydroxyguanosine. The peroxidase-catalysed oxidation of 8-hydroxyguanosine was also investigated and found to proceed by a mechanism identical to electrochemical oxidation. Purines and their nucleosides are one of the most important class of organic compounds being vital for normal cellular functions. It is therefore believed that the present study on electron transfer reactions of these compounds at different solid electrodes and the consequences of such reactions will provide fundamental information and guidance about their in vivo redox reactions.