ELECTROCHEMICAL INVESTIGATIONS OF SOME BIOLOGICALLY IMPORTANT PURINES AND THEIR DERIVATIVES

Abstract

The transfer of an electron between two chemical entities is one of the most fundamental processes in chemistry and biochemistry. In biological systems, oxidative processes are known to play a dominant role in energy conversion and substrate metabolism. Typical of these are those involved in catabolism of many biologically important organic compounds. Redox pathway of such type however, are rather complex due to enzyme interactions and are very difficult to study in their entirety. 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 biological transformations undergoing in the living systems. In view of the importance of electrochemical studies in elucidating the mechanism of enzyme-catalysed reactions our laboratory has been investigating the electrochemical behaviour of various biologically important compounds. The present dissertation deals with the electrochemical oxidation of a variety of purines and their nucleosides and nucleotides. The peroxidase/H202 catalysed oxidation of these compounds, wherever possible has also been studied. The work reported has been divided into five chapters. The first chapter of the thesis is "General Introduction" which provides a compendious review of the pertinent work and their significant results. The second chapter of the thesis deals with the electrochemical investigations of Uric acid (I) in micellar media. Investigations in micellar medium were performed as the microenvironment of micellar systems is analogous to that of the active and binding sites of enzymes in a physiological system. Hence, attempts were made to evaluate the effects of ionic and nonionic surfactants on the differential pulse voltammetric behaviour of uric acid. It was observed that the peak potential and peak current significantly alter in the presence of ionic surfactants. The studies indirectly supported the formation of a cationic free radical in the electrode reaction. (1) The third chapter of the thesis presents the electrochemical oxidation of 3,7-dimethylxanthine (II) and 3,7-dimethyluric acid (III) at different solid electrodes to investigate the effect of electrode material on the redox reactions. The electrodes chosen for the studies were pyrolytic graphite (PGE), glassy carbon (GCE) and platinum (Pt). p 7I^ o CIH^3 II N UN H-N"V V „n H-N H (ii) n~l |T >0 (in) CH3 H CH3 H Asingle well- defined anodic peak (Ia) was observed in the linear sweep voltammetry of 3,7-dimethylxanthine only at pH > 7.0 at PGE and GCE. At pH < 7.0, the oxidation peak appeared to merge with the background current. Oxidation peak was not observed at platinum electrode even at pH > 7.0. The Ep us pH plots at both the electrodes were linear in the pH range 7.0 to 10.7. (Hi) Cyclic sweep voltammetry at PGE and GCE at a sweep rate of 200 mV s"1 exhibited a well defined anodic peak Ia at pH >7.0. In the reverse sweep, a cathodic peak IIIC was observed and in the subsequent sweep towards positive potentials one more anodic peak (IIJ was noticed in the pH range 2.1-6.0. The nature of the electrode reaction was established as EC in which charge transfer is followed by competitive chemical reactions. Controlled potential electrolysis provided the value of n, number of electrons transferred in the redox reaction as 4.0 ± 0.2. Spectral changes during electrolysis were monitored to detect the formation of UV-visible absorbing intermediate. The decay of the intermediate generated during the oxidation was found to occur in pseudo first-order reaction. The products of electrolysis were separated by gel-permeation chromatography and characterized by mp, IR, H NMR, mass etc. and a plausible mechanism for the formation of products is also suggested. The effect of methyl groups on the oxidation of xanthine and uric acid has also been evaluated. It has been found that the presence of methyl groups at positions 3 and 7 in xanthine and uric acid makes the oxidation difficult and alters the redox behaviour by its electron donating nature resulting in the formation of different products. The enzymic oxidation of 3,7-dimethyluric acid has also been carried out using horseradish peroxidase type VIII enzyme and catalysed by hydrogen peroxide. It was interesting to observe that the electrochemical and peroxidase catalysed oxidations of 3,7-dimethyluric acid yielded intermediate and products, which were kinetically, spectrally and analytically identical. Purines occur in every living cell, usually as constituents of large molecules as nucleoside and nucleotides. In our continued study into the electrochemical and enzymic oxidation of purines, the effect of ribosophosphate substituent on (iv) the redox properties of uric acid, was studied. For this purpose, 9-(3-dribofuranosyluric acid 5'-monophosphate (UA-9R-5'-P, Compound IV) was selected for the present investigations and the results obtained forms the fourth chapter of the thesis. OH (IV) HO-POCl^ Electrochemical investigations of compound IV were carried out at PGE, GCE and Pt electrodes. The cyclic voltammograms of nucleotide (IV) showed a well- defined oxidation peak (Ia) in the pH range 2.1-10.0 at PGE and GCE. The Ep vs pH plot exhibited a break at around pH 6.0, which corresponded to the pKa of UA-9R-5'-P. In the reverse sweep, reduction peaks Ic, IIC and IIIC were observed. Peaks Ic and IIC formed quasi-reversible couples with peaks Ia and IIa observed in the subsequent sweep towards positive potentials. The formation of a UV-absorbing intermediate is noticed at around 290 nm during electro-oxidation which disappeared in a pseudo first-order reaction (k = 1.8-2.0 x 10" s" ). The major products of electro-oxidation were characterized and a tentative reaction mechanism has also been suggested. A comparative study of the electrochemical and enzymatic oxidation indicated that the two oxidations proceed by identical reaction mechanisms. Linear sweep voltammetric studies of compound IV were also performed in micellar medium using cationic, anionic and non-ionic surfactants. The effect (v) of various surfactants on the peak potential and peak currents of UA-9R-5-P were also monitored. It was observed that the oxidation peak (Ia) splitted in two well - defined peaks in presence of cationic surfactant at pH 3.2. No splitting was noticed in presence of anionic and non-ionic surfactants. The last chapter of the thesis is devoted to the electrochemical oxidation of a nucleoside of 6-mercaptopurine (V), which has been claimed as one of the most effective drug used in cancer therapy. HOCH^ OH OH Linear and cyclic sweep voltammetry of 6-mercaptopurine riboside at a sweep rate of 20 mV s"1 exhibited a single well defined oxidation peak (Ia). Effect of pH, concentration and sweep rate were studied and the results are presented in this chapter. On the basis of linear and cyclic sweep voltammetry, coulometry, spectral studies during electro-oxidation and product identification, a mechanism for the oxidation has been suggested. Thus, it can be inferred on the basis of these studies that if an electrochemically driven and enzymatically mediated redox reactions can be shown to yield the same products profile, then a careful investigation of such electrode processes can provide profound insights into the chemical mechanism associated with the biochemical reactions.