OXIDATION CHEMISTRY OF SOME PURINES AND THEIR NUCLEOSIDES AND NUCLEOTIDES

Abstract

Electrochemistry is the branch of chemistry that encompasses the interrelation of electrical and chemical effects. The all-important feature is the application of an electrochemical potential to monitor and control the extent of redox reactions of biologically important compounds. The importance of a redox reaction involves the response obtained when the electroactive species is converted in a heterogeneous electron - transfer reaction to the product. In biological systems, oxidative processes play an important role in energy conversion and substrate metabolism. Reactions in biological systems are usually complex in nature and the biologically active site of interest is probably deep in the molecular framework. Hence, understanding the transfer and storage of energy occurring via various metabolic processes at a molecular level is very difficult. Electrochemistry here acts as an excellent appliance to explore the mechanism underlying the couple of chemical processes for energy utilization in the living organism. Superficial similarities between enzyme catalyzed reaction and electrochemical reactions further create interest in such studies. In recent years, it has been clearly demonstrated that coupling of non-electrochemical techniques, such as 1HNMR, mass, molecular spectroscopy with electrochemical techniques has the potentiality to elucidate the mechanistic aspects of complex enzyme catalyzed biological transformations. In view of the importance of electrochemical techniques, our laboratory has been investigating the redox behaviour of various biologically important compounds such as uric acid, adenine, hydroxyadenines etc. Purines are constituents of well-known nucleic acids i.e. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which play an important role in the physiology of human system. Recently, electrooxidation of some purine nucleosides and nucleotides, which are carbohydrate derivatives of purines, was carried out in our laboratory due to their biological and clinical significance. The work presented in this thesis is a small part of the big proposal submitted by Prof. R.N. Goyal in connection with the redox chemistry of biomolecules. The present thesis deals with the electrochemical oxidation studies of methylated purines and N-oxides, nucleosides and nucleotides of adenine. For simplicity the results of the investigation are organized in the thesis as follows: The first chapter of the thesis is "General Introduction" and presents a brief review of the pertinent work and significant results of various compounds selected. The salient features of various techniques used in the investigation have also been highlighted. Xanthine, a dihydroxy derivative of purine, is the major end product of purine catabolism in man and other animals. In view of the importance of xanthine and methylxanthines in human physiology, the second chapter of the thesis has been devoted to extensive electrochemical investigations carried out on xanthine (I) and methylxanthines .The effect of methyl groups at position 1, 3, 7, 9 on the ease of oxidation of xanthine has also been evaluated. in HN 4 0' 3Tj H 9 (I) Cyclic voltammetry of xanthine and methylxanthines at pyrolytic graphite electrode (PGE) at a sweep rate of 100 mVs"1 exhibited a well-defined oxidation peak (lla) in the pH range 3.0 -10.0. In the reverse sweep, at least one cathodic peak was noticed in all the compounds. Some of the compounds have more than one reduction peaks. When the direction of sweep was again switched to positive potentials, a new oxidation peak la was observed in all the compounds. The Ep of oxidation peaks lla and la was dependent on pH and shifted to less positive potential with increase in pH. The Ep vs pH plots exhibited a break corresponding to the pKa of these molecules. The peak potential of the reduction peak was also dependent on pH and shifted to more negative potential with increase in pH. The UV spectral changes and kinetics of the decay of the UV-absorbing intermediate generated during the reaction were also studied. The products of electrooxidation were separated by gel-permeation chromatography and characterized by FT-IR, GC-Mass and Mass studies. Based on the experimental results, tentative mechanisms for the oxidation of these compounds have been suggested. The effect of methyl groups on the ease of oxidation of xanthine has also been evaluated. Studies indicated that it is not only electron releasing property of methyl group that affects oxidation but also changes in orientation and conformation due to these groups play a significant role. Electron density values, calculated at N atoms of various xanthines, indicate that presence of methyl IV group at N1 and N3 does not increase electron density significantly whereas maximum change in the electron density occurred when methyl group is present at N7 position. Apoor correlation between Ep and HOMO is observed probably due to adsorption complications. Purine nucleosides and nucleotides occur in biological systems, the behaviour of which is expected to be more complicated than their parent bases. Electrochemical behaviour of adenosine was therefore considered for investigation and results obtained are presented in the third chapter of the thesis. Adenosine (II), a purine nucleoside is claimed as a powerful natural vasodilator and is useful in the synthesis of a wide variety of anti-asthma and anti-AIDS drugs. Cyclic sweep voltammetry of adenosine at a sweep rate of 100 mVs"1 exhibited a single well-defined oxidation peak la at pyrolytic and glassy carbon electrode (GCE). At GCE the peak potential was at least 25-50 mV more positive than at PGE. When direction of sweep was reversed a cathodic peak llc was noticed at both the electrodes. The peak potentials of la and llc were dependent on pH. EC nature of the reaction was established in which electrode reaction is coupled with an irreversible follow-up chemical step. Coulometric studies indicated that the number of electrons involved in oxidation in acidic and neutral or alkaline media are 5.8 ± 0.2 and 5.0 ± 0.1 per mole respectively. Spectral changes during electrolysis were monitored to detect formation of a UVabsorbing intermediate. The decay of the UV-absorbing intermediate occurred in a pseudo-first-order reaction. The products of electrooxidation were separated by gel-permeation chromatography and characterized by m.p., TLC, IR, 1HNMR and mass spectra studies. A plausible mechanism has been suggested for the formation of the products. nh2 /N HOH2C ^ (ID 0 HO OH Fourth chapter of the thesis deals with the oxidation of adenine N(1)- oxide (III). The presence of N-oxide function introduces profound differences from the parent purine because it makes these compounds oncogenic in nature. NH2 N ^ ("I") H Thus, an attempt has been made in this chapter to study electrooxidation of this compound. In the pH range 1.4 -9.8 at 100 mVs"1, more than one oxidation peaks were noticed in the acidic pH range and a single oxidation peak in the neutral and alkaline range. When reverse sweep was applied a reduction peak IVC was observed. Ep vs pH plots, for both anodic and cathodic peaks, were linear. The kinetics of decay of the UV-absorbing intermediate generated during electrooxidation was studied and the decay occurred in a pseudo-first-order reaction. Products obtained from electrooxidation were separated by gelpermeation chromatography and characterized by 1HNMR, IR and mass spectra. VI Tentative mechanisms for the formation of products in acidic and neutral or alkaline medium have also been suggested. The last chapter of the thesis deals with the oxidation chemistry of a purine nucleotide, viz., adenosine monophosphate (IV). As purines are basic units of nucleic acids, they serve as energy storage compounds in the form of mono, di and triphosphates. Cyclic voltammetric studies at 100 mVs"1 exhibited a single well-defined oxidation peak la in the pH range 1.4 -9.8. On reversal of sweep, a bump was occasionally observed. Effect of pH, concentration and sweep rate were studied and results are presented in this chapter. On the basis of voltammetric, coulometric, spectral studies during electrooxidation and product identification, a mechanism for the oxidation has been suggested. nh2 N V> (IV) OH— P— OH2(> OH HO OH Purines, their nucleosides and nucleotides are thus one of the most important classes of organic compounds essential for normal cellular functions. It can be inferred that present studies at solid electrodes, coupled with other techniques provide profound insights into the mechanism of biochemical reactions.