OXIDATION CHEMISTRY OF SOME BIOLOGICALLY IMPORTANT MOLECULES

Abstract

The studies focused on the electrochemical behaviour of naturally occurring organic compounds present major challenges both from an electromechanistic and an analytical view point. In recent years, it has been clearly demonstrated that electrochemical studies can provide useful and often uniquely valuable insights into the generally complex mechanisms of enzymic and perhaps in vivo redox reactions. A combination of electrochemistry with other sophisticated techniques like molecular spectroscopy, mass spectrometry or HPLC has further been claimed as a powerful approach for elucidation of redox behaviour of biomolecules. For a number of years now this laboratory has been investigating the redox behaviour of a variety of compounds. Some of these compounds were studied in detail like uric acid, adenine, hydroxyadenines, etc. whereas others were studied superficially. As purines are the building blocks of two well known nucleic acids viz., RNA and DNA, the enzymic oxidation of these compounds was also studied wherever possible. Recently, electro-oxidation of Indole and its derivatives was initiated in our laboratory due to their biological and clinical significance in central nervous system. The work reported in the thesis is a small part of the big proposal submitted by Prof. R.N. Goyal in connection with redox chemistry of biomolecules. The present dissertation deals with the electrochemical oxidation studies of some alkyl substituted xanthines, indole and hydroxyindoles. The (ii) peroxidase catalyzed oxidation of these compounds wherever possible, has also been studied. The toxicity of oxidation products of indole and hydroxyindoles has also been examined in albino mice. The results of the investigations are organized in the dissertation as follows : The first chapter of the thesis is General Introduction and presents a brief review and significant results relevant to the present study. The chapter also highlights the salient features of the techniques used in the investigation. The second chapter of the dissertation describes electrochemical and enzymic oxidation of 1-methylxanthine (I) and 1-methyluric acid (II). Redox behaviour of these compounds were considered interesting because bacterial restriction enzyme system is found to contain a methylase which methylates specific base residues in the host DNA. (I) Linear sweep voltammetry of 1-methyluric acid exhibited a well defined anodic peak (IJ in the pH range 2.3-10.0 at a sweep rate of 10 mVs'1. Peak potential of peak Ia was dependent on pH and shifted to less possitive potential with increase in pH. A plot of Ep vs. pH exhibited two linear segments which meet at around pH 6.0. The point of intersection corresponds to the pKa value of 1-methyluric acid. In cyclic sweep voltammetry at sweep rate 100 mVs"1, 1-methyluric acid exhibited one anodic peak (IJ. In the reverse sweep two cathodic peaks Ic and H. were noticed. The nature of the electrode reaction (iii) was established as EC in which charge transfer is followed by irreversible chemical steps. 1-Methylxanthine (I) on the other hand exhibited a well defined oxidation peak. In the reverse sweep a cathodic peak was noticed and on futher changing the direction of sweep, one more anodic peak was observed at less positive potential (IIJ. Effect of sweep reversal potential was studied on peak IIa. Chronoamperometric studies were performed to determine the half life of diimine generated in the reaction. Controlled potential electrolysis indicated a 4e, 4H+ oxidation in the case of compound (I) and 2e, 2H+ for compound (II) in the entire pH range studied. The UV spectral studies suggested that UV-absorbing intermediate generated during electro-oxidation decayed in a pseudo first order reaction in both the compounds to give final products. Products of oxidation were separated using Gelpermeation chromatography and chracterized by ^NMR, mass, m.p. etc. as 1-methylalloxan and urea at pH 3.0 and hydantoin derivative at pH 7.0. Tentative mechanisms for the EC reaction have also been suggested. 5-Hydroxytryptamine has been found to function as neuro-transmitter in central nervous system long ago. The basic unit of 5-hydroxytryptamine is an indole nucleus. To systematically study the redox properties of this compound, it was considered desirable to first elucidate the electrochemical behaviour of its basic unit. Chapter third of the thesis deals with the electrochemical oxidation of Indole (III) at pyrolytic graphite electrode. Linear sweep voltammetry of Indole gave a well defined anodic peak I in the pH range 2.0-10.5. The peak potential was found to be independent below pH 6.0 and above pH 6.0 the E shifted to less positive potential with increse in pH. In cyclic voltammetry at the sweep rate of 100 mVs1 a well defined anodic peak was noticed and in the reverse sweep two cathodic peaks IIc and IIIc were found in the pH range 2.0-6.0. Both the cathodic peaks merged to a single peak in alkaline pH range. EC nature of the electrode reaction was established by the plots of AE /A log v vs. log v. The value of n, number of electrons involved in oxidation was found to be 1.0 ± 0.1 in the entire pH range. Spectral studies indicated that a UV-absorbing intermediate is generated during electro-oxidation which decays in first order reaction. The major product of electro-oxidation was characterized as a trimer using FT-IR, XHNMR and mass spectrum and a tentative mechanism is also proposed. The effect of oxidation product on the blood of albino mice was determined and the product was found much more toxic in comparison to indole. Fourth chapter of the thesis deals with the electrochemical investiations of hydroxyindoles in view of the formation of hydroxy derivatives during oxidation of 5-hydroxytryptamine. The hydroxy derivatives of 5- hydroxytryptamine have been found highly toxic and responsible for the damage of various types of neurons. Two hydroxyindoles viz., 4-hydroxyindole(IV) and 5-hydroxyindole(V) are selected for the present studies. H( ( IV ) ( V ) (v) It is expected that the oxidation behaviour of hydroxyindoles would provide deep insights about the oxidative mechanism of serotonin in central nervous system. Oxidation of 4-hydroxyindole was carried out at PGE in the pH range 2.1-10.7 and one well defined anodic peak was noticed. The peak potential of anodic peak was dependent on pH and shifted to less positive potential with increase in pH. In cyclic sweep voltammetry, one well defined oxidation peak Ia was observed and on reversing the sweep two cathodic peaks IIc and IIIc were noticed. On further reversing the potential sweep, anodic peaks IIa and IIIa were noticed which formed quasi reversible couples with peaks IIr and IIL The major product of the electrode reaction was found as a tetramer which was characterized by !HNMR, mass and related techniques. A tentative mechanism has also been suggested for its formation. Similar studies were also carried for 5-hydroxyindole. The effect of oxidation product on various blood parameters of albino mice was determined after intracranial injection at LD50 dose. The products were found to produce liver disorder and hypothyrodism. It is established on the basis of these studies that electrochemical studies when coupled with other related techniques provide deep insights into the redox chemistry of biomolecules.