PURINE-PYRIMIDINE BASE PAIR INTERACTION WITH SOME METAL IONS IN SOLUTION

Abstract

Nucleic acids are the naturally occurring polymers like proteins, biomolecules and largest macromolecules in the cell. They are very long, linear polymers, composed of many nucleotides. A nucleotide contains (1) a five-carbon sugar molecule (nbose or deoxyribose), (2) one or more phosphate groups and (3) anitrogenous base (purines and pyrimidines). The pynmidine has a single six-member ring containing two nitrogen atoms viz., uracil, cytosine and thymine, abbreviated as U, Cand T. Purine bases have a six-member pyrimidine ring fused to a five-member imidazole ring, the fused system containing four nitrogen atoms are e.g., adenine and guanine abbreviated as A and G. These different types of heterocyclic nitrogenous bases are found in two main types ofnucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The difference between these two types of nucleic acids lies in the fact that the sugar obtained from the former is nbose whereas that from the latter is 2'-deoxyribose, which lacks an -OH group at 2- position. Both these sugar units are found in furanose form and are linked through their p-1-postion to 1-position ofpyrimidines and 9-position ofpurines. A lot of works have been reported on the secondary structure of DNA. Quantitative analysis of the DNA hydrolysis has shown that in DNA, the ratios of Ato T and Gto C are 1:1. These results could be interpreted only if it is assumed that the bases are paired in DNA. Based upon these observations and assumptions and X-ray diffraction studies of DNA it was proposed that the DNA molecule actually consists of two separate polynucleotide strands leading in opposite directions and coiled about a common axis as right handed helices. The polynucleotide chains of the double helix are held together at definite intervals through hydrogen bonds which are not random but are specific between pairs of the bases. Thus Gofone coil is hydrogen bonded to Cofthe other and Aofone is hydrogen bonded to T of the other. The former pairing (G.C) involves three hydrogen bonds whereas the latter (AT) involves only two and a G.C pair has been reported to be more stable than an AT base pair. This is the most stable arrangement as no other pairing of these four bases can lead to such a strong bonding. Base pair as well as mispair formation and their possible incorporation in the DNA helix has also been observed to be assisted by the presence ofmetal ions. Metal cations in general stimulate the initiation of reaction, possibly by affecting the organization of the subunit of which the subenzyme is composed as it is not required to achieve binding of enzyme to the DNA. In brief, the biological functioning of nucleic acids involves the participation of metal ions either directly or indirectly, hence it is desirable to study the biochemical aspects concerned with metal nucleic-acid interactions. Though the structures of nucleic acids, viz., DNA and RNA and the primary roles of the nucleic acids and their derivatives as hereditary, determinants in biological reproduction and growth have been well established. But in order to propose the structure and reactions of nucleic acids and their constituents, it is necessary to have the knowledge of the sites and the thermodynamic quantities associated with the interaction ofthe protons and the metal ions with these substances. in In an attempt to obtain an insight concerning the metal ion interactions with the biologically important hgands, in the present work a detailed physico-chemical investigation of interaction of transition metal ions with guanine, cytosine, cytidine, 5-bromocytosine, 5-azacytosine and 5-fluorocytosine separately as well as in presence of each others have been undertaken. Apotentiometnc study has been performed to mvestigate the stability of the complexes which will help towards understanding the driving forces that lead to the formation of such complexes in biological systems. In the present thesis various metal ions viz., Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Ca(II), Ba(II) and Sr(II) have been chosen. The thermodynamic parameters at different temperature have been calculated and the presence of different complex species at different pH in solution have also been determined through species distribution plots by using BEST computer program. Further, for suggesting the binding sites of purines and pyrimidines in the metal complexes in solution, synthesis and characterization of some ternary metal complexes have been performed and the isolated complexes were screened for their antitumor activity against Dalton's lymphoma (DL) tumor systems both in vivo and in vitro. For the sake of the convenience, the work embodied in the thesis is presented in the following chapters: The first chapter of the thesis is general introduction and presents the up-to-date survey of literature related to the various punnes-pyrimidines, their haloderivatives, nucleosides and nucleotides. The different type of metal complexes IV both in solid and solution related to the present research have been posed in the context of the cited work. The solution and the thermodynamic studies of some metal complexes with cytosine and its derivatives are presented in chapter two. The stability constants of the bmary (1:1) complexes of Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Ca(II), Sr(II) and Ba(II) with cytosine, cytidine, 5-bromocytosine, 5-azacytosine and 5- fluorocytosine were determmed in an aqueous solution (Ionic strength, /= 0.1 M NaN03) using Bjerrum-Calvin's pH-titration technique as adopted by Irving and Rossotti at 25°, 35° and 45°C temperatures. The experimental pH titration data were analyzed with the aid of the BEST computer program in order to evaluate the formation constants of various intermediate species formed in the above binary systems. At all the temperature studied, the constants decrease in the order Cu(II) >Ni(ID >Zn(II) >Co(II) >Cd(II) >Ca(II) >Sr(II) >Ba(II). The enthalpy and entropy changes for the formation of above 1:1 binary complexes were calculated from temperature coefficient data. The relative small values of Mf\ coupled with large positive values of A5°f offers an evidence for the role of entropy as the primary factor favouring the formation ofthe neutral 1:1 metal ligand complexes. In chapter three of the thesis, the solution studies of nucleic acid base pair and mispair interaction with metal ion- a potentiometnc aspects are described. Potentiometnc equilibrium measurements were made for the interaction ofCo(II), Ni(II), Cu(II), Zn(II), Cd(II), Ca(II), Sr(II) and Ba(II) with purme (guanine) in 1:1 ratio and with pyrimidines (viz., cytosine, cytidine, 5-bromocytosine, 5- azacytosme and 5-fluorocytosine) as primary hgands in 1:1:1 ratio at 25, 35, 45 ± 0.1 °C temperatures and /- 0.1 MNaN03 in aqueous solution. The experimental pH-titration data were analyzed with the aid of the computer programme in order to evaluate the formation constants of various intermediate species formed and their relative distribution in ternary systems. The experimental conditions were selected such that self association of the nucleobases and their complexes due to stacking mteraction was negligibly small, i.e., the monomenc normal and hydroxo ternary complexes were studied. The enthalpy (AH°f) and entropy (AS0,) changes for the formation of binary and ternary complexes were calculated from temperature coefficient data. With the help of these thermodynamic parameters, we have evaluated AAH°t and AAS°f to explain the extra stability of these ternary complexes. The chapter four of the thesis describes the synthesis, structural and antitumor studies of some mixed ligand complexes isolated from the mteraction of metal and base pair/mispair interaction. Mixed-ligand complexes ofCo(II), Ni(II), Cu(II), Zn(II) and Cd(II) with cytosine/5-azacytosine/5-fluorocytosine as primary ligand and guanine as secondary ligand were prepared in aqueous ethanol solution at pH of about 7. The isolated complexes were characterized by various physicochemical methods and subjected to ascreenmg system of Dalton's lymphoma (DL) tumor cells both in vivo and in vitro. Some complexes exhibit significant antitumor activity.

The materials, reagents, theoretical treatment, experimental details, synthetic procedure and different type of spectroscopic measurements are described in Chapter five. The methods for testing the antitumor property ofthe complexes have also been included.