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Deuteration is the replacement of a hydrogen atom by deuterium atom in a molecule. The replacement begins at the most acidic hydrogen in the molecule. In ATP, the deshielded hydrogen is C8-H which is the first replaced during deuteration. During ATP deuteration some of the ATP is hydrolysed to ADP concurrently. Using kinetic analysis, it was confirmed that the ATP hydrolysis that occurs is 1st order in ATP concentration, while the hydrogen replacement is 2nd order. The ATP and its C8 deuterated analogue were tested against three enzymes shikimate kinase (SK), acetate kinase (AK) and glutamine synthetase (GS) to determine if a kinetic isotope effect (KIE) exists in these systems. With AK and GS, the KIED increased as the KIEH decreased, while with SK the KIED decreased as the KIEH increased as the concentration of the ATP or deuterated analogue increased. Deuteration of imidazole and purine compounds reduced the specific activity of AK or SK at low concentrations in an enzyme-catalysed reaction. From a library of imidazole-containing compounds that inhibited SK, three compounds were selected and their IC50 values were determined on the SK-catalysed reaction. These compounds show a differential potency and efficiency between their protonated and deuterated analogues when compared in a 1:1 mixture. Synthesized purines incorporating three different substituents at N-9 were tested against AK or SK for their ability to lower the specific activity of the enzymes used / Physics / M. Sc. (Physics)

The three-dimensional structures of biological macromolecules and molecular assemblies are becoming increasingly important with the changing methodologies of drug discovery. The structures aid in understanding of protein function at the molecular level: be it a macromolecular assembly, a cytosolic enzyme or an intermembrane receptor molecule. X-ray crystallography is the most powerful technique to obtain the three-dimensional structures of such molecules at or near atomic resolution. With such a wide-spread importance, crystallography is an integral part of structural biology and also of the current drug discovery programs. The present thesis mainly deals with application of the crystallographic techniques for understanding the structure and function of adenylosuccinate lyase (ASL) from bacterial pathogens Salmonella typhimurium and Mycobacterium tuberculosis as well as its non-pathogenic counterpart Mycobacterium smegmatis. Studies were also carried out to understand the structure-function relationship of the protease in the plant virus Sesbania Mosaic Virus (SeMV). The thesis has been divided into six chapters. The first chapter contains an introduction to nucleotide synthesis and ASL superfamily of enzymes known as the aspartase/fumarase superfamily based on the published literature. Chapter 2 provides the details of the techniques used for the investigations presented in this thesis. Chapters 3-5 deal with the structural and functional studies carried out on ASL from the three bacterial organisms. Chapter 6 deals with the simulation studies carried out on SeMV protease. Mechanism and importance of nucleotide synthesis is introduced in Chapter 1, with special emphasis on purine de novo and salvage pathways. ASL is introduced as an important enzyme for purine synthesis. Its superfamily, the aspartase/fumarase superfamily of enzymes is described in detail with respect to its structure, function and pathophysiology. Objectives of the present study are outlined towards the end of the chapter. The experimental and computational techniques utilized during the course of my research are described in Chapter 2. These techniques include gene cloning, protein expression and purification, kinetic and biophysical characterization of proteins, crystallization, X-ray diffraction, data collection and processing, structure solution, refinement, model building, validation and structural analysis, phylogenetic studies, molecular docking and molecular dynamic simulation studies. Adenylosuccinate lyase is an important enzyme participating in purine biosynthesis. With the emergence of drug resistant variants of various pathogens, ASL has been recognized as a drug target against microbial infections. Chapter 3 deals with the structural and functional characterization of ASL from Salmonella typhimurium. Two constructs of the StASL gene were cloned and expressed leading to the purification of truncated (residues 1-366) and full-length (residues 1-456) polypeptides. Crystallization of the two polypeptides resulted in three independent structures. The full-length structure was very similar to the E. coli ASL structure consistent with 95% amino acid sequence identity between the two polypeptides. However, the truncated structures showed large distortions, especially of the active site residues, accounting for the catalytic inactivity of the truncated polypeptide in spite of retaining all residues considered important for function. The full-length ASL was catalytically active. A unique feature observed in StASL, not reported in other ASLs, was its allosteric regulation by the substrate. Kinetic studies also revealed hysteretic behavior of the enzyme. The electron density map of the full-length structure showed two novel densities on the molecular 2-fold axis into each of which a molecule of cadavarine could be fitted. Docking studies revealed a ligand-binding site at the inter-subunit interface between the two observed densities which might represent a potential allosteric site. Combining the structural and kinetic results, a possible morpheein model of allosteric regulation of StASL was hypothesized. Chapter 4 deals with the crystallographic and kinetic investigations on ASL from Mycobacterium smegmatis and Mycobacterium tuberculosis. MsASL and MtbASL were cloned, purified and crystallized. The X-ray crystal structure of MsASL was determined at 2.16 Å resolution. It is the first report of an apo-ASL structure with a partially ordered active site C3 loop. Diffracting crystals of MtbASL could not be obtained and a model for its structure was derived using MsASL as a template. Most of the active site residues were found to be conserved with the exception of Ser 148 and Gly 319 of MsASL. Ser 148 is structurally equivalent to a threonine in most other ASLs. Gly 319 is replaced by an arginine residue in most ASLs. The two enzymes were catalytically much less active when compared to ASLs from other organisms. Arg319Gly substitution and reduced flexibility of the C3 loop might account for the low catalytic activity of mycobacterial ASLs. The low activity is consistent with the slow growth rate of Mycobacteria, their high GC containing genomes as well as with their dependence on other salvage pathways for the supply of purine nucleotides. Chapter 5 deals with the identification of the catalytic residues important for ASL catalysis in view of the earlier conflicting reports on the identity of these residues. pH-dependent kinetic studies were performed on full-length StASL. The theory behind these studies is also described in this chapter. Two residues with pKa values of 6.6 and 7.7 were identified as essential for the enzymatic activity. These results were interpreted along with structural comparison of MsASL and other superfamily enzymes with ordered C3 loops. They suggest that His 149 and either Lys 285 or Ser 279 of MsASL are the residues most likely to function as the catalytic acid and base, respectively. The final Chapter 6 of the thesis deals with the structural and dynamic studies carried out on Sesbania mosaic virus (SeMV) protease. The chapter begins with a general introduction to viruses, followed by a brief summary of SeMV. The goal of this study is to understand the interactions between the protease and VPg at a structural level using the information available from biochemical studies. Crystallographic studies initiated for the mutant H275APro and Y315APro were unsuccessful due to the insolubility of the proteins. Co-crystallization or soaking experiments of wild type protease with cognate peptides were unsuccessful due to the inability of the enzyme to bind to its substrates in the absence of VPg. Higher resolution structure of wild type protease did not yield any new insights when compared to the earlier reported structure determined at a lower resolution. In the absence of structural insights, molecular dynamic simulations were carried out on wild type protease structure and in silico generated mutants using GROMACS package. The studies showed the importance of flipping of residue Phe 301 and opening-closing of the loop region corresponding to residues 301-308 for the catalytic mechanism. The thesis concludes with Future perspectives of the various studies carried out on ASL and SeMV protease. The atomic coordinates determined from the work presented in this thesis have been deposited in the PDB and the assigned PDB codes are reported in the respective chapters. Publications cited in the thesis are listed in the Bibliography section.

Cette thèse s’inscrit dans le cadre de l’étude de la relation structure-activité d’analogues de ligands naturels de récepteurs nicotiniques et purinergiques. Ce travail se divise en deux parties.<p><p>Dans la première partie de cette thèse, nous avons réalisé la synthèse d’analogues de la 11-homosédinone, alcaloïde isolé de la plante Sedum acre, qui présente une activité agoniste sur différents récepteurs nicotiniques du système nerveux central. Les différents analogues ont été synthétisé par application de la méthoxylation anodique pour introduire succesivement deux substituants en postion 2 et 6 d’un noyau pipéridinique. Les analogues synthétisés se différencient par la nature du noyau aromatique, la présence d’un groupement méthyle sur l’atome d’azote de la pipéridine et l’oxydation du sustituant en position 2. Ce travail a notamment permis de montré l’importance du groupement N-méthyle vis-à-vis de l’activité des analogues. Nous avons également pu mettre en évidence que l’introduction d’un halogène sur le noyau aromatique diminuait l’activité de l’analogue sur le récepteur a7 tout en augmentant l’acitivité sur le récepteur a4b2 et que l’introduction d’un noyau furanique permettait d’augmenter la sélectivité vis-à-vis du récepteur a4b2 tandis que l’introduction sur le noyau aromatique d’un groupement nitro ou méthoxy conduit à une perte totale de l’activité.<p><p>Dans la seconde partie de cette thèse, nous avons réalisé la synthèse d’analogues de la dATP, afin d’évaluer leur effet agoniste sur le récepteur P2Y11, impliqué dans différents mécanismes de différentiation cellulaire, dont notamment celui de la maturation des cellules leucémiques HL60 en cellules de type neutrophile. Les analogues synthétisés se différencient de la dATP par la présence d’un groupement méthylène ou dichlorométhylène entre les phosphores b et g de la chaîne polyphosphate, ainsi que par l’estérification de l’alcool en position 3’ du sucre. Ce travail a pu confirmer que les analogues en série 2’-désoxy conduisent à de meilleures activités que ceux de la série 2’-OH. Nous avons également pu montrer que l’estérification de la position 3’ conduit à une diminution de l’activité agoniste, à l’exception du groupement a-naphtoyle qui conduit à une augmentation significative de l’activité sur P2Y11.<p><p> / Doctorat en sciences, Spécialisation chimie / info:eu-repo/semantics/nonPublished

The ribosome is a macromolecular machine that produces proteins in all kingdoms of life. The proteins, in turn, control the biochemical processes within the cell. It is thus of extreme importance that the machine that makes the proteins works with high precision. By using three dimensional structures of the ribosome and homology modelling, we have applied molecular dynamics simulations and free-energy calculations to study the codon specificity of protein synthesis in initiation and termination on an atomistic level. In addition, we have examined the binding of small molecules to riboswitches, which can change the expression of an mRNA. The relative affinities on the ribosome between the eukaryotic initiator tRNA to the AUG start codon and six near-cognate codons were determined. The free-energy calculations show that the initiator tRNA has a strong preference for the start codon, but requires assistance from initiation factors 1 and 1A to uphold discrimination against near-cognate codons. When instead a stop codon (UAA, UGA or UAG) is positioned in the ribosomal A-site, a release factor binds and terminates protein synthesis by hydrolyzing the nascent peptide chain. However, vertebrate mitochondria have been thought to have four stop codons, namely AGA and AGG in addition to the standard UAA and UAG codons. Furthermore, two release factors have been identified, mtRF1 and mtRF1a. Free-energy calculations were used to determine if any of these two factors could bind to the two non-standard stop codons, and thereby terminate protein synthesis. Our calculations showed that the mtRF’s have similar stop codon specificity as bacterial RF1 and that it is highly unlikely that the mtRF’s are responsible for terminating at the AGA and AGG stop codons. The eukaryotic release factor 1, eRF1, on the other hand, can read all three stop codons singlehandedly. We show that eRF1 exerts a high discrimination against near-cognate codons, while having little preference for the different cognate stop codons. We also found an energetic mechanism for avoiding misreading of the UGG codon and could identify a conserved cluster of hydrophobic amino acids which prevents excessive solvent molecules to enter the codon binding site. The linear interaction energy method was used to examine binding of small molecules to the purine riboswitch and the FEP method was employed to explicitly calculate the LIE b-parameters. We show that the purine riboswitches have a remarkably high degree of electrostatic preorganization for their cognate ligands which is fundamental for discriminating against different purine analogs.

Cytokinins are a large group of phytohormones. Since their discovery in the 1950s, they have shown to play a pivotal role in plant physiology. Most studies so far focused on cytokinin action mechanisms and their metabolic regulation. Identification of AtABCG14 and AtPUP14 as cytokinin-specific membrane carriers brought researchers' attention to cytokinin membrane transport, too. In this thesis, we performed experiments with radio-labelled cytokinin tracers. We show that trans-zeatin and isopentenyladenine, two major biologically active cytokinins, are readily transported across the plasma membrane in tobacco BY-2 cell suspension. Making use of mathematical modelling, we show that BY-2 cells possess a membrane transport system with an affinity toward cytokinins. Next, we show that atabcg14 and atpup14 mutations affect cytokinin metabolism in Arabidopsis thaliana plants. Keywords: cytokinin, Arabidopsis thaliana, tobacco BY-2 cell lines, membrane transport, purine permease, ATP-binding cassette, radio-labelling

Phosphatases of the haloacid dehalogenase superfamily are one of the cell's tools for dephosphorylation of many diverse endogenous and exogenous compounds. This work is aimed at enzymes Tt82 and cytosolic purine 5'-nucleotidase II (cN-II), two members of this large enzyme superfamily. The Tt82 originates in the hyperthermophilic archaeon Thermococcus thioreducens. Up to date, there is only a small amount of knowledge about properties and biological function of this enzyme. Based on its sequence and structure, it was predicted that the Tt82 should possess a phosphatase catalytic activity. Consequently, potential substrates of the Tt82 were proposed by the molecular docking. In this work, the phosphatase activity of the Tt82 was confirmed together with several of its substrates: AMP, D-glucose 1-phosphate, D-glucose 6-phosphate and p-nitrophenyl phosphate (pNPP). Activity towards AMP and pNPP was then characterized by steady-state kinetics at 37 řC and 60 řC. In consistence with its thermophilic origin, the Tt82 showed markedly higher activity towards both substrates at 60 řC. Nonetheless, the effectivity of the Tt82 catalytic activity towards these substrates was actually very low. This leads to assumption, that the identified substrates are probably not biologically relevant. On the other hand, it is quite...

Made available in DSpace on 2016-08-17T18:39:46Z (GMT). No. of bitstreams: 1 5245.pdf: 1568165 bytes, checksum: 9fc25ff9dc83339e50628c08854efd2c (MD5) Previous issue date: 2012-08-29 / Universidade Federal de Sao Carlos / Schistosimiasis mansoni is a neglected chronic parasitic disease that affects thousands of people worldwide, caused by the trematode Schistosoma mansoni. In the infected host the disease is characterized by the presence of granuloma, imunnopathological response of the cellular infiltration against egg antigens. Thus, the host-parasite relation favors hepatosplenomegaly, acite and hepatic fibrosis. Current chemotherapy is based on the use of Praziquantel (PZQ), used against all species of Schistosoma spp for over 30 years. The main issue is that the PZQ is practically inactive against immature schistosomula and favors the resistance growth of the existent lineages, which makes the study for new drugs and vaccines that can contribute to the control of this disease even more urgent. One of the paths on the search for new therapeutic targets is the study of essential enzymes to the S. mansoni. In particular, it is known that the enzymes Adenylate Kinase 1 and 2 (ADK), Uridine cytidine Kinase 1 and 2 (UCK), Hypoxanthine guanine phosphoribosyltransferase (HGPRT) e Purine nucleoside phosphorilase 1 (PNP) are found on the metabolic pathways of the parasite s nucleotide, participating in the metabolism of purines and pyrimidines. Our goals in this study were to assess the immunization with these enzymes, using the S. mansoni cercariae infected murine model, and subsequently analyze the acting in oviposition and growth of adult worms. Our results showed that the immunization in Balb/c mice with the UCK enzyme was capable of inducing a specific immune response, which favored a significant reduction of the parasitic load (adult worms). However, it was not possible to observe significant reduction in the number of eliminated eggs. Regarding the immunization with PNP and HGPRT enzymes, the mice had a reduction in the number of eggs per gram of feces. The data obtained are considered interesting and can be new targets for immunotherapy against schistosomiasis mansoni. Thereby, new assays must be made with different dosages of the enzymes for a better assessment on how these enzymes modulate the parasitic load through the eggs reduction, reduction in the adult worms retrieving, as well as the antibody levels during the murine infection by the S.mansoni. / A esquistossomose mansônica é uma doença parasitária, crônica e negligenciada que afeta milhares de pessoas ao redor do mundo, causada pelo trematódeo Schistosoma mansoni. No hospedeiro infectado a doença é caracterizada pela presença do granuloma, resultado imunopatológico do infiltrado celular contra antígenos dos ovos A quimioterapia atual é baseada no uso do Praziquantel (PZQ), usado contra todas as espécies de Schistosoma spp há mais de 30 anos. O principal problema é que o PZQ é praticamente inativo contra esquistossomulos imaturos e favorece o desenvolvimento de resistência das linhagens existentes. Um dos caminhos na busca por novos alvos terapêuticos é o estudo de enzimas que são essenciais para o S. mansoni. Em especial, sabe-se que as enzimas Adenilato Quinase 1 e 2 (ADK), Uridina Citidina Quinase 1 e 2 (UCK), Hipoxantina-guanina fosforibosiltransferase (HGPRT) e Purina Nucleosídeo Fosforilase 1 (PNP) são encontradas nas vias metabólicas de nucleotídeos do parasito, participando do metabolismo de purinas e pirimidinas. A estratégia de utilizar enzimas do parasito na esquistossomose mansônica murina foi de avaliar uma resposta induzida por estas enzimas quando aplicadas em camundongos BALB/c e desafiados com cercarias de S. mansoni. Desta forma, avaliamos a fase crônica de camundongos imunizados e infectados com S. mansoni, onde foram analisadas amostras parasitológicas, hematológicas, sorológicas e fluidos da cavidade peritoneal. Nosso objetivo neste estudo foi avaliar a imunização com essas enzimas, usando o modelo murino infectado com cercarias de S. mansoni e posteriormente avaliar a ação na oviposição e desenvolvimento de vermes adultos. Nossos resultados demonstraram que a imunização em camundongos Balb&#8725;c com a enzima UCK foi capaz de induzir uma resposta imune específica, a qual favoreceu a diminuição significativa da carga parasitária (vermes adultos). No entanto, não foi possível observar redução significativa no número de ovos eliminados. Em relação à imunização com as enzimas PNP e HGPRT os camundongos que receberam as imunizações com PNP e HGPRT tiveram redução no número de ovos por grama de fezes. Os dados obtidos são considerados interessantes e podem ser considerados novos alvos para a imunoterapia contra a esquistosomose mansônica. Dessa forma, novos ensaios deverão ser realizados com diferentes doses das enzimas para melhor avaliar como essas enzimas modulam a carga parasitária através da redução de ovos, diminuição na recuperação de vermes adultos, assim como os níveis de anticorpos durante a infecção murina pelo S. mansoni.

The thesis is divided into four chapters Chapter 1 provides a brief introduction to the metal-nucleic acid interactions, the role of synthetic models to understand them with both solution (potentiometric) and solid state (Crystallographic) studies. Further the work done in the area of nucleobase [purines and pyrimidines] metal complexes and nucleotide metal complexes are briefly reviewed. Chapter 2 contains an account of synthesis and characterizations of metal [Cu/Co] purine [adenine] complexes and is divided into two sections Viz., Section I and Section II. Section I Five crystals structures of copper adenine dimeric complexes are synthesized and characterized with 1, 10-phenanthroline as coligand. The first ternary [Cu2(phen)2(µ-ade)2Cl2].3H2O complex (2a) crystallizes in the orthorhombic space group Pna21. In the crystal structure of 2a it has been observed that the five and six membered rings of adenine are arranged in such a way that the five membered ring nitrogen atoms N9 and N9A are coordinated to Cu1 while the nitrogen atoms N3 and N3A are coordinated with Cu2 center. This is the first time such co-ordination is observed for the copper-adenine dimeric complexes, while the earlier report shows an alternate coordination. In the complex adenine-adenine dimer formation is observed, mediated via N-H···N hydrogen bond interactions which give rise to a corrugated sheet like pattern along the bc plane. The 1,10-phenanthroline rings and water molecules are packed in the grooves of these corrugated sheets via non covalent interactions. The second ternary [Cu2(phen)2 (µ-ade)(µ-Cl)Cl2].5H2O complex (2b) obtained under same reactant conditions, as 2a, by changing the ratio of the reactants, is the unique example of a dimeric copper complex with one adenine acting as a bridging ligand. The complex 2b crystallizes in the monoclinic centric space group P21/c. Interestingly, the crystal packing of complex 2b does not show any direct adenine-adenine hydrogen bond interactions as was seen for 2a, but adenine moieties of neighboring molecules interact indirectly, mediated via N-H···O and O-H···N hydrogen bonds through solvent water molecules forming a zig-zag pattern. It is interesting to note that two hydrogen bond networks are running across the body diagonal like “X” mediated by the nitrogen atoms of the adenine base and the chlorine atom, axially coordinated to copper centre. Similarly the water molecule O4 and N7 are involved in forming a four membered ring at the body center through the non covalent interactions. As seen for the complex 2a, complex 2b also depicts the presence of slipped π-π stacking intra and intermolecular interactions for the 1,10-phenanthroline rings. The third complex [Cu2(phen)2(µ-ade)2(H2O)2](ClO4)2 complex (2c), obtained by post synthetic modification of 2a, crystallizes in the monoclinic space group Cc. The adenine moieties forms a dimer mediated via N-H···N hydrogen bonds at the pseudo two fold and are connected to the neighboring dimers through the possible hydrogen bond between the nitrogen atom N1 and the axially coordinated oxygen atom O1 of the water molecule. The perchlorate anions are trapped in the pockets surrounded by the adenine and 1,10-phenanthroline moieties. The Nitrogen atom N6, N6A of the adenine bases forms hydrogen bond with N7, N7A of the five membered rings of adenine bases and the oxygen atom O4, O7 of both perchlorate ions, the other oxygen atoms O3, O5 from Cl1 and O8 of Cl2 are involved in C-H···O hydrogen bonds but the remaining oxygen atoms O6, O9 and O10 of the perchlorate ions are not involved in hydrogen bond network. Thus the dimerization involves axial oxygen atoms and the five and six membered nitrogen atoms N7 and N1. The 1,10-phenanthroline rings show both intra as well as intermolecular slipped π-π stacking interactions. The fourth complex [Cu2(phen)2(µ-ade)2(H2O)2](BF4)2 complex (2c), obtained by post synthetic modification of 2a, crystallizes in the monoclinic space group Cc. The adenine moiety forms intermolecular N-H···N hydrogen bonds with the neighboring adenine moieties at the pseudo two fold and is connected to the neighboring dimers through the N-H···O hydrogen bond via axial water molecule. The dimerization of the neighboring adenine moieties is favored through the hydrogen bond between the oxygen atom O2 of Cu2 and N1 of the six membered ring, in return the oxygen atom O1 of second molecule is hydrogen bonded to the nitrogen N7 of the five membered ring of the first molecule. Interestingly the three fluorine atoms F1, F2 and F3 are involved in hydrogen bond and in the second BF4 ion only two fluorine atoms F6 and F7 are involved where F1 and F6 acts as a bifurcated hydrogen bond acceptor while the remaining fluorine atoms are not taking part. Here too, as in the previous case of 2c 1,10-phenanthroline rings show both intra as well as intermolecular slipped π-π stacking interactions. The fifth complex [Cu2(phen)2(µ-ade)2(H2O)2](PF6)2 complex (2c), obtained by post synthetic modification of 2a, crystallizes in the monoclinic space group Cc. The adenine moiety forms intermolecular N-H···N hydrogen bonds with the neighboring adenine moieties at the pseudo two fold and is connected to the neighboring dimers through the N-H···O hydrogen bond via axial water molecule. As observed in the previous structure of 2c and 2d the dimerization of the neighboring molecule is favored through the hydrogen bond between the oxygen atom O2 of Cu2 and N1 of the six membered ring, in return the oxygen atom O1 of second molecule is hydrogen bonded to the nitrogen N7 of the five membered ring of the first molecule. Interestingly the nitrogen atom N6 of the six membered ring is involved in four hydrogen bonds, Where one H is hydrogen bonded to N1 of the neighboring base while the second hydrogen atom is being shared by three fluorine atoms belonging to the second PF6 ion and in turn all these three fluorine atoms acts as bifurcated acceptor of the hydrogen bond with the carbon atoms of 1,10-phenanthroline. It is noteworthy that the fluorine atoms F3, F4, F5 and F6 are involved in single hydrogen bonds with the 1,10-phenanthroline carbon atoms. At the same time the rest of the fluorine atoms are not involved in any non covalent interactions. Here too, as in the previous cases of 2c and 2d 1,10-phenanthroline rings show both intra as well as intermolecular slipped π-π stacking interactions. The complexes 2c, 2d and 2e are isostructural. All the three complexes crystallized in the noncentric space group Cc as the precursor complex 2a [Pna21] with the difference being the nature of the complex, 2a being neutral whereas 2c, 2d and 2e are complex salts. All the three complexes have similar bond lengths between the coordinating atoms and the central copper metal but they differ in the angles subtended by the ligands at the copper centres which are also reflected in the dihedral angle between the planes of the coordinating ligands. Though the molecular structure of the three complexes differs only in the nature of the counter ion, the crystal packing analysis reveals the finer differences. The interaction of adenine with neighboring adenine is same for the three complexes 2c, 2d and 2e but differs from the precursor complex 2a. Section II covers the synthesis and characterization of cobalt adenine binary and ternary complexes with 1,10-phenanthroline and 2,2’-bipyridyl as coligands for the ternary complexes. The first binary [Co2(µ-Hade)2(µ-H2O)2(H2O)4](NO3)4·2H2O complex (2f) crystallizes in the centric space group P21/c. Though there were four water molecules, coordinated to the metal Co centres, available for intra molecular hydrogen bond interactions with the base nitrogen atoms the orientation of the coordinated bases is not favorable to enable the C-H···O hydrogen bond formation, but intermolecular hydrogen bonds were observed. The structure is stabilized mainly through the O-H···O and N-H···O hydrogen bond interactions between the neighboring molecules via nitrate ions. Interestingly there is an absence of any direct adenine-adenine interactions. The terminally coordinated water molecule O2 forms hydrogen bond with nitrate anion on both sides, which in turn the nitrates hold the bases of two different molecules as the network is running -N6-O10-O9-O2-O5-N6-. Both the nitrate anion oxygen atoms are involved in hydrogen bond where all the oxygen atoms are bifurcated acceptor. The nitrate ions with nitrogen atoms N10 and N11 are making a nine and eight membered ring through hydrogen bond with adenine nitrogen atoms [N6 and N7] and coordinated water molecules [O2 and O3] respectively. The second binary [Co(Hade)2(H2O)4]SO4·5H2O complex (2g) crystallizes in the centric space group P21/n. Interestingly, only one adenine [N3A] is involved in forming the O-H···N intramolecular hydrogen bond with the water molecule while the adenine on other side is not in favorable orientation. All the water molecules coordinated to the metal center are involved in forming hydrogen bonds where O1, O2 and O4 form two hydrogen bonds while, O3 forms three hydrogen bonds. The water molecule and sulphate ions are trapped in between the adenine bases and forming an interesting network of hydrogen bond running in opposite directions. In general the sulphate and the water molecule are holding the symmetry related molecules connecting the nitrogen atoms N6 and N7 of the adenine. The crystal structure of 2g shows the presence of intermolecular π-π stacking interaction between the six membered rings of the neighboring adenine molecules along a axis. These stacked adenine moieties looks like a zig- zag pattern when viewed down a axis. Here too as in previous case of 2f there are no adenine-adenine interactions present. It is noteworthy that both of these complexes[differing only in the nature of salts i.e. CoNO3 and CoSO4] differ in the adenine coordination to the cobalt centre [N9 and N3 co-ordination in 2f; N9 coordination in 2g]. The third ternary [Co2(µ-ade)2(µ-OH)2(phen)2](NO3)2·6H2O complex (2h) was synthesized by a one pot reaction and crystallizes in the triclinic space group P-1. Though there are two hydroxyl ions coordinated to the metal centre there is no favorable intramolecular hydrogen bond formation. The adenine moieties of 2h interact with each other forming a dimer at the inversion centre, which looks like a zig -zag sheet pattern, via N-H···N hydrogen bond. In addition to this the hydroxyl O1 forms hydrogen bond with water oxygen and the oxygen atom of the disordered nitrate anion. These chains are further linked to neighboring chains by N-H···O hydrogen bond and a slipped π-π interaction between the 1,10-phenanthroline rings forming a sheet like pattern. The fourth ternary [Co2(µ-ade)2(µ-OH)2(phen)2](OTs)2·6H2O complex (2i) , was also synthesized by a one pot reaction and crystallizes in the triclinic space group P-1. Similar to previous case though there are two hydroxyl groups bridging the metal centres as dimers, no intramolecular hydrogen bonds were observed. The adenine moieties interact with each other forming a zig-zag pattern via N-H···N hydrogen bond like in the previous structure 2h. Interestingly, contrary to the previous case where two such zig- zag sheets interacted with each other through slipped π-π stacking between the 1,10-phenanthroline rings, no such interaction was found among the neighboring sheets. Instead, the 1,10-phenanthroline rings interact with tosylate counter ion through C-H···O hydrogen bonds. Down the c axis projection, at the inversion centre tosylate ion and the water molecules form an eight membered ring where the water oxygen O1W acts as a donor in the two hydrogen bonds and the oxygen atom O2 of the tosylate acts as bifurcated acceptor. On the other side, the tosylate oxygens form a twelve membered ring with the water oxygen atom O2W. Thus, eight membered and twelve membered rings are formed alternately and both are subtending an angle of 113°. It is noteworthy that the tosylate ion is parallel to the adenine base while perpendicular to the 1,10-phenanthroline rings favoring the π-π and C-H···π stacking interactions between the neighboring zig zag chains. The fifth ternary [Co2(µ-ade)2(µ-OH)2(bpy)2](NO3)2·6H2O complex (2j) synthesized via one pot reaction and crystallizes in the triclinic space group P21/n. Similar to previous two cases there are two hydroxyl groups bridging the metal centres as dimers, no intramolecular hydrogen bonds were observed in the present case. The adenine moieties interact with each other forming a zig-zag pattern via N-H···N hydrogen bond as observed in the previous two structures 2h and 2i. The adenine also interacts with nitrate ion through N-H···O hydrogen bond. The nitrate groups are oriented parallel to the adenine base. The adenine base nitrogen atom N6 is involved in holding the neighboring adenine nitrogen atom N7 in addition to the nitrate oxygen atoms O3 and from the same nitrate the other oxygen atoms O4 is involved in hydrogen bond with the carbon atom C8 thus forming a nine membered ring. These chains interact with the parallel chains by slipped π-π stacking interaction similar to that observed in complex 2h. Chapter 3 describes the syntheses and characterizations of copper pyrimidine [uracil, cytosine and thymine] ternary complexes with 1,10-phenanthroline as coligand. The first polymeric [Cu(phen)(µ-ura)(H2O)]n·H2O complex (3a) crystallizes in the monoclinic space group P21/c. The protons of the water oxygen O1W is oriented towards the uracil rings enabling O-H···O intramolecular hydrogen bonds with O2 as a bifurcated bond acceptor of the uracil on either sides and the chain extends to infinity along the c axis. The structure is stabilized by slipped π-π stacking interactions between the 1,10-phenanthroline rings of neighboring polymeric chains. Each polymeric chain also interacts through C-H···O hydrogen bond between the neighboring chains. The second polymeric [Cu(phen)(µ-ura)(H2O)]n·MeOH complex (3b) is isostructural to (3a) and crystallizes in the monoclinic space group P21/c. Similar to 3a the coordinated water oxygen O1w is oriented towards the uracil rings enabling O-H···O intramolecular hydrogen bonds with O2, as a bifurcated hydrogen bond acceptor, of the uracil on either sides and the chain extends to infinity along the c axis. The structure is stabilized by slipped π-π stacking interactions between the 1,10-phenanthroline rings of neighboring polymeric chains. Each polymeric chain also interacts through C-H···O hydrogen bond between the neighboring chains. Both these complexes differ only in the lattice solvent molecule i.e. water for 3a and methanol for 3b. These complexes are the first example of direct uracil to metal coordination structurally characterized. Also, both the ring nitrogen atoms N1 and N3 are involved in coordination to the metal. The third polymeric [Cu4(cytosine)3Cl3(OH)2]n·14H2O complex 3c is the first polymeric complex known with cytosine and 1,10-phenanthroline as coligands. It crystallizes in the orthorhombic centric space group Pbca. Out of the four, three copper centres adopts square pyramidal [4+1] geometry {τ = 0.17 [Cu1], 0.028 [Cu3] and 0.053 [Cu4]}, whereas the fourth copper centre exhibits distorted trigonal bypyramidal [3+2] geometry. {[τ = 0.66 [Cu2]}. Two copper centres Cu1 and Cu3 have same co-ordination environment viz., the basal plane of the square pyramid is formed by cytosine [N1and N1A], 1,10-phenanthroline [N7, N8 and N11, N12] and chlorine ligands [Cl1, Cl3] while the axial site is occupied by other chlorine atom [Cl2] which act as a bridge between Cu1 and Cu3 in the polymeric chain. The cytosine ring attached to Cu1 and Cu3 act as tridentate ligand co-ordinating to two other copper centres [Cu2, Cu4] via O2, O2A and N3, N3A respectively. Thus remaining three sites of Cu2 are occupied by 1,10-phenanthroline [N9, N10] and a bridged hydroxyl [O1D] moiety. The hydroxyl moiety [O1D] acts as a bridging ligand between Cu2 and Cu4. Thus the basal plane of the trigonal bipyramid for Cu2 is formed by N9, O2 and O2A while axial sites are occupied by N10 and O1D. The basal plane for Cu4 is formed by N3, N3A, O1D and N3C [from third cytosine ligand] while the axial site is occupied by a hydroxyl ion [O1]. The structure is stabilized by slipped π-π intra molecular stacking interactions between the 1,10-phenanthroline rings. The cytosine moieties interact with each other through bifurcated N-H···O hydrogen bond where the proton of N6c is involved with O2 and O2A of the other two cytosine moieties coordinated to the same copper centre. The neighboring chains of the polymer are linked by inter molecular slipped π-π stacking interactions between the cytosine ring attached to Cu4 and the 1,10-phenanthroline rings. The chains are also connected through C-H···Cl hydrogen bonds where the chlorine atom Cl4 is involved in the bifurcated hydrogen bond one as intramolecular and the second as intermolecular. Both the Nitrogen atoms [N6, N6A] of different cytosine are involved in the noncovalent interactions, with the water [O41, O10W] as intermolecular hydrogen bond as well as intramolecular hydrogen bond with chlorine atoms [Cl4, Cl4* (* symmetry generated)] respectively. The water molecules pack between the polymeric chains via noncovalent interactions. Thus this complex is the first example of its kind where all the possible binding modes of cytosine are utilized. The fourth [Cu2(Phen)2(thy) (µ-OH)2(H2O)].HCO3·4.5H2O complex (3d) obtained as the minor product along with 3e crystallizes in the triclinic space group P1 with two molecules in the asymmetric unit. The structure displays the presence of a pseudo centre of inversion between the two molecules. But careful analysis of the structure reveals that the two different tautomeric forms of thymine are coordinated to the two copper centres in each molecule, thus making it a cocrystal. The molecule shows the presence of O-H···O intramolecular hydrogen bond between the thymine oxygen and the bridged hydroxyl ion. The structure is stabilized by slipped π-π stacking and C-H···π interactions between the 1,10-phenanthroline rings of neighboring molecules. The molecules also interact with solvent molecules and counter ions through non covalent C-H···O interactions. The fifth [Cu2(Phen)2(thy)(µ-OH)2(H2O)]Cl·3H2O complex (3e) which was the major product along with 3d also crystallizes in the triclinic space group P1 with two molecules in the asymmetric unit. The difference between 3d and 3e is the change in the nature of counter ion i.e. HCO3- for 3d and Cl- for 3e. Similar to 3d the two different tautomeric forms of thymine are coordinated to the two copper centres in each molecule, thus making it a cocrystal. The molecule shows the presence of O-H···O intramolecular hydrogen bond between the thymine oxygen and bridged hydroxyl ion. The structure is stabilized by slipped π-π stacking and C-H···π interactions between the 1,10-phenanthroline rings of neighboring molecules. The molecules also interact with solvent molecules and counter ions through non covalent C-H···O and C-H···Cl interactions. The sixth Cu(phen)(thy)2 complex (3e) was obtained just by changing the pH in the reaction condition for 3d and 3e and crystallizes in the monoclinic centric space group C2/c. Here a different tautomer of thymine other than that observed for 3d and 3e was coordinated to the central copper metal. The structure is mainly stabilized by slipped π-π stacking between the 1,10-phenanthroline rings of neighboring molecules as well as between the thymine rings. The thymine molecules also interact with neighboring thymine molecules through non covalent N-H···O interactions. These thymine thymine interactions were absent in 3d and 3e. Chapter 4 presents the synthesis and characterization of ternary copper 5’-Adenosine monophosphoric acid (5’-AMP)/ 5’-cytidine monophosphoric acid (5’-CMP) complexes with 2,2’-bipyridine/1,10-1,10-phenanthroline as coligands. The first Cu(bpy)(5’-AMP)2·2H2O complex (4a), obtained at pH = 3.0, crystallizes in the triclinic space group P1 with two molecules in the asymmetric unit Viz., complex A and Complex B. The phosphate group of 5’-AMP which has two protons in the uncoordinated state gets monodeprotonated at one hydroxyl group during the complex formation and is co-ordinated to the copper centre. Thus in each complex the charge on the central copper atom is balanced by 5’-AMP monodeprotonated ligand. The environment around both copper centres is same, Cu1 and Cu2 exhibits square planar geometry. The least square plane analysis reveals that the ribose sugar moieties adopt envelope conformation. The ΦCN angle, which is the torsion angle of the base with respect to sugar, are 84(2)°, 41(2) ° for complex A and - 43(2)°, 47(2) ° for complex B suggesting a anti conformation about the glycosyl bond for all the four 5’-AMP ligands. All the four ribose ring are puckered with one carbon atom of the ring,[C4’ and C3’A for complex A, C4’B and C3’C for complex B], displaced from the best four atom plane of furanose ring on the same side as C5’. [C4’ = -0.539(2) Å, C3’A = - 0.539(2) Å for complex A; C4’B = 0.509(17) Å, C3’C = 0.535(20) Å for complex B], suggesting in each complex, the confirmation of the ribose sugar of two 5’-AMP ligands are different. [C4’ endo and C3’A endo for complex A; C4’B endo and C3’C endo for complex B] Both the complexes A and B are stabilized by C-H···O intramolecular interaction between the adenine base and the phosphate oxygen atom. The structure is stabilized through a complicated network of C-H···O and N-H···O hydrogen bond interactions between the neighboring molecules where the oxygen atoms of the water molecules are involved in forming the network of bifurcated hydrogen bond. The adenine rings interact with each other through the N-H···N hydrogen bonds forming a dimer between the N6-N7 and N7-N6 similar to the base pairing observed in the DNA molecule, in addition to this the atom N6 is involved in forming a bifurcated hydrogen bond with the O7 atom of the phosphate group. Additionally, there is a presence of slipped π···π stacking interaction, between the bipyridine rings and adenine rings in a -B-A:A-B- fashion [B= 2,2’-bipyridine and A:A= adenine adenine adduct]. The second {Cu2(bpy)2(µ-5’-AMP)2(H2O)2·2[Cu(bpy)(5’-AMP)(H2O)2]·10H2O} complex (4b) is a cocrystal obtained at pH = 6.0, crystallizes in the monoclinic space group C2. The crystal structure of 4b can be described as a cocrystal made up of one dimeric [complex D] and two monomeric [complex M] copper (II) complexes. Both the complexes are ternary with 5‘-AMP and 2,2’- bipyridine as co ligands. These complexes are neutral in nature with the charge on the copper centres balanced by the 5’-AMP ligands. The asymmetric unit consists of half of this two component cocrystal system. The basal plane for the monomeric complex M is formed by two nitrogen atoms [N10A, N11A] from the 2, 2’-bipyridine , one water molecule [O1A] and a phosphate oxygen atom [O9A] from one of the 5’-AMP ligand, while the axial site is occupied by the other water molecule, O1W. The basal plane for the dimeric complex D is formed by two nitrogen atoms [N10, N11] from the 2, 2’- bipyridine , and two phosphate oxygen atom [O9 andO7] from two bridging 5’-AMP ligand, while the axial site is occupied by the other water molecule O2A. The 5’-AMP ligand bridges the two copper centres to form the dimeric complex. It is noteworthy that both the axial water molecules of complex D are on the same side. The least square plane reveals that the ribose sugar moieties adopt envelope conformation. The ΦCN angle, which is the torsion angle of the base with respect to sugar, 72(1)° for complex D and 77(1)° for complex M, suggest an anti conformation for both the complexes about the glycosyl bonds. The ribose rings are puckered in both complex D and M, with C3’ and C3’A displaced from the best four atom plane of furanose ring. C3’ deviates from the sugar plane by 0.604(13) Å which is opposite to C5’, imply C3’ exo conformation for the ribose ring. While for the ribose moiety in complex M, C3’A deviates from the sugar plane by 0.585(11)Å which is on the same side of C5’, confirm C3’A endo conformation for the ribose ring. The conformation around the C4’-C5’ bond described by the angles ΦOO [O1’-C4’-C5’-O5’= -60(1)°] and ΦOC [C3’-C4’-C5’-O5’= -179.8(9)°] is gauche trans, a rare conformation, for the complex D while around the C4’A-C5’A bond the angles ΦOO [O1’A- C4’A-C5’A-O5’A= -59(1)°] and ΦOC [C3’A-C4’A-C5’A-O5’A = 57(1)°] suggest the commonly observed gauche gauche conformation. The structure is stabilized through the extensive network of C-H···O and N-H···O hydrogen bond interactions between the neighboring molecules. The adenine rings interact with each other through the N-H···N hydrogen bonds forming a dimer between N6-N7 and N7- N6, mimicking the base pair observed in the DNA molecule, in addition to this N6 is involved in the formation of a bifurcated hydrogen bond with the O8 atom of the phosphate group. Additionally, there is a presence of slipped π···π stacking interaction, between the bipyridine rings and adenine rings in a -B-B-A:A-B-B- fashion [B= bipyridine and A:A= adenine adenine adduct]. The third [Cu2(bpy)2(µ-5’-AMP)2]·14H2O complex 4c crystallizes in the triclinic space group P1 with one molecule in the asymmetric unit. The complex is neutral in nature with the charge on the copper centres being balanced by the 5’-AMP ligands. It is noteworthy that both the axial water molecules of complex are on the opposite side to each other which is in contradiction to the orientation of the water molecule in dimeric complex D of the molecule 4b. The least square plane analysis of the ribose sugar moiety reveals that the sugar moiety adopts envelope conformation. The ΦCN angle, which is the torsion angle of the base with respect to sugar, is 2(4)° for one 5’-AMP ligand and 69(4)° for other 5’-AMP ligand, suggesting an anti conformation for both the complexes about the glycosyl bonds. The ribose rings are puckered in both the ligands, with C3’ and C2’A displaced from the best four atom plane of furanose ring. C3’ deviates from the sugar plane by -0.624(3)Å which is on the same side of C5’, reveals C3’ endo conformation for the ribose ring. While for the other ribose moiety, C2’A deviates from the sugar plane by 0.509(3)Å which is on the same side of C5’, confirms C2’A endo conformation for the ribose ring. The conformation around the C4’-C5’ bond described by the angles ΦOO [O1’-C4’-C5’-O5’= - 76(3)°] and ΦOC [C3’-C4’-C5’-O5’= 41(3)°] is gauche gauche for one of the 5’-AMP ligand. Also around the C4’A-C5’A bond the torsion angles ΦOO [O1’A-C4’A-C5’A-O5’A= -59(2)°] and ΦOC [C3’A-C4’A-C5’A-O5’A = 59(3)°] suggest the commonly observed gauche gauche conformation for the other 5’-AMP ligand. The complex is stabilized by C-H···O and N-H···O intramolecular interactions between the adenine base and the phosphate oxygen atom. The phosphate oxygen atoms O8 and O8A become bifurcated by hydrogen bonding to O1W and O4W. In turn by symmetry relation it forms a sheet like structure extending to infinity. The adenine also interacts with the bipyridine ring with slipped π···π stacking interaction. The structure is stabilized by extensive net work of C-H···O and N-H···O hydrogen bond interactions between the neighboring molecules. The adenine rings interact with each other through the N-H···N hydrogen bonds forming a dimer between N6-N7 and N7- N6, mimicking the base pair observed in the DNA molecules, in addition to this N6 is involved in the formation of a hydrogen bond with the O8 atom of the phosphate group. Very interestingly, the axially coordinated water molecules O1A, O2A along with the phosphate oxygen atoms O8, O8A and water molecules O1W, O4W form a six membered ring in the chair conformation of a cyclohexane ring through hydrogen bonds mediated by the water molecules. Additionally, there is a presence of slipped π···π stacking interaction, between the bipyridine rings and adenine rings in a –B-B-A:A-B-B- fashion [B= bipyridine and A:A= adenine adenine adduct]. This is similar to previous two structures. All the three structures show the presence of different coordinating nature of phosphate groups obtained just by varying the pH conditions. The presence of cocrystal suggests that more than one type of coordination can exists at the same time. The fourth [Cu2(bpy)2(µ-5'CMP)(µ3-5'CMP)(Cl)]n·3H2O polymeric complex (4d) crystallizes in the Orthorhombic space group P212121. The polymer can be described as follows. There are two 5’-CMP ligand in the asymmetric unit viz., I and II. I acts as bidentate bridging ligand co-ordinating through base [N3] and phosphate oxygen [O9] to Cu1 and Cu2 respectively. II acts as a tridentate ligand co-ordinating to Cu1 through phosphate oxygen [O7A] while to Cu2 through the base [N3A] and phosphate oxygen [O9A]. Thus ligand I connects Cu1 and Cu2 forming a chain along the a axis while this chain is extended in b axis direction via ligand II. The least square plane analysis of the ribose sugar moiety reveals that both sugar moieties adopt envelope conformation. The ΦCN angle, which is the torsion angle of the base with respect to sugar, are 40.0(8)° [for ligand I] and 19.2(8)° [For ligand II] suggesting an anti conformation for both sugar moieties about the glycosyl bond. Both the ribose ring adopt a puckered confirmation with C2’ and C3’A displaced from the best four atom plane of furanose ring by 0.511(7) Å and 0.461(7) Å for ligand I and II respectively. Both the atoms C2’ and C3’A are on the same side as C5’, hence the conformation is C2’ endo [for ligand I] and C3’A endo [for ligand II] respectively. The conformation around the C4’-C5’ bond described by the angles ΦOO [O1’-C4’-C5’-O5’= -86.0(6)°{for I} and O1’A-C4’A-C5’A-O5’A= -72.8(2)°{for II}] and ΦOC [C3’-C4’-C5’-O5’= 33.9(8)°{for I} and C3’A-C4’A-C5’A-O5’A = 45.6(6)°{for II}] is gauche gauche for both the ribose rings in the polymeric complex. The polymeric strand is stabilized by N-H···O intramolecular interaction between the cytosine base and the phosphate oxygen atom. The cytosine base also interacts with the axial Chlorine atom to form N-H···Cl hydrogen bond. The structure is stabilized through the extensive network of N-H···O, C-H···O and O-H···O hydrogen bond interactions between the water molecules and polymerizing, making the sheets to run in third direction. The chlorine atom Cl1 at the same time along with the water molecule O1W and O8W of the phosphate group forms an envelope shape five membered ring [Cl1-O2W-O8-O1W-O3W-Cl1] via hydrogen bond. Thus the water molecules, the phosphate oxygen atoms, the chlorine atoms and the nitrogen atoms of the base make the network of hydrogen bonds in three dimension. In the three dimensional network the copper atoms, the base and the sugar with the phosphate are running anti parallel direction pushing the bipyridyl ring on the outer side, thus remaining as the back bone of the sheet. Additionally, there is a presence of slipped π···π stacking interaction, both intra and inter strand, between the 2, 2’-bipyridine rings. Thus the bipyridine rings, stacked

Purines are organic compounds with miscellaneous functions that are found in all living organisms in complex molecules such as nucleotides, nucleosides or as purine bases. The natural balance of purine levels is maintained by their synthesis, recycling and degradation. Excess purines are excreted in the urine as uric acid. Purine nucleotides may be recycled by salvage pathways catalysing the reaction of purine base with phosphoribosyl pyrophosphate. A completely new central molecule of purine metabolism, inosine monophosphate, can be synthesized from precursors during the de novo purine synthesis (DNPS). DNPS involves ten steps catalysed by six enzymes that form a multienzymatic complex, the purinosome, enabling substrate channelling through the pathway. DNPS is activated under conditions involving a high purine demand such as organism development. Currently, three DNPS-disrupting disorders have been described: ADSL deficiency, AICA-ribosiduria and PAICS deficiency. All three disorders are caused by genetic mutations leading to the impaired function of particular enzyme causing insufficient activity of respective DNPS step, manifested biochemically by accumulation of substrate of deficient enzyme, biologically by disruption of purinosome formation and clinically by unspecific neurological features,...