This dissertation describes the benzylation of adenine under basic conditions, the unequivocal determination of the identity of the products of this reaction, an exploration of the effect of solvent on the reaction, a thorough computational study of the reaction mechanism and an investigation into the hydrogen-deuterium exchange reaction of the N-benzyladenine products and related compounds. The preferential sites of alkylation of adenine under basic conditions in DMSO were proven to be the N9 and N3 positions. X-ray crystal structures were obtained for both compounds. Formation of the N9-benzyladenine product is the most favoured in polar aprotic solvents, such as DMSO, and as the proportion of polar protic solvents, such as water, increases, so does the formation of the N3-benzyladenine product. Characteristic 1H NMR  chemical shifts of the purine ring protons and HMBC 1H-13C correlation NMR spectroscopy were useful tools to assign the 1H and 13C NMR spectra chemical shifts and confirm that the solution structures were the same as the isolated crystals. Simulating the SN2 mechanism for the N1-, N3-, N7- and N9-pathways computationally, employing DMSO as the simulated solvent, resulted in ambiguous results when considering the electronic energies of initial, TS and final products alone. However, a novel approach was developed (employing IQA-defined energy terms) to study fragment interactions along the reaction paths. It provided a full explanation of the reaction mechanism and yielded results which supported the N3/N9 positions of alkylation over the N1/N7 sites. The preference for the sites of alkylation occurs after the transition state, in which the N1/N7 reaction paths fail to proceed favourably to the end product, N1- and N7-benzyladenine, respectively. The N9-pathway dominates the N3-pathway at the product formation step, which corresponds to the N9- benzyladenine being the major product, as shown in Figure 1, and the N3-benzyladenine being the minor product from the benzylation of adenine. The faster rate of deuteration at the C8 position of N9-benzyladenine as compared to the deuteration rates at the C2 and the C8 of N3-benzyladenine, have shown support for a sp3 mediated mechanism and a carbene mediated mechanism of deuteration based on the “push” and “pull” mechanisms proposed for the C8 proton transfer of ATP in kinase enzymes. The deuteration of the C8 proton of 2,6-dichloropurine derivatives supports the existence of the carbene mediated mechanism since these compounds lack the amine moiety necessary for the sp3 mediated mechanism. These results demonstrate how experimentation and computation have led to greater insights into the reactivity of adenine and its derivatives. This strategy provides a useful platform for future research into adenine reaction mechanisms and the role adenine plays in kinase catalysis. / Dissertation (MSc)--University of Pretoria, 2015. / National Research Foundation (NRF) / Chemistry / MSc / Unrestricted

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Covalent binding of dinuclear metal carboxylate compounds to purine DNA nucleobases has been shown to be a source of anticancer activity, and has resulted in intense research to understand the coordination of metal complexes to DNA. This investigation focuses on the formation of dirhenium metal:dinucleotide complexes of purine nucleobases. To our knowledge, complexes formed by the reaction of dinuclear rhenium metal compounds with dinucleotides have not been reported in the literature.

In chapter 1, we describe development of a mild and efficient method for SNAr iodination of 6-chloropurine 2'-deoxynucleosides and nucleosides. Our studies demonstrate that 6-iodopurine nucleosides are excellent substrates for certain transition metal-catalyzed cross-coupling reactions. In chapter 2, we describe synthesis of protected 6-fluoro, 6-chloro, 6-bromo and 6-sulfonylpurine nucleosides. Comparisons among 6-fluoro-, 6-chloro-, 6-bromo, 6-iodo and 6-sulfonylpurine nucleosides for SNAr reactions with various N, O and S nucleophiles were investigated. Our results demonstrate that the 6-fluoropurine nucleoside is the best substrate for SNAr reactions among the four 6-halopurine nucleosides with oxygen, sulfur and aliphatic amine nucleophiles, and also with an aromatic amine plus TFA as a catalyst. However, the 6-iodopurine nucleoside is the best substrate for the aromatic amine without acid. With oxygen and sulfur nucleophiles, the 6-sulfonylpurine nucleoside reacted even faster than the 6-fluoropurine nucleoside. In chapters 3 and 4, nickel- and palladium-based systems with imidazolium-carbene ligands can catalyze efficient Suzuki cross-couplings of arylboronic acids and 6-[(imidazol-1-yl)-, (1,2,4-trizaol-4-yl), fluoro, alkylsulfanyl and alkylsulfonyl]purine 2'-deoxynucleosides and nucleosides to give the corresponding 6-arylpurine products.

Clostridioides difficile is a bacterial pathogen that causes pseudomembranous colitis and the majority of antibiotic-associated diarrheal cases. Broad-spectrum antibiotic usage disrupts the normal gut microbiota and thereby compromises colonization resistance, the main defense against C. difficile infection. Treatment options are limited to vancomycin, fidaxomicin, and the fecal microbiota transplant. Addressing the scarcity of these therapeutics, we documented two explorations in C. difficile drug discovery: (i) evaluation of antibacterial and toxin-suppressing activity of (+)-puupehenone and similar derivatives, and (ii) clarification of a discrepancy in the hypothesized mechanism of auranofin against C. difficile. A better understanding of how C. difficile colonizes and thrives in the gut can greatly benefit therapeutic development. Interestingly, C. difficile can scavenge nutrients such as amino acids and possibly even purines during infection. Amino acids including proline and glycine act as substrates for Stickland metabolism, a bioenergetics scheme that partially relies on enzymes containing selenium in the form of selenocysteine (e.g., D-proline reductase and glycine reductase). Purines such as xanthine and uric acid can be degraded by bacterial molybdenum hydroxylases harboring an uncharacterized form of selenium, though the role of these enzymes in C. difficile physiology is poorly understood. Selenium likely plays a key role in the scavenging of these nutrients during C. difficile infection. Our investigation of these selenium-dependent enzymes revealed two new findings in C. difficile biology: (i) a link between proline-dependent growth and D-proline reductase, characterized as an energy "addiction," and (ii) a previously uncharacterized selenium-dependent pathway involved in the catabolism of xanthine and uric acid. Overall, these physiological analyses of C. difficile provide promising candidates for therapeutics and key information regarding the organism's nutrient preferences.

La grande variété de propriétés biologiques associées au noyau purine en fait une structure privilégiée pour la conception et la synthèse de nouvelles molécules à visée thérapeutique. Cette spécificité est étroitement liée à la grande diversité de substituants pouvant être introduits sur les différentes positions du noyau purine et en particulier sur C2, C6, C8 et N9. Par conséquent, le développement de méthodes de fonctionnalisation rapides de cette famille de composés est d’un grand intérêt synthétique. Nous nous sommes focalisés sur la formation de liaisons C-C et C-N sur les positions 6 et 8 du noyau purine pour pouvoir présenter de nouveaux outils de synthèse permettant d’introduire une plus grande diversité fonctionnelle. D’une part, nous avons étudié la fonctionnalisation directe de liaisons C-H de purines, sujet encore peu exploré. En effet, de nos jours, le traditionnel couplage croisé (Negishi, Suzuki-Miyaura), utilisé pour la création de liaisons C-C, se voit de plus en plus concurrencé par ces réactions puisqu’elles ne nécessitent pas la préparation d’un partenaire organométallique. Ce sont des réactions dites à économie d’atomes. En nous basant sur l’expérience du laboratoire dans le domaine de la fonctionnalisation directe de liaisons C-H, nous avons envisagé l’alcénylation et l’alcynylation directes en position 8 de la purine, les motifs alcényle et alcynyle étant présents dans certaines purines d’intérêt biologique. D’autre part, nous nous sommes intéressés à deux méthodes de couplage croisé pallado-catalysé permettant la formation de liaisons C-N et C-C : le couplage de Buchwald – Hartwig entre une 8-iodopurine et des amides ou des amines aromatiques, et le couplage de Liebeskind – Srogl entre une 6-thioétherpurine et divers acides boroniques. / Purine is the most widely distributed N-heterocycle scaffold in the nature and its derivatives are well known for their biological and fluorescent properties. These characteristics are linked to the diversity of substituents that can be introduced, especially on the C-2, C-6, C-8 and N-9 positions. Therefore, the development of methods for rapid functionalization of this family of compounds represent a valuable asset. We focused on the formation of C-C and C-N bonds at positions 6 and 8 of the purine ring in order to provide new synthesis tools allowing the introduction of functional diversity. On the one hand, we studied the direct functionalization of C-H bonds of purines, subject still little explored. Indeed, nowadays, traditional cross-coupling reactions (Negishi, Suzuki-Miyaura), used for the creation of C-C bonds, are increasingly challenged by these reactions since they do not require the preparation of an organometallic partner. Their advantage lies in step and atom economy. Based on previous experience in our laboratory in the field of direct functionalization of C-H bonds, we envisioned direct alkenylation and alkynylation at position 8 of the purine, knowing that alkenyl and alkynyl patterns are found in purines of biological interest. On the other hand, we were interested in two pallado-catalyzed cross-coupling methods for the formation of C-N and C-C bonds : Buchwald – Hartwig coupling between 8-iodopurine and aromatic amines or amides, and Liebeskind – Srogl coupling between 6-thioétherpurine and a range of boronic acids.

(a) The Robins reagent [2-acetamido-6-O-(diphenylcarbamoyl)purine] was utilized for glycosylation under Lewis acid conditions. Regioselectivity of glycosylation depends on the glycosyl donor and its 2-O- or 2-N-protecting group. Regioselective N9 glycosylation of 2-acetamido-6-O-(diphenylcarbamoyl)purine with problematic glucosamine has been accomplished by protecting the amino function as a phthalimido group with consequent stabilization of the oxocarbenium cation, and lowering the activation energy by introduction of trichloroacetimidate at the anomeric carbon. (b) 6-Heteroaryl functions [6-(1,2,4-triazol-4-yl) and 6-(imidazol-1-yl)] were introduced into purine derivatives for regioselective N9 alkylation. The regiospecificity of alkylation mainly results from steric effects due to the coplanar conformation of the two linked heterocyclic rings governed by conjugation. Several of the obtained acyclic derivatives showed antiviral and antitumor activities. (c) Glycosylation of purine derivatives with 2-deoxy-3,5-di-O-(p-toluoyl)-a-D-erythro-pentofuranosyl chloride using the sodium salt method usually gave a mixture of both anomers. Lipophilic groups were introduced into the imidazole ring of 6-(imidazol-1-yl)purine derivatives to increase the solubility of the sodium salts in moderately polar solvents. Differential solvation effects in binary solvent mixtures were utilized to improve the stereoselectivity of glycosylation. The stereoselectivity varied with the sizes of lipophilic groups and the polarity of solvents. With the propyl group, and in CH3CN/toluene (1:1) and/or CH3CN/CH2Cl2 (1:1), regiospecfic and highly stereoselective glycosylation of purines with 2-deoxy-3,5-di-O-(p-toluoyl)-a-D-erythro-pentofuranosyl chloride was achieved. (d) Using the above method, a low cost and efficient synthesis of 2-chloro-2'-deoxyadenosine (2-CdA, cladribine) was accomplished with an overall yield of 48% from inexpensive guanosine and 57% from 2,6-dichloropurine. 2-Chloro-6-(2-propylimidazol-1-yl)purine was prepared either from guanosine in a yield of 61% in 5 steps or from 2,6-dichloropurine in a yield of 72% in one step. Coupling of this 2-chloro-6-heteroarylpurine with 2-deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentofuranosyl chloride in binary solvent mixtures, followed by activation of imidazolyl as a better leaving group via benzylation at N3 and then ammonolysis gave cladribine in good yield (79%) for 3 steps. Analogs of purine derivatives with lipophilic groups (butyl, pentyl and 2-phenylpropyl) worked almost as well.

Le cancer est la plus grande cause de mortalité après les maladies cardiaques. Les cellules cancéreuses sont issues des cellules saines dont les systèmes de multiplications et de régulations ont été annihilés. Elles se multiplient anarchiquement et forment des tumeurs. Nous nous sommes donc intéressés au cancer de la vessie qui en termes d’incidences dans les pays industrialisés est le quatrième cancer chez l’homme et le neuvième chez la femme. Parmi les nombreux récepteurs surexprimés dans les cellules tumorales de vessie, la kinase Tyro3 s’est révélée indispensable à la survie de ces cellules tumorales. Le premier objectif de cette thèse est de synthétiser un inhibiteur sélectif de Tyro3 possédant un noyau purine. Une première stratégie est orienté vers la synthèse d’un inhibiteur de type I alors que la seconde stratégie est menée vers la synthèse d’un inhibiteur de type II. Deux composés montrent une activité intéressante à 1 µM sur Tyro3 et semblent adopter un profil d’inhibition de type II. Le second objectif de la thèse présente une approche méthodologique pour la substitution de la position 2 et 8 du noyau purine. Une première partie présente la double lithiation des positions 2 et 8 de la 6-chloro-9-(tétrahydro-2H-pyran-2-yl)-9H-purine suivie d’une substitution avec différents électrophiles. Le dérivé 2,8-diodé obtenu est engagé dans une réaction de Sonogashira conduisant à des dérivés 2-alcynyl-8-iodo ou 2,8-dialcynyle. Une seconde partie traite de l’amidation et amination pallado-catalysées de la 8-iodo-6-(phénylsulfanyl)-9-(tétrahydro-2H-pyran-2-yl)-9H-purine. / After heart diseases, cancer is the most important cause of death. Cancerous cells are normal cells which multiplication and regulation system have been affected. They anarchically grow and give tumors. We have investigated in bladder cancer which is fourth cancer among men and ninth cancer among women in industrial countries. Amongst overexpressed receptors in bladder cancerous cells, Tyro3 seems to be essential for the survival of bladder cancerous cells. First goal of this thesis is to synthesize a potent and selective inhibitor of Tyro3 with a purine scaffold. Two approaches have done. The first approach is the synthesis of a type I inhibitor whereas the second approach is the synthesis of a type II inhibitor. Two compounds have shown an interesting activity against Tyro3 at 1 µM and they seem to be type II inhibitors. The second part of the thesis was the functionalization of position 2 and 8 of purine scaffold. We show first the double deprotonation by lithium species of 6-chloro-9-(tétrahydro-2H-pyran-2-yl)-9H-purine following by substitution with different electrophiles. Obtained 2,8-di-iodine compound is engaged in Sonogashira reaction which gives 2-alkyne or 2,8-di-alkyne compunds. And, we investigate in palladium catalyzed amidation and amination of 8-iodo-6-(phénylsulfanyl)-9-(tétrahydro-2H-pyran-2-yl)-9H-purine.

Plasmodium falciparum est responsable de la forme la plus grave de paludisme avec plus de 600 000 décès par an. L'absence de vaccin efficace, combinée à l'émergence de résistances aux traitements récurrents, exige le développement de nouvelles molécules. Afin de limiter ces résistances, il est nécessaire de cibler de nouvelles voies métaboliques indispensables à la survie du parasite. Ce travail de thèse repose sur l'étude de deux voies métaboliques essentielles au parasite que sont la voie de recyclage des bases puriques et la voie de biosynthèse des ancres glycosylphosphatidylinositol (GPI).En ce qui concerne la voie de recyclage des bases puriques, la détermination des structures cristallines de l' « IMP specific 5‘-nucleotidase » (PfISN1) associée aux études biochimiques et biophysiques (SAXS, EM, MALS…), a permis de préciser le mécanisme d'action fournissant ainsi une base solide pour la mise au point d'inhibiteurs. Une banque de plus 3000 composés a été criblée par Fluorimétrie à Balayage Différentiel et les effets des molécules sélectionnées seront évalués sur l'enzyme et sur la croissance du parasite en culture.Quatre cibles thérapeutiques potentielles appartenant à la voie de biosynthèse des ancres GPI ont été sélectionnées. L'utilisation de plusieurs systèmes d'expression disponibles au laboratoire (bactérie, levure, acellulaire en germe de blé) ou via des plateformes européennes pour l‘expression en cellules de mammifères HEK293T (Oxford), de cellules BHK21 transfectées avec le virus de la vaccine modifié, T7-MVA, (Strasbourg) ou la plateforme ESPRIT (Grenoble) ont permis de passer outre les difficultés rencontrées pour exprimer les protéines d'intérêt. L'une des quatre cibles, la mannose-1-phosphate guanylyltransférase (PfMPG), a pu être exprimée de manière suffisante quantitativement et qualitativement pour une caractérisation biochimique et structurale. Une analyse par SAXS et cristallographie aux rayons X a été réalisée / Plasmodium falciparum is responsible for the most severe form of malaria with more than 600,000 deaths per year. The lack of an effective vaccine, combined with the emergence of drug resistant parasites, necessitates the development of new drugs. In order to limit these resistances, it is necessary to target new metabolic pathways essential for parasite survival. This thesis work is based on the study of two metabolic pathways essential to the parasite, the purine salvage pathways and the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway.Concerning the purine salvage pathway, the determination of the crystal structures of the IMP -specific 5'-nucleotidase (PfISN1) associated with biochemical and biophysical studies (SAXS, EM, MALS, etc.) have allowed to propose a reaction mechanism, thereby providing a solid basis for the conception and development of inhibitors. A library of more than 3000 compounds was screened by Differential Scanning Fluorimetry and the selected molecules will be evaluated for their inhibitory effect on the enzyme and on the growth of parasites in culture.Four potential therapeutic targets belonging to the GPI anchor biosynthesis pathway were selected. The use of several in-house available expression systems (bacteria, yeast, and acellular wheat germ) as well as European platforms for the expression in HEK293T mammalian cells (Oxford), in BHK21 cells transfected with the modified vaccinia virus, T7-MVA, (Strasbourg) or the ESPRIT platform (Grenoble) has allowed us to overcome the difficulties encountered on obtaining the selected protein targets. One of the four targets has been expressed in sufficient amount and quality for biochemical- and structural characterization, namely the mannose-1-phosphate guanylyltransferase (PfMPG). SAXS and X-ray crystallography analyses have been carried out

Previous evidence has shown that the simple sequences microsatellites and poly-purine/poly-pyrimidine tracts (PPTs) could be both a cause, and an effect, of meiotic recombination. The causal link between simple sequences and recombination has not been much explored, however, probably because other evidence has cast doubt on its generality, though this evidence has never been conclusive. Several questions have remained unanswered in the literature, and I have addressed aspects of three of them in my thesis. First, what is the scale and magnitude of the association between simple sequences and recombination? I found that microsatellites and PPTs are strongly associated with meiotic double-strand break (DSB) hotspots in yeast, and that PPTs are generally more common in human recombination hotspots, particularly in close proximity to hotspot central regions, in which recombination events are markedly more frequent. I also showed that these associations can't be explained by coincidental mutual associations between simple sequences, recombination and other factors previously shown to correlate with both. A second question not conclusively answered in the literature is whether simple sequences, or their high levels of polymorphism, are an effect of recombination. I used three methods to address this question. Firstly, I investigated the distributions of two-copy tandem repeats and short PPTs in relation to yeast DSB hotspots in order to look for evidence of an involvement of recombination in simple sequence formation. I found no significant associations. Secondly, I compared the fraction of simple sequences containing polymorphic sites between human recombination hotspots and coldspots. The third method I used was generalized linear model analysis, with which I investigated the correlation between simple sequence variation and recombination rate, and the influence on the correlation of additional factors with potential relevance including GC-content and gene density. Both the direct comparison and correlation methods showed a very weak and inconsistent effect of recombination on simple sequence polymorphism in the human genome.Whether simple sequences are an important cause of recombination events is a third question that has received relatively little previous attention, and I have explored one aspect of it. Simple sequences of the types I studied have previously been shown to form non-B-DNA structures, which can be recombinagenic in model systems. Using a previously described sodium bisulphite modification assay, I tested for the presence of these structures in sequences amplified from the central regions of hotspots and cloned into supercoiled plasmids. I found significantly higher sensitivity to sodium bisulphite in humans in than in chimpanzees in three out of six genomic regions in which there is a hotspot in humans but none in chimpanzees. In the DNA2 hotspot, this correlated with a clear difference in numbers of molecules showing long contiguous strings of converted cytosines, which are present in previously described intramolecular quadruplex and triplex structures. Two out of the five other hotspots tested show evidence for secondary structure comparable to a known intramolecular triplex, though with similar patterns in humans and chimpanzees. In conclusion, my results clearly motivate further investigation of a functional link between simple sequences and meiotic recombination, including the putative role of non-B-DNA structures.