Nicotine and nornicotine are the two main alkaloids that accumulate in Nicotiana tabacum L. (tobacco), and nornicotine is the N-demethylation metabolite of nicotine. Nicotine is synthesized in the root, and probably primarily in the root tip. Both nicotine and nornicotine exist as two isomers that differ from each other by the orientation of H atom at the C-2' position on the pyrrolidine ring. (S)-nicotine is the dominant form in tobacco leaf and the enantiomer fraction of nicotine (EFnic), the fraction of (R)-enantiomer over the total nicotine, is approximately 0.002. Despite considerable efforts to elucidate nicotine and nornicotine related metabolism, a comprehensive understanding of the factors responsible for regulating the variable EF for nornicotine (0.04 to 0.75 ) relative to nicotine has been lacking. The objectives of these investigations were to understand the mechanisms behind the discrepancy. There are three nicotine demethylases reported to be active in tobacco. In vitro recombinant CYP82E4, CYP82E5v2 and CYP82E10 demethylated (R)-nicotine three, ten and ten-fold faster than (S)-nicotine, respectively, and no racemization was observed in either nicotine or nornicotine during demethylation. To confirm these in vitro results, the accumulation and demethylation of nicotine enantiomers throughout the growth cycle and curing process were investigated. Scion stock grafts were used to separate the contributions of roots (source) from leaves (sink) to the final accumulation of nicotine and nornicotine in leaf. The results indicate that nicotine consists of 4% of the R enantiomer (0.04 EFnic) when synthesized. However, (R)-nicotine is selectively demethylated by CYP82E4, CYP82E5 and CYP82E10, resulting in an approximate 0.01 EFnic and 0.60 EFnnic in the root. After most of (R)-nicotine is demethylated in root, nicotine and nornicotine are translocated to leaf, where nicotine is further demethylated. Depending on the CYP82E4 activity, an EFnnic of 0.04 to 0.60 is produced and only 0.2% of the remaining nicotine in the leaf is (R)-configuration.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Les pesticides et toxiques de guerre organophosphorés sont responsables d'intoxications qui se révèlent préoccupantes pour les autorités sanitaires. La recherche de solutions thérapeutiques pour pallier le manque de moyens efficaces pour contrer ces intoxications apparaît comme essentielle. L'acétylcholinestérase (AChE), enzyme de régulation de l'influx nerveux, en est la cible principale.Au cours de cette thèse, nous avons, d'une part, mis en place une stratégie de conception de molécules capables de réactiver les cholinestérases dites vieillies. Dans ce but, plusieurs dizaines de molécules ont été conçues et synthétisées. Leur évaluation in silico par docking moléculaire et in vitro par mesure d'affinité pour l'enzyme et par étude cristallographique n'a pas permis d'obtenir la réalkylation espérée, mais ce travail nous a offert quelques nouvelles perspectives.D'autre part, nos travaux de cristallographie de l'inhibition de la butyrylcholinestérase humaine par les agents V et le sarin montrent que cette cholinestérase a une énantiosélectivité altérée pour ces inhibiteurs chiraux par rapport à l'AChE humaine. Ceci implique la nécessité de quantités d'enzyme plus importantes pour atteindre la capacité protectrice désirée et donc un surcoût non négligeable. L'AChE humaine serait finalement un bioépurateur de neurotoxiques organophosphorés économiquement plus viable. / Organophosphate pesticides and chemical warfare are responsible for poisoning that pose to health security issues. The research of therapeutic solutions to overcome the lack of effective means to counter these poisonings is essential. Acetylcholinesterase (AChE), the enzyme involved in the regulation of nerve impulses, is the main target. ln this thesis, we have, first, set up a strategy for designing molecules that can reactivate aged cholinesterases. To this purpose, severa! dozen molecules have been designed and synthesized. Evaluation in silico by molecular docking and in vitro by rneasuring affinity for the enzyme and crystallographic study did not observe the desired realkylation. But this work opened new perspectives.Secondly, our crystallographic work on the inhibition of human butyrylcholinesterase by V-agents and sarin shows that cholinesterase has altered enantioselectivity for these chiral inhibitors of human AChE. This implies that larger amounts of enzyme are required for the desired protection and thus, a significant additional cost. Human AChE seems finally a more suitable neurotoxic bioscavenger.

Neste trabalho foi avaliada a seletividade (quimio ou enantiosseletividade) de quatro enzimas Baeyer-Villiger mono-oxigenases (BVMOs: PAMO, PAMO M446G, HAPMO e CHMO) frente a substratos contendo boro ou selênio. Inicialmente uma série de boro-acetofenonas foram submetidas à bio-oxidação catalisada por estas BVMOs. A enzima CHMO mostrou quimiosseletividade para transformação da ligação C-B em detrimento da reação de Baeyer-Villiger. Enquanto PAMO e PAMO M446G catalisaram a oxidação de ambas as funções em substratos 4-substituídos e a seletiva transformação de C-B no caso de substratos 3-substituídos. A enzima HAPMO levou a reação de Baeyer-Villiger e a transformação da ligação C-B em todos os casos. Quando alquenos contendo boro foram utilizados como substratos, somente aqueles que continham uma porção fenila em sua estrutura foram oxidados por BVMOs. Em nenhum dos casos foi observada reação de epoxidação e todas as enzimas levaram a transformação da ligação C-B em C-O. Compostos quirais contendo boro foram submetidos a reações com as BVMOs na tentativa de transformação enantiosseletiva. PAMO e PAMO M446G foram as melhores enzimas levando, na maioria dos casos, a satisfatória oxidação dos substratos. Entretanto, somente um composto pôde ser oxidado com boa enantiosseletividade (e.e 82-91%). Compostos quirais contendo o átomo de selênio também foram alvos de estudo com BVMOs. Novamente a enzima PAMO se mostrou a melhor opção dentre as enzimas testadas e somente quando R2 e R1 = Ph houve boa enantiosseletividade na oxidação (e.e 97 %). / In this work we evaluated the selectivity (chemo or enantioselectivity) of four Baeyer-Villiger mono-oxigenases (BVMOS: PAMO, M446G PAMO, HAPMO and CHMO) in the presence of boron-containing or selenium-containing compounds. Initially, a series of boron-acetophenones were submitted to oxidation reactions mediated by BVMOs. The enzyme CHMO was chemoselective leading only to C-B bond transformation instead Baeyer-Villiger reaction. However, PAMO and PAMO M446G mediated both oxidations in 4-substituted substrates, and only the C-B transformation in 3-substituted substrates. The enzyme HAPMO leading to Baeyer- Villiger reaction and C-B transformation in all cases. When boron-containing alkenes were the substrates, only compounds with phenyl moiety in the structure were oxidized by BVMOs. It was observed only the C-B transformation and none of the epoxidation reaction. Chiral boron compounds were submitted to BVMOS mediated reactions in an attempt of enantioselective transformation. PAMO and M446G PAMO showed the best results leading, in most cases, to a satisfactory oxidation. However, only one compound was oxidized with great enantioselectivity (82-91% ee). Selenium-containing chiral compounds were also tested in reactions mediated by BVMOs. Again, PAMO showed the best results among BVMOs tested, but only when R2 e R1 = Ph the reaction occurred with great enantiosselectivity (97 % ee).

Biocatalysis represents an attractive route to the production of various compounds which are difficult or impossible to synthesise and isolate using traditional chemical synthesis. In particular, the production of chiral molecules is a function ideally suited to biocatalysis, due to the natural stereospecificity of enzymes. The synthesis of such chiral molecules is essential in the production of pharmaceuticals, additives for the food and drinks industry and the creation of specialist polymers. CYP105AS1, isolated from Amycolatopsis orientalis, is a cytochrome P450 enzyme which produces the inactive 6-epi-pravastatin of the blockbuster anti-cholesterol drug pravastatin. Previous directed evolution efforts have engineered this enzyme to produce a five-point mutant, known as P450prava, which partially reversed the stereospecificity of the enzyme to produce a majority pravastatin product mixture. This thesis details work to use structure-led engineering approaches to redesign the active site of P450prava to introduce stringent stereospecificity. A combinatorial approach of manual and computational rational design was pursued, leading to the creation of a novel T95F/V180M double mutant of P450prava. This double mutant was found to have successfully eliminated the unwanted 6-epi-pravastatin enantiomer from the product mix, leaving a pure pravastatin product. P450prava was also shown to bind and hydroxylate other statin substrate molecules, demonstrating its versatility in the production of drug metabolites and other high-value oxyfunctionalised molecules. This property, along with its proven tolerance of significant active site engineering efforts, demonstrates the viability of the P450prava as a platform for the creation of novel biocatalysts for the production of various hydroxylated products from diverse substrate molecules.

Neste trabalho, várias reações de Heck foram investigadas utilizando principalmente métodos baseados na Teoria do Funcional de Densidade (DFT). Na primeira parte foi proposto um novo ciclo catalítico de Heck, com etapas aniônicas e neutras combinadas desencadeado por um complexo de paládio (Pd) suportado por um ligante carbeno N-heterocíclico (NHC) inédito. Posteriormente, a influência estérica do ligante NHC foi investigada na seletividade da reação de Heck inicialmente estudada. Para o NHC pouco volumoso, a componente eletrônica era majoritária comparada às contribuições de caráter não-covalente. Entretanto, quando o NHC com maior impedimento estérico foi analisado, somente alguns funcionais de densidade (TPSS-D3, ωB97x-D, BP86-D3, e M06-L), juntamente com o método de função de onda DLPNO-MP2, foram capazes de predizer a tendência de seletividade experimental. Por fim, a natureza do metal foi analisada por meio de uma comparação entre catalisadores NHC de níquel (Ni) e Pd nas reações de Heck. Os resultados teóricos forneceram indícios mecanísticos para o entendimento da menor atividade catalítica usualmente encontrada nos processos envolvendo complexos NHC de Ni e da necessidade experimental da formação de complexos de Ni catiônicos para atingir uma maior eficiência catalítica nessas reações. Na segunda parte deste estudo, dois exemplos marcantes da literatura sobre as reações de Heck-Matsuda enantiosseletivas foram teoricamente investigadas. Os cálculos DFT mostraram a influência crucial do substrato (olefina) na seletividade destas reações. Em um dos casos a seletividade da reação foi aprimorada através dos dados mecanísticos fornecidos pelos cálculos DFT. / In this work, several examples of Heck reactions were investigated using mostly Density Functional Theory (DFT) methods. At the first part, a new Heck catalytic cycle was proposed with combined anionic and neutral steps initiated by a newly N-heterocyclic carbene (NHC) based palladium (Pd) complex. Posteriorly, the influence of steric demanding of NHC ligand was investigated on selectivity of Heck reaction initially studied. In the case of small NHC ligand, the electronic component is more important than the noncovalent contributions. However, when the crowded NHC ligands were studied only selected density functionals (TPSS-D3, ωB97x-D, BP86-D3, e M06-L), and the wavefunction based method DLPNO-MP2, were capable to predict the experimental selectivity trends reported. Finally, the metal nature was analyzed by a comparison of the nickel (Ni) and Pd catalyzed Heck reactions. The theoretical results provided mechanistic insights that help to understand the low catalytic activity usually reported when Ni catalysts were used and the experimental requirement of cationic intermediates to achieve some efficiency for NHC-Ni-catalyzed Heck coupling. At second part, two representative examples from literature about the enantioselective Heck-Matsuda reactions were theoretically investigated. DFT calculations shown the crucial influence of substrate (olefin) on the selectivity of these reactions. One of cases studied, the selectivity of reaction was improved by the DFT results

Ce mémoire de thèse se concentre sur le développement de réactions multicomposants énantiosélectives de dérivés 1,3-Dicarbonylés en présence d'un organocatalyseur, en vue de préparer des motifs polyhétérocycliques fusionnés.Dans un premier temps, nous décrivons nos résultats initiaux sur une réaction multicomposants énantiosélective avec des énals et des amines primaires simples. Au vu des faibles énantiosélectivités obtenues, des amines fonctionnalisées ont ensuite été introduites dans ces réactions, permettant ainsi de synthétiser des pyrrolopiperazines et d'autres molécules hétérocycliques polyfonctionnalisées énantioenrichies, toutes obtenues avec des rendements intéressants et des énantiosélectivités élevées.Ayant utilisé avec succès des β-Cétoamides α-Méthyléniques dans ces réactions multicomposantes, nous avons réalisé par ailleurs que leur simple addition de Michael sur des oléfines pauvres en électrons n'avait jamais été décrite en version organocatalysée. Nous avons donc étudié leur réaction avec des nitrooléfines en présence d’organocatalyseurs chiraux, et les produits attendus ont alors été obtenus avec de bons rendements et d'excellentes diastéréo- et énantiosélectivités. / This thesis focuses on the development of enantioselective multicomponent reactions with 1,3-Dicarbonyls in the presence of an organocatalyst, to synthesize fused polyheterocyclic motives.At first, we describe our initial results on an enantioselective multicomponent reaction with enals and simple primary amines. In view of the low enantioselectivities achieved, functionalized amines were then introduced in these reactions, thereby synthesizing enantioenriched pyrrolopiperazines and other polyfunctionalized heterocyclic molecules, all obtained with attractive yields and high enantioselectivities.Having successfully used methylene β-Ketoamides in these enantioselective MCRs, we realized also that their simple Michael addition to electron-Poor olefins had never been described in organocatalytic conditions. We therefore studied their reaction with nitroolefins in the presence of various chiral organocatalysts, and the expected products were pleasingly obtained with high yields, excellent diastereo- and enantioselectivities.

Ce mémoire de thèse se concentre sur la mise au point de nouvelles réactions mettant en jeu des composés 1,3-cétoamides en organocatalyse énantiosélective, enfin d’accéder à des structures inaccessibles jusqu’alors, ainsi que sur le développement d’une nouvelle synthèse de composés 1,3-dicarbonylés originaux et leur utilisation dans des réactions orgcanocatalysées.Dans un premier temps, nous décrivons tous nos essais de fonctionnalisation stéréosélective en des 1,3-cétoamides acycliques non substitués, par réaction de Mannich énantio- et diastéréosélective, par réaction d’amination énantiosélective et par réarrangement de Lossen énantiosélectif. Puis, nous abordons comment les 1,3-cétoamides nous ont permis de réaliser la synthèse multi-composés régio-, énantio-, et diastéréocontrôlée de 1,2,3,4-tétrahydropyridines, motif qui n’avait jamais été obtenu en organocatalyse auparavant et que la synthèse d’une autre famille d’hétérocycles complexes, les 2,6-DABCO.Dans un second temps, nous détaillons la mise au point d’une nouvelle méthode de synthèse, basée sur l’addition de nucléophiles silylés sur des intermédiaires -oxocétènes, générés par réarrangement de Wolff initié par irradiation micro-ondes. Ainsi, des 1,3-cétoaldéhydes masqués des 1,3-cétoamides primaires, des composés bicycliques fusionnés furan-3-ones et un 1,3-cétoazoture d’acyle masqué, ont pu être synthétisés. L’utilisation en organocatalyse énantiosélective de ces substrats originaux est également détaillée. / This thesis focuses on the development of new reactions using 1,3-ketoamides in enantioselective organocatalysis to access structures never obtained before and on the development of a new synthesis of original 1,3-dicarbonyl compounds and their use in organocatalyzed reactions.Firstly, we describe all our attemps of -functionalization of acyclic unsubstituted 1,3-ketoamides by enantio- and diastereoselective Mannich reaction, by enantioselective amination reaction and by enantioselective Lossen rearrangement. Then, we address how 1,3-ketoamides allow us to achieve the regio- enantio- and diastereoselective multicomponent synthesis of 1,2,3,4-tetrahydropyridines, a scaffold never reached previously as well as the synthesis of another family of complex heterocycles, 2,6-DABCO.Secondly, we detail the development of a new synthetic methodology, based on the addition of silylated nuclophiles to -oxoketene intermediates, generated by Wolff rearrangement triggered by microwave irradiation. This way, masked 1,3-ketoaldehydes, primary 1,3-ketoamides, fused bicylic furan-3-ones and a masked 1,3-ketoacyl azide have been synthesized. The use of these original substrates in enantioselective organocatalysis will be discussed as well.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Neste trabalho foi avaliada a seletividade (quimio ou enantiosseletividade) de quatro enzimas Baeyer-Villiger mono-oxigenases (BVMOs: PAMO, PAMO M446G, HAPMO e CHMO) frente a substratos contendo boro ou selênio. Inicialmente uma série de boro-acetofenonas foram submetidas à bio-oxidação catalisada por estas BVMOs. A enzima CHMO mostrou quimiosseletividade para transformação da ligação C-B em detrimento da reação de Baeyer-Villiger. Enquanto PAMO e PAMO M446G catalisaram a oxidação de ambas as funções em substratos 4-substituídos e a seletiva transformação de C-B no caso de substratos 3-substituídos. A enzima HAPMO levou a reação de Baeyer-Villiger e a transformação da ligação C-B em todos os casos. Quando alquenos contendo boro foram utilizados como substratos, somente aqueles que continham uma porção fenila em sua estrutura foram oxidados por BVMOs. Em nenhum dos casos foi observada reação de epoxidação e todas as enzimas levaram a transformação da ligação C-B em C-O. Compostos quirais contendo boro foram submetidos a reações com as BVMOs na tentativa de transformação enantiosseletiva. PAMO e PAMO M446G foram as melhores enzimas levando, na maioria dos casos, a satisfatória oxidação dos substratos. Entretanto, somente um composto pôde ser oxidado com boa enantiosseletividade (e.e 82-91%). Compostos quirais contendo o átomo de selênio também foram alvos de estudo com BVMOs. Novamente a enzima PAMO se mostrou a melhor opção dentre as enzimas testadas e somente quando R2 e R1 = Ph houve boa enantiosseletividade na oxidação (e.e 97 %). / In this work we evaluated the selectivity (chemo or enantioselectivity) of four Baeyer-Villiger mono-oxigenases (BVMOS: PAMO, M446G PAMO, HAPMO and CHMO) in the presence of boron-containing or selenium-containing compounds. Initially, a series of boron-acetophenones were submitted to oxidation reactions mediated by BVMOs. The enzyme CHMO was chemoselective leading only to C-B bond transformation instead Baeyer-Villiger reaction. However, PAMO and PAMO M446G mediated both oxidations in 4-substituted substrates, and only the C-B transformation in 3-substituted substrates. The enzyme HAPMO leading to Baeyer- Villiger reaction and C-B transformation in all cases. When boron-containing alkenes were the substrates, only compounds with phenyl moiety in the structure were oxidized by BVMOs. It was observed only the C-B transformation and none of the epoxidation reaction. Chiral boron compounds were submitted to BVMOS mediated reactions in an attempt of enantioselective transformation. PAMO and M446G PAMO showed the best results leading, in most cases, to a satisfactory oxidation. However, only one compound was oxidized with great enantioselectivity (82-91% ee). Selenium-containing chiral compounds were also tested in reactions mediated by BVMOs. Again, PAMO showed the best results among BVMOs tested, but only when R2 e R1 = Ph the reaction occurred with great enantiosselectivity (97 % ee).