A substrate structure rule to predict the enantioselectivity of Candida rugosa lipase (CRL) towards $ alpha$-substituted carboxylic acids is proposed. Lipase from Candida rugosa resolved methyl $ alpha$-substituted phenylacetates -5. These substrates were examined to understand the influence of steric and the electronic nature of the substituents at the chiral center on the enantioselectivity. Crude CRL was partially enantioselective towards substrates -3, and highly selective towards and, E $>$ 100. Partial purification of crude CRL by 2-propanol extraction and separation by an ion exchange chromatography, dramatically improved the enantioselectivity towards -3, E $>$ 100. One main lipase fraction, F$ sb{ rm C}$ was obtained with 58 fold purification and 62% yield. This improvement in enantioselectivity was achieved with a simple dialysis against deionized water or 50% 2-propanol extraction. Lauric acid (10 mM), partially reversed the increase in the enantioselectivity. The absolute configuration of the favored enantiomer for crude and partially purified lipase preparations, was S. Other lipases with opposite stereoselectivities were not identified. / Based on literature and our results, it is believed that the size of the substituents at the stereogenic carbon center can account for the enantioselectivity of CRL catalyzed hydrolysis. The opposite configuration is not preferred when the electronic nature of the medium size substituent is varied. The rule can predict the selectivity data for partially purified CRL preparations. We believe that the accuracy of the rule can be improved if more examples of partially purified CRL reactions are examined.

In order to extend application of the gel-degradation mechanism of delignification, model compounds of the general structure (3,4-dimethoxyphenyl)-CH(OR)R$ sp prime$ were treated at room temperature with increasing charges of molecular Cl$ sb2$ in glacial HOAc. / Catalytic amounts of Cl$ sb2$ cleaved benzyl aryl ether bonds (OR=2-methoxy-4-methylphenoxy) before any ring chlorination occurred. Yields of cleavage decreased in the order: R$ sp prime$=H; R$ sp prime$=$-$CH$ sb2$-2-methoxyphenoxy; R$ sp prime$=$-$CH(CH$ sb2$OH)-2-methoxyphenoxy. These results support the concept that the delignification of wood pulp by chlorine results from benzyl aryl ether cleavage. The conditions of the reaction, as well as control reactions with hydrochloric acid, suggest that the cleavage is due to conventional acid hydrolysis induced by chlorine. The experimental evidence does not rule out another possible mechanism in which the cleavage is initiated by molecular chlorine. / Benzyl ether links were generally stable in models of the benzyl alkyl type, whose primary reactions were ring chlorination. A large excess of chlorine caused side-chain displacement, hitherto considered the primary reaction in the solubilization of lignin.

The use of chemically modified oligonucleotides (AON) to selectively inhibit the expression of genes involved in pathological processes, offers a rational approach for the prevention and treatment of some disorders. Phosphorothioate oligonucleotides (PS-DNA), in which one of the non-bridging oxygen atoms of the internucleotide linkage is replaced by a sulfur atom, have been extensively studied and are among the most widely used "antisense" oligonucleotides (AON). However, the isomeric analogue, in which one of the two bridging oxygen atom is replaced by sulfur has not been extensively investigated, as such, this prompted our interest towards the construction of oligonucleotides containing the modified 3'-S- and 5'-S-phosphorothiolate linkages. An oligodeoxynucleotide (DNA) strand containing a single and three consecutive 3'-and 5'-S-phosphorothiolate internucleotide linkages as well as a chimeric oligonucleotide, composed of 2'-OMe-RNA 'wings' with a central hexanucleotide ("gap") composed of 3'-deoxy-3'-thiothymidine (T3'S) residues were successfully prepared via the phosphoramidite approach. (Abstract shortened by UMI.)

A green stereoselective total synthesis of cardiobutanolide is proposed, starting from 1,6-dibromohexa-2,4-diene. The synthetic route consists of only 7 steps in total, where each step is performed in aqueous media, and no protecting groups are used. One of the key steps in the synthesis involves a combination of cyanide substitution and intramolecular Pinner reaction in one pot. Studies dealing with reaction process monitoring, tests of catalysts, and condition optimization are reported. A "no purification" process for the sequence of the first four steps of the synthesis is described. The possibility of achieving the synthetic goal is discussed. / Une synthèse totale verte du cardiobutanolide est proposée à partir de 1,6-dibromohexa-2,4-diène. La voie de synthèse se compose de seulement 7 étapes au total, où chaque étape est réalisée en milieu aqueux, et aucun groupe protecteur n'est utilisé. L'une des étapes clés dans la synthèse implique une combinaison de la substitution de cyanure et de la réaction intramoléculaire Pinner dans un pot. Le suivi de la réaction, les tests de catalyseurs et l'optimisation des conditions sont décrits. Un processus "sans purification" pour la séquence des quatre premières étapes de la synthèse est décrit. La possibilité d'atteindre l'objectif de synthèse est discutée.

Aminoglycosides are broad-spectrum antibiotics that target the A-site of bacterial 16S ribosomal RNA. Their use, however, is increasingly threatened by the rapid spread of resistance. One of the most common determinants of aminoglycoside resistance in bacteria is the expression of a class of enzymes known as aminoglycoside 6′-N-acetyltransferases (AAC(6′)s). These enzymes use acetylcoenzyme A (AcCoA) to acetylate most aminoglycosides at the 6′-NH2, thus decreasing their affinity for RNA and leading to bacterial resistance. One strategy pursued by the Auclair research group to overcome aminoglycoside resistance is to develop AAC(6′) inhibitors. Chapter 2 of this thesis describes enzymatic studies with the group's first generation of inhibitors, aminoglycoside-CoA bisubstrates and truncated analogs. The bisubstrates exhibited potent nanomolar competitive inhibition of the Enterococcus faecium isoform AAC(6′)-Ii and proved to be useful mechanistic and structural probes. They did not however show activity in cells. Enzymatic studies with truncated bisubstrates allowed extensive SAR studies and led to the discovery of a second generation of inhibitors, one of which is capable of blocking aminoglycoside resistance in cells expressing AAC(6′)-Ii. To improve the potency of this compound, a series of derivatives with various amide groups replacing the ester functionality were synthesized. It was hypothesized that this modification would increase potency and biological stability. To our surprise however these small changes had a negative impact on the interaction between the inhibitors and AAC(6′)-Ii. Chapter 3 describes our second approach to counter aminoglycoside resistance, which involved the synthesis of new aminoglycosides designed to be active against resistant bacterial strains. A series of neamine N-6′-acylated derivatives were designed to retain the hydrogen bonding ability of 6′-N to RNA, yet disrupt binding to AAC(6′)-Ii. Some of these compounds showed moderate but not clinically relevant activity in cells.Finally, the third approach elaborated in Chapter 4 is to design prodrugs with dual, resistance inhibition and antibacterial activities. The goal was to develop aminoglycoside N-6′ derivatives as prodrugs that were expected to be extended to bisubstrate analogs by the CoA biosynthetic enzymes in bacteria. The resulting bisubstrates were expected to not only block aminoglycoside resistance via AAC(6′) inhibition, but to also kill bacteria by blocking the fatty acid biosynthetic pathway. A small series of aminoglycoside derivatives was thus synthesized. Unexpectedly none of them showed direct antibacterial activity. Preliminary biological studies however suggest that even in the absence of in vitro AAC(6′) inhibition, these molecules can potentiate the activity of aminoglycosides against an aminoglycoside resistant strain. To our knowledge they are the first reported prodrugs able to block aminoglycoside resistance in cells. / Les aminoglycosides sont des antibiotiques à large spectre qui ciblent le site A de l'ARN ribosomique 16S dans la bactérie. Leur usage, par contre, est de plus en plus menacé à cause de l'étendue rapide de la résistance contre les aminoglycosides. La classe d'enzyme aminoglycoside-6′-N-acétyltransférases (AAC(6′)) est l'un des détermiants de résistance le plus commun. Ces enzymes utilisent acétylcoenzyme A (AcCoA) pour acétyler plusieurs aminoglycosides à la position 6′-NH2, ce qui diminue leur affinité pour l'ARN et la résistance en résulte. Une des stratégies privilégiée par notre groupe de recherche pour combattre la résistance aux aminoglycosides est décrite au Chapître 2. Elle consiste à développer des inhibiteurs contre AAC(6′). La première génération d'inhibiteurs, des bisubstrats de type aminoglycoside-CoA, a démontré une inhibition compétitive nanomolaire in vitro. Quoiqu'ils fussent utiles comme éléments de recherche structurale et mechanistique, ils ne démontrent aucune activité biologique. L'étude subséquente d'analogues de bisubstrats raccourcis a permis d'établir des relations structure-activité et a mené à la découverte d'une seconde génération d'inhibiteurs, incluant une molécule avant la capacité de bloquer la résistance aux aminoglycosides dans les cellules bactériennes exprimant l'AAC(6′)-Ii. Pour améliorer l'efficacité de ce composé, nous avons synthétisé une troisième génération d'inhibiteurs dont l'ester a été remplacé par divers groupements amide. L'hypothèse était que l'amide serait plus stable, menant à des composés plus stables et plus efficace, biologiquement parlant. À notre surprise, ces modifications ont diminué de façon significative l'interaction entre les inhibiteurs et AAC(6′)-Ii. Le troisième chapître de cette thèse explore une autre stratégie pour contrer la résistance envers les aminoglycosides. Elle vise à créer des agents antibactériens actifs contre les bactéries résistantes. Une série de dérivés de néamine acylés à la position 6′-N ont été envisagés afin de retenir l'habileté de l'amine 6′ à faire un lien hydrogène avec l'ARN tout en perturbant l'adhérance envers l'AAC(6′). Certains composés ont démontré des propriétés antibactériennes modérées dans les cellules mais sans potentiel clinique. Enfin, notre troisième approche vise à créer des pro-médicaments possédant à la fois la capacité de contrer la résistance ainsi que des propriétés antibactériennes. Notre but était de développer des dérivés d'aminoglycoside qui seraient transformés en analogues de bisubstrats par les enzymes bactériennes impliquées dans la biosynthèse de CoA. Les bisubstrats résultant pourraient non seulement bloquer la résistance en inhibitant l'enzyme AAC(6′), mais aussi avoir des propriétés antibactériennes en affectant la biosynthèse des acides gras. Une série de ces dérivés d'aminoglycosides a été synthétisée et aucun produit ne montre d'activité antibacterienne. Par contre, des résultats préliminaires suggèrent que même si ces molecules n'inhibitent pas l'enzyme AAC(6') purifiée, elles possèdent l'habileté d'augmenter l'action des aminoglycosides dans les bactéries résistantes. Selon nos connaissances, il s'agit des premiers pro-médicaments capablent de bloquer la resistance aux aminoglycosides dans les cellules.

Enzymes are increasingly applied to organic synthesis because of their high enantioselectivity, chemoselectivity, and environmental friendliness. Hydrolytic enzymes are the largest class used for biotransformations, and subtilisin is one of the commonly used serine proteases for organic synthesis. We hypothesized that we could expand the usefulness of subtilisin to hydrolyze unnatural substrates. The X-ray crystal structure of subtilisin shows that it has a large open active site. Previous workers focused on hydrolysis of natural substrates and close analogs, but the open active site suggests that subtilisin should accept bulky substrates that do not resemble peptides. / The Diels-Alder reaction is important in organic synthesis because it has the potential to create several stereocenters in a single step. We resolved a bulky secondary alcohol ester of a spiro chiral auxiliary for the Diels-Alder reaction on a gram scale using subtilisin Carlsberg. The reaction proceeded with high enantioselectivity and yield. Cholesterol esterase showed high enantioselectivity but with the opposite enantiopreference, consistent with mirror image arrangement for the active sites of subtilisins and lipases/esterases. We also used molecular modeling to identify the molecular basis of enantioselectivity of subtilisin Carlsberg toward this secondary alcohol. / Resolution of tertiary alcohol esters is difficult because they are also very bulky, but important because there are only a few synthetic and biocatalytic methods to prepare enantiopure tertiary alcohols. We discovered several proteases that hydrolyze esters of tertiary alcohols, one of which was subtilisin Carlsberg. This is the first examination of protease hydrolysis of tertiary alcohol esters. Substrate studies and molecular modeling explained their reactivity and enantioselectivity. / Sulfinamidies are also bulky substrates, but surprisingly subtilisin catalyzed a catalytic promiscuous reaction of N-acyl sulfinamides. Subtilisin Carlsberg-catalyzed hydrolysis of N-acyl sulfinamides favored cleavage of the sulfinamide (S(O)-N) bond with a minor amount of the expected carboxamide (C(O)-N) bond. The sulfinamide hydrolysis was enantioselective and confirmed by product isolation from the S-N sulfinamide cleavage. In contrast, the related subtilisins BPN' and E favored the C-N carboxamide hydrolysis. / Further examination using electrospray-mass spectrometry revealed a sulfinyl-enzyme intermediate located at the active site, analogous to an acyl-enzyme. This suggested an analogous mechanism to the amide/ester hydrolysis. Substrate variation indicated a substrate reversal was responsible for the change in reactivity by binding in the S1 acyl pocket. Three mutations of subtilisin BPN' towards subtilisin Carlsberg increased the S-N hydrolysis by 14-fold, indicating that only a few mutations were responsible for the catalytic promiscuity. The substrate specificity and mutagenesis were consistent with a reversed orientation for the sulfinyl hydrolysis reactions. / The active site of subtilisin is indeed versatile for unnatural substrates and unnatural reactions. We expanded the applications of subtilisin to resolve bulky substrates, such as chiral auxiliaries and tertiary alcohol esters, and also to perform a new catalytic promiscuous sulfinamide S-N bond hydrolysis. Based on these results, subtilisin should prove useful for future resolutions of bulky substrates, and also in catalytic promiscuous reactions where unusual reactive centers are hydrolyzed.

A model study for the synthesis of the fungal metabolites phomoidride A and B is presented. Building on previous studies from our group, the carbocyclic core of these molecules was constructed using a [6+4] cycloaddition between tropone 41 and the silyl enol ether of 2-cyclopenten-1-one. Efforts to install the C-17 phomoidride sidechain on substrates derived from this carbocycle met with limited success. This led us to revise our strategy by employing the novel 5-substituted cyclopentadiene 118. In contrast to the known instability of 5 substituted cyclopentadienes towards [1,5] hydrogen shifts, the 2-silyloxy moiety of 118 imparted remarkable thermal stability on the diene. A practical method was developed for the multigram-scale synthesis of cyclopentenone 132, a useful synthon and precursor to 118. Diene 118 underwent smooth [6+4] cycloaddition with tropone 41 at 23°C to give 133 in 51-54% yield.* / Attempts to install the C-17 stereocenter of the phomoidrides through hydroboration of alkene 120 were thwarted by low yields. Remarkably, significant amounts of Markovnikoff products (9-23%) were isolated after the hydroboration of 120 and related alkenes with BH3·DMS. We undertook a series of DFT studies (B3LYP16-31G*) to understand the origins of reduced anti-Markovnikoff selectivity. These studies suggested that the bridging ketones and remote diene both contributed to this phenomenon. These studies led us to synthesize carbocycle 158. In line with predictions, hydroboration of saturated diketone 158 was completely selective for the anti-Markovnikoff product.* / The C-17 sidechain of the phomoidrides was successfully extended through a conjugate addition reaction. Compound 189, which contains the correct stereochemistry of the C-9 and C-17 sidechains, represents our furthest progress to date on the molecules.* / *Please refer to dissertation for diagrams.

A new series of vitamin D analogs were synthesized with a saturated seco B-ring and several potential aromatic A-ring mimics. These new A-ring/seco B-ring analogs examine the possibility of forming positive electrostatic interactions in the seco B-ring portion of the vitamin D nuclear receptor, the effect of increasing interactions between the ligand and Tryptophan 286 as well as the ability of aromatic rings at miming the natural A-ring. A central core based on the natural C,D-ring was developed, which allows divergent analogs synthesis for fast screening of side chain and A-ring mimics. / Une nouvelle série d'analogues de la vitamine D a été synthétisée avec un cycle B secondaire saturé, ainsi qu'avec divers composés aromatiques pouvant potentiellement imiter le cycle A naturel. Cette série d'analogues évalue la possibilité de faire des interactions positive dans la région du cycle B secondaire du récepteur nucléaire de la Vitamine D, l'effet d'amplifier l'interaction entre le ligand et Tryptophane 286 ainsi que la possibilitée d'imiter le cycle A naturel avec des cycles aromatique. Un composé basé sur les cycles C et D de la vitamine D a été développé, ce composé permet l'obtention rapide d'analogues visant à évaluer des imitations de la chaine latérale et du cycle A naturel.

AbstractRuthenium-Catalyzed C-C Bond Formation via Functional-Group Directed C-H Bond ActivationXiangyu GuoAdvisor: Prof. Chao-Jun LiMcGill UniversityThis thesis is an investigation on the formation of carbon-carbon (C-C) bonds in the presence of ruthenium catalyst.In the first part of this thesis, oxidative dehydrogenative coupling reactions for carbon-carbon (C-C) bond formation are described. A ruthenium-catalyzed dimerization of 2-phenylpyridine derivatives is demonstrated to synthesize biaryls using iron(III) chloride as the terminal oxidant. In addition, the oxidative cross coupling of arenes and cycloalkanes is also illustrated, achieving a unique para-selectivity.In the second part of the thesis, a ruthenium-catalyzed olefination via decarbonylative addition of aldehydes to terminal alkynes is described. Conjugated and isolated C=C bonds can be chemoselectively generated in two catalytic systems starting from aromatic and aliphatic aldehydes. The method provides an alternative synthesis of C=C bonds from direct C-H bond addition to triple bonds. / RésuméRuthenium-Catalyzed C-C Bond Formation via Functional-Group Directed C-H Bond ActivationXiangyu GuoSuperviseur: Prof. Chao-Jun LiUniversité McGillCette thèse est le résultat de la recherche sur la formation de liaisons carbone-carbone (C-C), catalysé par le ruthénium. La première partie de cette thèse expose les résultats sur la formation de liaison carbone-carbone (C-C) par la réaction de couplage oxydant par déshydrogénation. La synthèse de composés biaryl par l'utilisation d'un catalyseur de ruthénium a permis la dimérisation des dérivés de la 2-phénylpyridine en présence de chlorure de fer (III) comme oxydant terminal. En outre, l'oxydative cross-coupling entre arènes et cycloalcanes, a montrer une notable, para-sélectivité. La seconde partie de cette thèse, décrit les résultats obtenue sur la réaction d'oléfination decarbonylative entre un aldéhyde et un alcyne vrai, catalyser par le ruthénium. En partant d'aldéhydes aromatiques ou aliphatiques et par l'utilisation de deux systèmes catalytiques, la synthèse chemioselective de double liaison C=C conjuguée ou isolée ont pu être réalisé. Cette réaction fournit ainsi, une intéressante alternative à la synthèse de doubles liaisons C=C par la directe addition de liaison C-H sur une triple liaison.

Cytochrome P450 enzymes (CYPs or P450s) form a ubiquitous family of heme-dependent monooxygenases known mainly for their role in xenobiotic metabolism and their remarkable ability to regio- and stereoselectively oxidize inactivated C-H bonds, a feat that is difficult to achieve by chemical methods. Unfortunately, our ability to study and exploit these enzymes as in vitro biocatalysts has been limited by their low activity, low stability and poor product predictability. This thesis focuses on the study of human drug metabolizing P450 isoforms, namely CYP2E1, CYP3A4 and CYP2D6 because of their exceptional ability to accept a large variety of substrates. In Chapter 2, we demonstrate the utility of "type II ligands" as chemical auxiliaries for biocatalysis with human CYP2E1. We show that linking the chemical auxiliary nicotinate to a variety of short hydrocarbon substrates can promote their oxidation with predictable regioselectivity at the secondary aliphatic or alkenyl C-H bond furthest from the auxiliary. The origin of this selectivity was rationalized through docking studies of our auxiliary-substrate compounds with reported X-ray crystals structures of CYP2E1. These results not only confirm the general utility of the chemical auxiliary approach pioneered by our lab to direct the predictable oxidation of inactivated C-H bonds by P450 enzymes, but also provide a system with complementary regioselectivity.A short study of the effects of macromolecular crowding on the activity of human CYP3A4 and CYP2D6 is presented in Chapter 3. We found that certain crowding agents were not detrimental to enzyme activity while others had a negative effect. Moreover, certain conditions (initially tested as controls) that improved enzymatic activity were uncovered. In Chapter 4, the non-covalent oriented-immobilization of CYP3A4 via its C-terminal histidine-tag is described. We show that immobilization on Ni-NTA agarose resin via this strategy has no detrimental effect on enzyme activity or stability. The lyoprotectant properties of Ni-NTA were also investigated. In Chapter 5, we designed and characterized a mutant of CYP3A4 that retains its enzymatic activity upon modification with a variety of fluorescent maleimide dyes via a single cysteine residue on its surface, namely C64. We also show that the activity of this mutant is preserved upon immobilization onto solid supports via this same cysteine residue. Finally, results of a preliminary feasibility study towards applying this immobilization strategy to eventual single-molecule fluorescence microscopy studies are presented. / Les enzymes cytochrome P450 (CYPs ou P450) forment une famille omniprésente de mono-oxygénases possédant un noyau hème au site-actif. Ces enzymes sont surtout connues pour leur rôle dans le métabolisme de produits pharmaceutiques et pour leur capacité remarquable à oxyder les liens C-H non-activés de façon régio- et stéréosélective. Malheureusement, notre capacité d'étudier et d'utiliser ces enzymes comme biocatalyseurs in vitro est limitée par leur faible activité, instabilité et une incapacité des connaissances actuelles à prédire leurs produits.Dans le chapitre 2, nous démontrons l'utilité des ligands de type II comme auxiliaires chimiques pour la biocatalyse avec CYP2E1 humaine. Nous démontrons que l'auxiliaire chimique nicotinate, lorsque lié à une variété d'hydrocarbures courts, peux promouvoir leur oxydation avec une régiosélectivité prévisible pour le lien C-H secondaire aliphatique ou alcényle le plus éloigné de l'auxiliaire. L'origine de cette sélectivité a été rationalisée à l'aide de «docking» moléculaire de nos composés auxiliaire-substrats à l'intérieure de structures cristallines de CYP2E1 publiées par d'autres chercheurs. L'utilité d'auxiliaires chimiques pour contrôler la régiosélectivité des enzymes P450 avait déjà été démontrée par notre groupe de recherche. Les résultats présentés dans ce chapitre offrent non seulement une confirmation du potentiel de cette stratégie, mais aussi un système complémentaire pour l'oxydation prévisible de liens C-H non-activés par les enzymes P450. Ces résultats confirment également la généralité de l'approche mis au point dans notre laboratoire qui décrit l'utilisation d'une auxiliaire chimique pour diriger l'oxydation prévisible de liens C-H non-activés par les enzymes P450.Une étude des effets de l'encombrement macromoléculaire sur l'activité enzymatique des CYP3A4 et CYP2D6 humaines est présentée dans le chapitre 3. Nous avons trouvé que leur activité demeure inchangée par la présence de certains agents encombrants alors que d'autres ont un effet négatif. De plus, certaines conditions (testées initialement comme contrôle) qui améliorent l'activité enzymatique ont été découvertes.Dans le chapitre 4, l'immobilisation orienté non-covalente de CYP3A4 par son étiquette de type his-tag C-terminale est décrite. Nous démontrons que son immobilisation sur une résine Ni-NTA à base d'agarose via cette stratégie n'a aucun effet négatif sur l'activité ou la stabilité de l'enzyme. Les propriétés lyoprotectrices de cette résine ont aussi été investiguées.Dans le chapitre 5, nous concevons et caractérisons un mutant actif de CYP3A4 lors de modifications avec une variété de maléimides fluorescentes à l'endroit d'un unique résidu cystéinique à sa surface, soit le C64. Nous démontrons aussi que ce mutant préserve son activité lorsqu'immobilisé sur des supports solides par ce même résidu cystéinique. Finalement, les résultats d'études préliminaires sont présentés qui envisagent l'application de cette stratégie d'immobilisation envers des études éventuelles de spectroscopie de fluorescence à la résolution d'une seule molécule.