The Diels-Alder reaction is one of the most powerful reactions available to synthetic chemists. Novel catalysts for this reaction are constantly being evaluated, thus creating a need for high-throughput assays. In this thesis, three strategies were tested in the design of novel fluorescence-based assays to monitor Diels-Alder reactions. The first strategy relied on intermolecular FRET pairing using fluorescence donors as dienes and fluorescence acceptors as dienophile (or vice versa). The second strategy utilized the dienophile naphthalene fumarate ethyl ester which contains a quenched fluorescent moiety. This dienophile was expected to regain its fluorescence upon Diels-Alder reaction with a diene. The third strategy involved a masked 7-hydroxycoumarin fluorophore linked to a dienophile. The fluorophore was positioned so that after Diels-Alder reaction it could be easily eliminated. The third strategy was the most promising even though several problems had to be overcome.

A new, general method for the asymmetric synthesis of quaternary carbons is described. This technique involves stereocontrolled generation of alpha,alpha-disubstituted amide enolates via reduction of bicyclic alpha,alpha-disubstituted alpha-thioglycolate lactams using one-electron transfer reagents. A model of the geometric constraints necessary to reductively prepare alpha,alpha-disubstituted amide enolates with control of enolate E/Z stereochemistry is described and several chiral auxiliaries were prepared to test this design. / Successful preparation, trapping, isolation and characterization of alpha,alpha-disubstituted amide enolates is described. This reductive enolization method allows, for the first time, the stereoselective preparation of both E- and Z-acyclic alpha,alpha-disubstituted amide enolates. / Reactivity and selectivity of alpha,alpha-disubstituted amide enolates in alkylation with alkyl halides is explored. Dichotomous reactivity is observed for both E- and Z-alpha,alpha-disubstituted amide enolates. Z-alpha,alpha-Disubstituted amide enolates are shown to alkylate with high levels of stereoselectivity; E-alpha,alpha-disubstituted amide enolates alkylate with poor stereoselectivity. / Methods for isolation of the quaternary carbon-containing adducts are described. Conditions for the determination of the enantiomeric excess of chiral, non-racemic neopentyl alcohols were developed and are also described. / Development of a facile, environmentally friendly, modular synthesis of a model second-generation chiral auxiliary using simple, inexpensive starting materials is described.

This thesis is separated into two parts. In part A, a novel 5-alkyl-2-silyloxy cyclopentadiene is presented. Racemic and enantioselective syntheses of the new cyclopentadiene have been developed. This new cyclopentadiene is significantly more stable towards 1,5-H shifts than most cyclopentadienes. Through the use of computational modeling, it was determined that the added stability comes from the electron donation of the 2-silyloxy group into the LUMO of the diene. The novel diene was used for Diels-Alder cycloadditions at room temperature with a variety of dienophiles. An europium Lewis acid was found to be compatible with the diene while activating dienophiles for cycloaddition. It was also determined that an enantioenriched diene will generate a cycloaddition product with no erosion in enantiomeric excess. Stabilization by a silyloxy group was also used to generate a stable 1-alkyl-3-silyloxy cyclopentadiene that underwent smooth Diels-Alder reaction. The 5-alkyl-2-silyloxy cyclopentadiene was used as a cycloaddition partner in the synthesis of part of the natural product palau'amine. Through a Diels-Alder/oxidative cleavage sequence, the E-ring stereoarray of the originally proposed structure of Palau'amine was obtained. / In part B, the synthesis and purification of a novel multi-action drug, triciferol, are presented. Triciferol was designed to combine a vitamin D3 framework and a metal binding group with histone deacetylase inhibition properties. The secosteroidal core of triciferol is obtained from degradation of vitamin D2. The vitamin D A-ring is obtained from (-)-quinic acid and is appended to the core by a Horner reaction. The unsaturated side chain is built by sequential Wittig reactions and terminated by a metal-binding hydroxamic acid. This conjugated hydroxamic acid group was found to be susceptible to exposition to trace metals. The final purification of triciferol could only be accomplished using reverse phase silica chromatography. Triciferol was found to be an effective bifunctional agent, acting both as an agonist of the vitamin D receptor and an inhibitor of histone deacetylase. Moreover, it proved to be a potent anti-cancer drug in vitro. / Cette thèse comprend deux parties. Dans la partie A, un nouveau 5-alkyl-2-silyloxy cyclopentadiène est présenté. Deux synthèses de ce nouveau cyclopentadiène ont été développées : l'une racémique et l'autre énantiosélective. Le nouveau cyclopentadiène est significativement plus stable vis-à-vis de la migration 1,5 d'hydrogène que la plupart des cyclopentadiènes connus. À l'aide de la modélisation par ordinateur, la stabilité du nouveau cyclopentadiène a été attribuée à une donation électronique du groupe 2-silyloxy au LUMO du diène. Le nouveau cyclopentadiène a été utilisé lors de cycloadditions Diels-Alder à température ambiante avec une sélection de diénophiles. Un acide de Lewis à base d'europium, permettant d'activer les diénophiles sans décomposer le diène, a été trouvé. Il fut aussi déterminé qu'une version énantioenrichie du cyclopentadiène génère un produit de cycloaddition sans aucune détérioration de l'excès énantiomérique initial. La stabilisation par un groupe silyloxy a aussi été appliquée avec succès à un cyclopentadiène 1-alkyle-3-silyloxy, lui permettant d'encourir un Diels-Alder sans problème. Le 5-alkyle-2-silyloxy cyclopentadiene a été utilisé lors d'une cycloaddition menant à la synthèse d'une partie de la palau'amine, un produit naturel. La stéréochimie de l'anneau E de la structure originale de la palau'amine a été atteinte grâce à une séquence de Diels-Alder / clivage oxydatif. / Dans la partie B, la synthèse et la purification d'un nouveau médicament bi-fonctionnel, le triciferol, sont présentées. Le triciferol a été conçu afin de combiner la structure de la vitamine D3 à un groupe chélateur de métaux, ayant la capacité d'inhiber les HDAC. Le centre sécostéroïde du triciferol est obtenu par dégradation de la vitamine D2. L'anneau A est synthétisé à partir d'acide quinique et est attaché au centre sécostéroïde grâce à une réaction de Horner. La chaine insaturée est construite par réactions de Wittig et est terminée par un acide hydroxamique chélateur. Cet acide hydroxamique insaturé se décompose facilement en présence de métaux ; la purification finale du triciférol doit être effectuée par une chromatographie en phase inverse. Le triciférol a les capacité d'un médicament bi-fonctionnel : c'est un agoniste du récepteur de la vitamine D et un inhibiteur des enzymes HDAC. fr

Calcitriol (1alpha,25-(OH)2 D3, 1,25D) is known for its calcium regulatory function, but also for being a powerful inhibitor of cell growth in a variety of normal and neoplastic cells. As such, it is a lead structure in the development of new cancer drugs. The goal of our project is to identify analogues of calcitriol which bind tightly to the vitamin D receptor (VDR) yet do not exhibit unwanted hypercalcemic effects. The development of novel analogues using conventional chemistry and dynamic chemistry was investigated. First, the numerous building blocks required for the exploration of the different libraries were created through the use of conventional techniques. We then developed different synthetic schemes based primarily on thiol alkylation and amide couplings, enabling rapid parallel synthesis of potential 1,25D analogues. Preliminary investigations allowed us to implement with our collaborators a set of biological assays to evaluate our compounds. Although some analogues appeared to bind to the VDR, it was found that none of the 8 analogues tested in the preliminary studies seemed to be vitamin D receptor (VDR) agonists. In a second part, our research focused on the study of dynamic combinatorial chemistry as a tool for the synthesis of 1,25D analogues. Our first dynamically-generated libraries were created via thioester exchange. It was found that the rate of thioester exchange was dependent on the nature of the thiols involved and the acyl portion of the thioesters. Aliphatic acid thioesters exchanged more slowly than aromatic acid thioesters. The use of branched (alpha-substituted) thiols also slowed the rate compared to primary ones. However, the use of large concentration of aliphatic or aromatic thiols could accelerate the thioester exchange. When thioesters were placed in presence of protein targets in a dynamic system, their inherent electrophilic nature rendered them prone to chemical decomposition (hydrolysis and acylation of protein nucleophilic residues) at basic pHs. Nonetheless, we created thioester libraries of up to 40 members from as few as 7 building blocks under near neutral conditions. We showed that these dynamic libraries ere influenced by the presence of proteins. We have not been able to confirm if thioester libraries were influenced exclusively by the VDR via its binding site. Some of our observations challenged the viability of the thioester exchange as a reversible process in the context of VD3 analogue synthesis. Finally, we created dithiol building blocks, amenable to disulfide exchange in dynamic libraries. Chemically stable, these disulfide entities easily generated libraries of at least 30 members from as few as 7 building blocks. Our preliminary disulfide libraries seemed uninfluenced by VDR's presence. These results still allowed us to understand better the characteristics and limitations of dynamic systems applied to the development of novel vitamin D analogues. / Calcitriol (1alpha,25-dihydroxyvitamine D3, 1,25D) est connu pour sa fonction dans la régulation du métabolisme du calcium, mais aussi comme étant un puissant inhibiteur de la prolifération cellulaire dans un éventail de cellules normales et néoplastiques. Comme tel, il représente le point de départ pour le développement de nouvelles molécules anticancéreuses. Le but de notre projet est d'identifier des analogues du calcitriol capables de se lier au récepteur de la vitamine D (RVD) sans causer d'hypercalcémie. Le développement de nouveaux analogues de la vitamine D3 a été exploré en utilisant la chimie conventionelle et la chimie dynamique. Dans un premier temps, de nombreux synthons nécessaires à la création des différentes librairies ont été fabriqués par des techniques de synthèse traditionnelles. Ensuite, des routes de synthèse basées sur l'alkylation de thiols et la formation d'amides ont été élaborées, permettant une synthèse en parallèle efficace d'analogues de la vitamine D. En collaboration avec nos partenaires, des études préliminaires ont permis d'établir la validité d'une série de tests pour évaluer l'activité biologique de nos analogues sur le récepteur de la vitamine D. Bien que plusieurs composés semblent se lier au récepteur, aucun des 8 analogues évalués de façon préliminaire n'ont révélé une activité agoniste du RVD. Dans un deuxième temps, nous avons axé notre recherche sur l'étude de la chimie combinatoire dynamique (CCD) comme outil pour la création et l'évaluation d'analogues de la vitamine D. La CCD est basée sur le fait qu'une protéine peut influencer la composition de bibliothèques dynamiques en favorisant la création de ses propres ligands, et ce, proportionnellement à leur constante d'affinité. Les premières bibliothèques dynamiques furent créées à partir d'échanges entre thioesters. Il a été évalué qualitativement que la vitesse de ce processus est dépendante de la nature des thiols impliqués ainsi que de la nature de la portion acyl des thioesters. L'échange des thioesters d'acides aliphatiques est plus lent que celui des thioesters d'acides aromatiques. Aussi, plus les thiols sont encombrés stériquement, plus le processus est lent. Toutefois, l'utilisation de grandes concentrations de thiols aromatiques et aliphatiques peut accélérer le processus d'échange. Lorsque des thioesters sont utilisés en présence de protéines à pH basique, leur caractère électrophilique les rend propices à la dégradation chimique par hydrolyse ou par acylation des fonctionnalités nucléophiles des protéines. Néanmoins, à pH neutre, des bibliothèques de thioesters possédant plus de 40 composantes furent facilement réalisées à partir de seulement 7 synthons. Il a pu être démontré que la composition de ces bibliothèques est influencée par la présence de protéines. Cependant, nous n'avons pas été en mesure de confirmer si le RVD a véritablement influencé la nature des bibliothèques de thioesters grâce à son site de liaison. Certaines de nos observations mettent en doute l'utilisation de la transthioestérification comme processus dynamique viable pour la création d'analogues de la vitamine D en présence de protéines.Finalement, des synthons dithiols furent créés et leur combinaison a permis la formation de bibliothèques dynamiques de plus de 30 composés, grâce à l'échange de disulfures. Les études préliminaires ont montré que les bibliothèques de disulfures ne semblent pas être influencées par la présence du RDV. Ces résultats ont permis de connaître les caractéristiques et les limites de ces systèmes dynamiques appliqués au développement de nouveaux analogues de la vitamine D.

Despite significant progress in the field of organic chemistry, the asymmetric formation of all-carbon quaternary centers still represents a considerable challenge. Our group previously reported the stereocontrolled generation and alkylation of alpha,alpha-disubstituted amide enolates to form quaternary centers. This reaction takes place via the reduction of bicyclic alpha,alpha-dialkylated alpha-thioglycolate lactams using one-electron transfer reagents. / This method suffered from several limitations to practicality, thus this research project has focused on the development of a second-generation chiral auxiliary system which would overcome the first generation auxiliary shortcomings. / The design and synthesis of a new chiral auxiliary system is described. The design is modular and allows the auxiliary to be synthesized in a few shorts steps from commercially available materials. Alkylation of the new auxiliary, reductive enolization and the subsequent final alkylation to form quaternary centers is achieved with high levels of stereocontrol for both enolate intermediates. Two sets of conditions are described to cleave the auxiliary and liberate the products containing the quaternary centers. / The methodology that has been developed is versatile and represents a practical solution to the enantioselective formation of quaternary carbon centers. It also overcomes all the shortcomings of the first generation.

The X-ray crystal structure of the protease subtilisin shows its active site is on the surface. It binds substrate in an extended conformation and reacts only with soluble substrates. Subtilisin has a large nonpolar pocket (the S1 pocket) to bind acyl group and a shallow crevice (the S 1' pocket) to bind one substituent of a secondary alcohol group, while the other substituent remains in solvent. The reactivity and enantioselectivity of subtilisin toward nonpolar secondary alcohol esters is low. Based on its structure, we hypothesized that subtilisin would be highly reactive if the acyl group anchored the substrate to the active site and highly enantioselective if there was a large hydrophobicity difference between alcohol substituents. / To test our first hypothesis, we show that an anchoring acyl group increases protease reactivity and extends subtilisin E to a new class of substrates, N-acyl sulfinamides. Subtilisin E did not catalyze hydrolysis of N-acetyl arylsulfinamides, but did catalyze a highly enantioselective hydrolysis of N-dihydrocinnamoyl arylsulfinamides. The N-dihydrocinnamoyl group mimics phenylalanine and thus binds the sulfinamide to the active site. We then use the 3-(3-pyridyl)propionyl group as anchoring group to increase substrate solubility. We use this group to resolve multi-gram quantities of three important compounds and isolate the enantiomers without the use of chromatography. / Next, we use different anchoring acyl groups to extend several proteases to tertiary alcohol esters. We show that a syn-pentane-like interaction destabilizes the transition state for reaction of tertiary alcohols, but that the addition of an anchor group that binds substrate to the protease stabilizes transition state and enables proteases to catalyze hydrolysis of tertiary alcohol esters. / To test our second hypothesis, we show subtilisin enantioselectivity stems from a favourable hydrophobic interaction between nonpolar substituent and S1' pocket residues and favourable solvation of polar substituent in water. The enantioselectivity of a series of secondary alcohols in water varied linearly with the difference in hydrophobicity (logP/P0) of the substituents. The larger the logP/P0 difference the higher the enantioselectivity. Consistent with our hypothesis, the enantioselectivity of subtilisin toward N-acyl arylsulfinamides is high because of the large difference in substituent hydrophobicity.

There are some examples where conformational restriction has been introduced into nucleotides with the sole purpose of increasing the thermodynamic stability of the duplex or triplex between the modified oligonucleotide and its complement. This thesis explored the hypothesis of incorporating a cyclopropane moiety into the internucleotide linkage. This research demonstrates the successful synthesis of a novel cyclopropanated sulfonamide dinucleotide 19.(DIAGRAM, TABLE OR GRAPHIC OMITTED...PLEASE SEE MAI) / This novel molecule has been synthesized by reacting an $ alpha, beta$ unsaturated phenylselenoyl nucleotide derivative 7 with an active methylene sulfonamide nucleotide 17 under Michael addition conditions. Binding studies concerning the cyclopropanated sulfonamide dinucleotide 19 are pending.

In order to make use of enzymes in unnatural applications more efficiently, novel enzymes have to be developed and they may arise from an existing enzyme with similar performance. The fast way to improve the performance of the enzyme is by directed evolution. It alters a naturally existing protein, without in depth knowledge of the starting enzyme and produces mutants containing the required activities. / The lipase from Bacillus thermocatenulatus is an alko-thermophilic enzyme. Optimization of the lipase production is carried out for use with the new screening method using a pH indicator to evaluate hydrolytic activities. This newly cloned lipase is next characterized with two substrate libraries made up of commercially available general and enantiomerically pure esters. An important chiral synthon solketal is identified as the directed evolution target molecule for this lipase. By modifying with error-prone PCR, the enantioselectivity of the lipase towards solketal n-octanoate is improved.

The introduction of enantioselectivity into zinc catalyzed homoaldol reactions is described. Coordinating ligands such as bis-oxazolines and beta-amino alcohols inhibit the addition of zinc homoenolates to aldehydes while Lewis acids such as titanium (IV) isopropoxide and boron trifluoroetherate promote this reaction. Notably, the addition of zinc homoenolates to benzaldehyde mediated by chiral titanium (IV) alkoxides yields homoaldol adducts with moderate enantioselectivity. / The development of titanium catalysis of the homoaldol addition of silyloxyalkoxycyclopropanes to aldehydes is also described. The methodology features alkoxytitanium (IV) triflates as catalysts, which mediate homoenolate addition to a range of carbonyl substrates. The preparation of such titanium triflates by a new procedure, namely the treatment of titanium (IV) alkoxides with trimethylsilyltriflate, affords more reactive catalysts than those prepared by conventional methods. In addition, the catalyst system is stable at elevated temperatures for an extended period of time. / Titanium (IV) triflates derived from a variety of alkoxide ligands as well as ligands with sulfide linkages are effective catalysts for this reaction. Efforts directed towards extending this catalytic process to a highly enantioselective protocol are detailed. Moderate enantioselectivity is obtained in homoaldol reactions mediated by a tridentate alkoxide derived titanium tiflate.

Pseudo-dynamic combinatorial chemistry (pDCC) combines the synthesis, screening and destruction of combinatorial libraries to kinetically resolve inhibitors based on their affinity for a target. In our proof-of-principle studies, a library of dipeptides was formed in the presence of a target, carbonic anhydrase (CA), and a destruction mechanism, a protease. Since the target and the protease were separated by a dialysis membrane, only the proportion of dipeptides that was not bound to the target was available for destruction and therefore, the rate of hydrolysis of the pseudo-dynamic combinatorial library (pDCL) could be correlated to the librarie's relative affinity for the target. The first set of proof-of-principle pDCC experiments were found to be flawed. Rather than reflecting the binding affinity of the library for the target, the final product distribution reflected the protease's substrate specificity. The main problems with these pDCLs were insufficient and imbalanced rate of destruction of the peptides in the absence of the target, pH drift, and insufficient permeability across 1000 MWCO cellulose ester membranes. A dipeptide amide based pDCL that was efficiently cleaved by thermolysin was designed. The new pDCL could be used in pH 7.5 75 mM HEPES, 16.6 mM CaCl2 buffer, provided that the chambers were limited by 3500 MWCO membranes. A 4th generation pDCC experiment that evolved reflecting the library's affinity for the target was performed. In order to study pDCC behavior a simplified pDCC mimic (pDCCm) was designed. In pDCCm, synthesis was replaced by a static library of compounds and destruction by dilution. The design of a pDCCm based kinetic model led to a / La chimie combinatoire pseudo dynamique (pDCC) combine la synthèse, le criblage et la destruction de libraires combinatoires afin de sélectionner cinétiquement des inhibiteurs sur la base de leur affinité pour une cible. Nos études préliminaires ont été basées sur une librairie de dipeptides formés en présence de l'anhydrase carbonique (CA) comme cible et détruits grâce à une protéase. Étant donné que la cible et la protéase étaient séparées par une membrane de dialyse, seuls les dipeptides non complexés avec la cible étaient susceptibles d'être détruits. Ainsi, la vitesse d'hydrolyse des membres de la librairie combinatoire pseudo-dynamique (pDCL) peut correspondre aux affinités relatives des molécules pour la cible. Les premières expériences prototypes de pDCC étaient défectueuses. Plutôt que de refléter l'affinité des membres de la librairie pour la cible, la distribution finale des produits reflétait la préférence de la protéase pour les substrats. Les principaux problèmes de ces pDCL étaient: une vitesse de destruction des peptides insuffisante et inégale en l'absence de la cible, une déviation progressive du pH et une perméabilité insuffisance au travers les membranes d'ester de cellulose 1000 MWCO. Une pDCL basée sur des amides dipeptidiques, aptes à être efficacement clivés par thermolysine a été élaborée, et utilisée avec succès. Afin d'étudier les pDCC, une expérience modèle simplifiée (pDCCm) a été créée. Dans ce modèle, la synthèse a été remplacée par une librairie statique de molécules et la destruction par une dilution. Le design d'un modèle cinétique basé sur la pDCCm a mené à la cr