The biosynthesis of three natural products, L-2-amino-4-pentynoic acid (propargylglycine) (1), 3-(3-isocyanocyclopent-2-enylidene) propionic acid (2), and (+)-(alpha)-(S)-amino-(2,5-dihydro-5(S)-methyl)-furan-2(R)-acetic acid (3) (furanomycin), produced by an unidentified Streptomyces, Trichoderma hamatum, and Streptomyces threomyceticus respectively, have been investigated. Although two prospective labeled precursors of the amino acid (1), {1-('14)C}-norvaline and {1-('14)C}-allylglycine were synthesized, and procedures for the isolation, detection and degradation of (1) devised, the elucidation of the biogenesis of (1) was hampered due to lack of production of (1) by the microorganism. The isonitrile acid (2) has been shown by using ('14)C- and ('3)H-labeled precursor incorporation experiments to be derived from the amino acid tyrosine which undergoes an oxidative cleavage of the aromatic ring followed by recyclization. In addition to specific incorporation studies with {('14)C}- and {('3)H}-labeled precursors, experiments using {('2)H}, {('18)O}, and {('13)C}-labeled precursors in conjunction with ('2)H-NMR and ('13)C-NMR have been undertaken to investigate the biosynthetic origin of the amino acid (3). The evidence indicates that (3) arises from one unit of propionic acid and two units of acetic acid.

C. roseus is the source of the important anticancer indole alkaloids, vincristine and vinblastine. Hairy root cultures are an attractive alternative for the production of plant derived secondary metabolites. C. roseus hairy root cultures were established in our laboratory by A. rhizogenes mediated transformation of C. roseus seedlings prior to the beginning of this project. The objectives of this project were twofold: (i) Alkaloid analysis of C. roseus hairy root cultures, (ii) Scaleup of hairy root cultures from shake flasks to bioreactors. An extraction method was adapted for the rapid quantification of C. roseus hairy root extracts, and was used for quantifying alkaloid yields from five different root clones. Optimum analytical wavelengths were chosen for quantification after analyzing chromatograms at five different wavelengths using PDA detection. Extracts were analyzed using different analytical techniques--GC/MS, MS, HPLC-PDA and TLC--and several alkaloids were identified. The presence of ajmalicine, akuammine, apparicine, catharanthine, hoerhammericine, lochnericine, serpentine and tabersonine was confirmed. The presence of vindoline--an important precursor of vinblastine and vincristine--could not be confirmed although GC/MS indicated the presence of vindoline fragment ions. A novel method based on non-linear regression analysis was developed for online measurement of oxygen uptake rates. Thus, mass transfer rates could be adjusted in bioreactor cultures to provide sufficient oxygen. Three different bioreactor configurations--two recirculation reactors (60 mL, 1L) and a 500 mL spinner flask--were designed for scaleup studies. Growth, nutrient utilization and alkaloid productivities were measured. This is the first study where several biochemical parameters have been measured in multiple bioreactor configurations and found to be comparable--without exception--to shake flask cultures. Cultures were cultivated in different gas phase environments--'air' and '2.5% CO$\sb2$'--in each bioreactor configuration. This is the first study of the effect of CO$\sb2$ on hairy roots cultivated in liquid culture. A beneficial effect ($>$98% confidence) of CO$\sb2$ was observed on biomass accumulation. Possible metabolic importance of CO$\sb2$ on heterotrophic plant cultures is discussed.

Enantiomerically-pure compounds are becoming increasingly important particularly to the pharmaceutical industry. Enzymes are useful tools in the synthesis of such compounds. To better understand how lipases discriminate between enantiomers of chiral secondary alcohols we obtained x-ray crystal structures of covalent complexes of Candida rugosa lipase with the transition-state analogs (1R)-menthyl hexylphosphonate and (1S)-menthyl hexylphosphonate. These compounds are transition-state analogs for the hydrolysis of menthyl esters. We observed that the transition-state analog of the unfavored (1S)-enantiomer of menthol disrupted the hydrogen bond between N$ varepsilon$2 of histidine 449 and the menthol oxygen. His 449 is part of the catalytic triad in CRL. This may account for the slower reaction of the (1S)-enantiomer of menthol. The crystal structures also identified binding site regions for secondary alcohols in CRL. These regions are common among many lipases and esterases and account for their common enantiopreference toward secondary alcohols. / Lipases and esterases can resolve compounds with phosphorus and sulfur stereocenters by hydrolysis of a pendant acetoxy group. Both CRL and cholesterol esterase have high selectivity for (2-acetoxy-1-naphthyl)methylphenylphosphine oxide. They resolved this substrate with and E of 81 and 32 respectively. A synthetic scale resolution of this substrate with subsequent recrystallization and chemical transformation followed by stereospecific reduction gave both enantiomers of (2-methoxy-1-naphthyl)methylphenyl-phosphine with 96-97% ee. This chiral phosphine is potentially useful in asymmetric syntheses. / CE is the most selective enzyme for the sulfur substrates tested but these enantioselectivities were moderate, E's ranged from 5 to 25. From the CE resolution of the phosphorus and sulfur compounds and others we propose an empirical model that predicts which enatiomer reacts faster. The model is based on the size of the substituents and their conformational preferences. / Crude Aspergillus niger resolves esters of pipecolic acid with an E of 20 $ pm$ 4. A simple partial purification of ANL by fractional precipitation with ammonium sulfate increased the enantioselectivity to $>$100. The partially purified ANL can be used in a synthetic scale resolution of ($ pm$)-n-octyl pipecolate to give (S)-($-$)-pipecolic acid (93% ee) and (R)-(+)-pipecolic acid (97% ee).

The retention behaviour of 16 phenothiazines and structurally-related drugs on a $ beta$-cyclodextrin-bonded phase column was studied with respect to pH, mobile phase composition and column temperature. Both isocratic and gradient-elution separations of these compounds were investigated. / The enantiomers of twelve racemic dinitrophenyl amino acid derivatives were separated on a $ beta$-cyclodextrin-bonded phase column. The effects of pH, methanol and triethylammonium acetate (TEAA) buffer concentrations on the retention and resolution were investigated. The chiral recognition mechanism was studied by means of UV-visible, circular dichroism and proton nuclear magnetic resonance spectroscopic methods. / A multiple-interaction type of chiral stationary phase was developed by bonding $ beta$-cyclodextrin to silica gel and modifying the cyclodextrin cavity by flexibly capping its primary hydroxyl or small side. These modified $ beta$ cyclodextrin stationary phases contain a hydrophobic cavity, capable of inclusion complexation; aromatic groups, capable of $ pi$-$ pi$ interaction; and polar hydrogen-bonding sites, capable of forming hydrogen-bonding with the polar functional groups of the solutes. These stationary phases exhibit a high stereoselectivity toward a wide variety of chiral compounds. The preparation and properties of these modified $ beta$-cyclodextrin stationary phases are described. The enantiomeric separation of amino acids and their derivatives, of carboxylic acids, of phenothiazine drugs, and of other chiral compounds are reported. The effects of mobile phase composition on the retention and resolution are discussed.

Breast cancer remains one of the most persistent threats to women's health in the Western world. The selective estrogen receptor modulator (SERM) tamoxifen is a front-line treatment for the disease, but suffers from a high rate of acquired resistance and an increased risk of endometrial cancer. As such, improved small molecule inhibitors with the ability to overcome antiestrogen resistance while limiting adverse side effects are valuable pharmaceutical targets. Histone deacetylase inhibitors (HDACi) have recently emerged as versatile anticancer agents with antiproliferative activity in breast cancer cells. Combination therapy of SERMs and HDACi has demonstrated enhanced cytotoxicity and the ability to restore tamoxifen sensitivity to antiestrogen-resistant cancer cell lines. This research project describes a novel approach to overcoming antiestrogen resistance by combining the anticancer properties and cooperative activity of SERMs and HDACi into compact, merged bifunctional ligands. Previous research by the Gleason laboratory indicated that HDACi functionality, in the form of zinc-chelating hydroxamic acids, could be integrated into the polar side chain of SERMs while maintaining antiestrogenicity at micromolar concentrations. Building from these preliminary results, a series of novel hybrid molecules was designed with zinc-binding groups incorporated into the aliphatic side chain or the triphenylethylene core structure of the active tamoxifen metabolite 4-hydroxytamoxifen (4-OHT). The resulting structures were investigated in silico using the molecular docking program FITTED. Twelve hybrid compounds were then synthesized by geminal Suzuki coupling and McMurry coupling procedures. Despite the propensity of the compounds to isomerize around the central double bond, the majority of the syntheses proceeded diastereoselectively and HPLC purification furnished the final products as a single isomer or in good diastereomeric excess. The HDAC inhibition and ER antagonism of the bifunctional molecules were evaluated in collaboration with the Mader laboratory of the Université de Montréal. Side chain-substituted hybrid compounds demonstrated good affinity for the ER binding pocket and nanomolar ER inhibitory activity. In addition, long-chain chimeric ligands possessing amide linkers exhibited significant HDAC 6 inhibition, with one compound displaying inhibitory activity at sub-micromolar concentrations. However, these side-chain substituted hybrids also exhibited substantial ER agonism in the absence of estradiol, which limited their effectiveness as antiestrogens. Conversely, a compound containing a phenyl hydroxamic acid in the aromatic core of 4-OHT acted as a full antagonist at micromolar concentrations and displayed no estrogenic behaviour. The core-substituted hybrid also exhibited modest HDAC inhibition, though it lacked the strong activity of its side chain counterparts. The synthesized SERM/HDACi molecules therefore displayed encouraging results and provided important structural insight into the future creation of bifunctional ligands.A brief side project is also described as a contribution towards the total synthesis of (R)-puraquinonic acid. The enantioselective synthesis of this natural product was previously accomplished by the Gleason group using a novel lactam auxiliary alkylation sequence to set the molecule's sole stereocenter. However, the specific rotation of the final product was opposite that expected and contradicted previous stereochemical assignments of the alkylation sequence. To address this inconsistency, a convergent stereochemical proof was completed which allowed direct comparison of alkylation products to auxiliary-based Mannich addition products that could be characterized by X-ray diffraction. The absolute stereochemistry of the sequence was thereby unambiguously assigned and strongly supported the conclusion that our group had successfully synthesized (R)-puraquinonic acid. / Le modulateur sélectif des récepteurs des œstrogènes (SERM) tamoxifène est le traitement principal pour le cancer du sein, mais souffre d'un taux élevé de résistance acquise et augmente le risque de developer un cancer de l'endomètre. Par conséquence, de nouvelles molécules antiestrogénique avec la capacité de surmonter la résistance aux antioestrogènes et de limiter les effets secondaires indésirables sont nécessaires. Les inhibiteurs d'histones déacétylase (HDACi) ont récemment emergés comme des agents possédant une activité antiproliférative contre les cellules du sein cancéreuse. La thérapie combinatoire des SERMs et HDACi démontre non seulement une cytotoxicité améliorée, mais aussi la capacité de restaurer la sensibilité envers tamoxifène dans les lignées cellulaires de cancer résistantes aux antioestrogènes. Ce projet décrit une nouvelle stratégie pour combattre la résistance antioestrogène en combinant l'activité des SERMs et des HDACi par la fusion des pharmacophores pour produire des ligands bifonctionnels.Les recherches précédentes par le laboratoire du Dr. Gleason ont indiqué que la fonctionnalité HDACi - sous la forme d'acides hydroxamiques - peut être intégrée dans la chaîne polaire des SERMs tout en conservant une antiestrogenicité modérée. Basé sur ces résultats préliminaires, une série de nouvelles molécules hybrides a été conçue avec des groupes se liant au zinc incorporés soit dans la chaîne latérale ou dans la structure centrale triphényléthylène du métabolite actif de tamoxifène, 4-hydroxytamoxifène (4 -OHT). Ces molécules ont été étudiés in silico à l'aide de FITTED, un programme de modélisation moléculaire. Douze molécules hybrides ont été synthétisées par des reactions de Suzuki et des réactions de McMurry. La majorité des synthèses ont pu être accomplies d'une manière diastéréosélective et la purification par CLHP a fourni les produits finaux sous forme d'isomère pure ou dans de bons excès diastéréoisomèriques.Les molecules bifonctionnelles ont été évaluées pour l'inhibition des HDACs et l'antagonisme envers le recepteur d'oestrogens (ER) en collaboration avec le laboratoire du Dr. Mader à l'Université de Montréal. Les hybrides possédant une acide hydroxamique sur la chaîne laterale ont démontré une bonne affinité pour ER de l'ordre nanomolaire. De plus, les ligands chimériques à longue chaîne possédant un amide ont démontré une inhibition importante envers HDAC 6 à une concentration micromolaire. Cependant, ces hybrides ont également démontré une activité agoniste substantielle de ER, qui limité leur efficacité comme antioestrogènes. Par contre, un hybride contenant un acide hydroxamique dans la structure centrale aromatique de 4-OHT a agi comme une antagoniste à des concentrations micromolaires, sans comportement oestrogénique. Les molécules ciblant SERM/HDACi offrent donc des résultats initiaux encourageants et des leçons importantes pour la création de nouveaux ligands bifonctionnels.Un bref projet contribuant à la synthèse totale de l'acide (R)-puraquinonique est aussi décrit. La synthèse énantiosélective de ce produit naturel a été accompli par le groupe Gleason avec l'aide d'une séquence d'alkylation utilisant un auxiliaire chirale bicyclique pour établir l'unique stéréocentre de la molécule. Toutefois, le pouvoir rotatoire spécifique du produit final était l'inverse de ce qui était attendu, et en contradiction avec la stéréochimie précédente de la séquence d'alkylation. Pour prouver la stéréochimie de notre processus d'alkylation, une voie de synthèse convergente a été accomplie qui a permis une comparaison directe des produits d'alkylation avec des produits d'addition de Mannich dont on était capable de caractériser par diffraction aux rayons X. La stéréochimie absolue de la séquence a été ainsi assignée sans ambiguïté et procure une confirmation que notre groupe avait réussi à synthétiser l'acide (R)-puraquinonique.

The mevalonate pathway is important in all eukaryotic cells. One of the key enzymes in this pathway is the human farnesyl pyrophosphate synthase (hFPPS), which has a pivotal role in cell prenylation. Nitrogen containing bisphosphonates (N-BPs) are a class of drugs that have been shown to effectively inhibit hFPPS and improve the survival of patients with various cancers, including multiple myeloma. However, the current drugs on the market (Risedronate and Zoledronate) interact with the active site of hFPPS sub-optimally and suffer from poor oral bioavailability and soft tissue exposure. A new series of inhibitors have been designed that can potentially interact with both the GPP and IPP sub-pockets of hFPPS. Based on a computational model, substituted benzimidazole- and indole-based inhibitors were designed to bind in both sub-pockets of the active site cavity of hFPPS. The benzimidazole-based inhibitors were found to be chemically unstable due to a reversible Michael reaction. Thus all efforts were subsequently turned towards the synthesis of indole-based inhibitors. These compounds were chemically stable when the bisphosphonate moiety was placed at C-3. The compounds were tested by DSF and in an inhibition enzymatic assay. The 2-substituted indoles were found to be active and provide a new structural class of hFPPS inhibitors. / La voie du mévalonate est importante pour toutes les cellules de type eucaryote. La farnésyle pyrophosphate synthase humaine (FPPSh) est une des enzymes clés pour cette voie qui possède un rôle pivot dans la prénylation cellulaire. Les molécules azotées contenant des biphosphonates (N- BPS) appartiennent à une classe de médicaments pouvant inhiber efficacement FPPSh et améliorer le taux de survie de patients atteints de plusieurs types de cancers, incluant notamment le myélome. Cependant, les médicaments en circulation sur le marché pharmaceutique (Risedronate et Zoledronate) interagissent inefficacement avec le site actif de FPPSh et offrent à la fois une mauvaise biodisponibilité orale et une exposition des tissus mous. Une nouvelle série d'inhibiteurs a donc été proposée pouvant potentiellement interagir avec les sous cavités GPP et IPP de FPPSh. Les inhibiteurs dont la structure est basée sur le motif benzimidazole substituée en position 2 et indole présentent ce critère et ont donc été désignés pour ce projet. Bien que les inhibiteurs possédant le motif benzimidazole aient été synthétisés, il a malheureusement été impossible d'isoler les produits de réactions du fait de la réversibilité de la réaction de Michael. Nos efforts se sont alors tournés vers la synthèse d'inhibiteurs possédant le motif indole. Ces composés sont bien plus stables lorsque le groupement biphosphonate est placé au C-3. Ces molécules ont été évaluées par DSF et des essais enzymatiques. A notre plus grand plaisir, les indoles substituées en positions 2 se sont révélées être des molécules meneuses pour une nouvelle classe d'inhibiteurs de FPPSh.

In the field of molecular discovery, virtually screening large libraries of compounds proved to be often more cost-efficient than the traditional experimental approaches. In fact, it has now become common practice thanks to the virtual screening tools available to chemists in the pharmaceutical industry, specifically docking. Most docking programs do not account for the dynamics associated with protein-ligand binding whether it is protein flexibility or the inclusion of displaceable water molecules. FITTED1.0 was developed to include these specific two features and has been validated on a testing set of 33 protein-ligand complexes. Further developments were needed to increase the speed of FITTED to enable its application as virtual screening tool. This enhanced version, FITTED1.5, has been applied to the screening of the Maybridge library onto the HCV polymerase and revealed FITTED’S ability to identify active substances. With this and other successful applications of FITTED, a comparative study was performed against other docking programs, with a specific interest in the effect of the ligand and protein input conformation and the inclusion of bridging water molecules on the accuracy of docking programs. All three had major effects on accuracy and led to suggestions on how to better conduct comparative studies. In parallel, we applied our expertise in the virtual screening area to the field of asymmetric catalyst development and led to the creation of ACE1.0. When creating a tool for predicting steroselectivities, one has to describe the transition state with great accuracy although within a reasonable amount of time. To tackle this problem, ACE creates the transition states from linear combinations of reactant and product interactions. A genetic algorithm is then exploited as a conformational search engine to optimize the TS structure. ACE has been applied to the Diels-Alder cycloaddition and the proline-catalysed aldol reactions and has showed good correlatio / Dans le domaine pharmaceutique, le criblage virtuel de large bibliothèques de molécules est une alternative moins couteuse et souvent au moins aussi efficace que le criblage à haut débit. D’ailleurs, le développement de tels outils –et plus particulierement de méthodes de "docking"– a permis au criblage virtuelle de devenir pratique courante dans l’industrie pharmaceutique. Cependant, la plupart des méthodes de docking ne prennent pas en compte la dynamique des complexes protéine/ligand et plus spécifiquement la flexibilité des protéines et la présence de molécules d’eau nécessaires à une liaison optimale. Dans cette optique, FITTED1.0 a été développé et validé sur un jeu de 33 complexes protéine/ligand. Ainsi, FITTED1.0 permet de modéliser des complexes ternaires protéine/ligand/eau entièrement flexibles. D’autres développements ont ensuite été nécessaires pour en accroître la rapidité et permettre son utilisation pour le criblage de larges bibliothèques. Cette version améliorée, FITTED1.5, a été appliquée au criblage de la bibliothèque Maybridge sur la polymérase du virus de l’hépatite C et a permis la découverte de deux nouveaux inhibiteurs. Après ces résultats très encourageants, une étude comparative a été entreprise visant spécifiquement à évaluer l’impact des données d’entrées sur le pouvoir prédictif des programmes de docking les plus couramment utilisés incluant FITTED2.6. Nous avons alors démontré que la présence d’eau, la conformation du ligand et de la protéine au départ du calcul ont un impact majeur. En parallèle, nous avons bénéficié de notre expertise pour développer un second outil de criblage virtuel ACE1.0 mais cette fois appliqué au criblage de catalyseurs asymétriques. Dans le domaine de la catalyse asymétrique, il nous fallait prédire la structure et l’énergie potentielle des états de transition et ce, dans un temps raisonnable. Pour ce faire, ACE cr

Thesis (Ph.D.)--Lehigh University, 2007.

Thesis (Ph. D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 68-01, Section: B, page: 0304. Advisers: Irwin Kuntz; Thomas James.

Thesis (Ph. D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6642. Adviser: Rodney Kiplin Guy.