Monooxygenase Diversity and Oxygen Activation within Polyketide Antibiotic Biosynthesis

Molecular oxygen (O2) is a key component in cellular respiration and aerobic life. Through the redox potential of O2, the amount of free energy available to organisms that utilize it is greatly increased. Yet, due to the nature of the O2 electron configuration, it is non-reactive to most organic molecules in the ground state. For O2 to react with most organic compounds it must be activated. By activating O2, oxygenases can catalyze reactions involving oxygen incorporation into organic compounds. The oxygen activation mechanisms employed by many oxygenases to have been studied, and they often include transition metals and selected organic compounds. Despite the diversity of mechanisms for O2 activation explored in this thesis, all of the monooxygenases studied in the experimental part activate O2 through a transient carbanion intermediate. One of these enzymes is the small cofactorless monooxygenase SnoaB. Cofactorless monooxygenases are unusual oxygenases that require neither transition metals nor cofactors to activate oxygen. Based on our biochemical characterization and the crystal structure of this enzyme, the mechanism most likely employed by SnoaB relies on a carbanion intermediate to activate oxygen, which is consistent with the proposed substrate-assisted mechanism for this family of enzymes. From the studies conducted on the two-component system AlnT and AlnH, both the functions of the NADH-dependent flavin reductase, AlnH, and the reduced flavin dependent monooxygenase, AlnT, were confirmed. The unusual regiochemistry proposed for AlnT was also confirmed on the basis of the structure of a reaction product. The mechanism of AlnT, as with other flavin-dependent monooxygenases, is likely to involve a caged radical pair consisting of a superoxide anion and a neutral flavin radical formed from an initial carbanion intermediate. In the studies concerning the engineering of the S-adenosyl-L-methionine (SAM) dependent 4-O-methylase DnrK and the homologous atypical 10-hydroxylase RdmB, our data suggest that an initial decarboxylation of the substrate is catalyzed by both of these enzymes, which results in the generation of a carbanion intermediate. This intermediate is not essential for the 4-O-methylation reaction, but it is important for the 10-hydroxylation reaction, since it enables substrate-assisted activation of molecular oxygen involving a single electron transfer to O2 from a carbanion intermediate. The only role for SAM in the hydroxylation reaction is likely to be stabilization of the carbanion through the positive charge of the cofactor. Based on the DnrK variant crystal structure and the characterizations of several DnrK variants, the insertion of a single amino acid in DnrK (S297) is sufficient for gaining a hydroxylation function, which is likely caused by carbanion stabilization through active site solvent restriction. Despite large differences in the three-dimensional structures of the oxygenases and the potential for multiple oxygen activation mechanisms, all the enzymes in my studies rely on carbanion intermediates to activate oxygen from either flavins or their substrates. This thesis provides interesting examples of divergent evolution and the prevalence of carbanion intermediates within polyketide biosynthesis. This mechanism appears to be recurrent in aromatic polyketide biosynthesis and may reflect the acidic nature of these compounds, propensity towards hydrogen bonding and their ability to delocalize π-electrons.

Comparative electron impact and fast atom bombardment mass spectrometric studies of some HMPA adducts of phenyltin and phenyllead halides and studies of strong hydrogen bonding by FAB-MS

The fragmentation patterns and mass spectra of some phenyl tin and -lead halide adducts with hexamethylphosphoramide are compared by subjecting them t~ electron impact and fast atom bombardment ionization in a mass spectrometer. This comparison is restricted to the metal-containing ions. Ligand-exchange mechanisms of some of the metal-containing species are explored by FAB-MS. Several moisturesensitive organo-metallics and H-bonded systems have been examined by FAB for attempted characterization, but without any success. Scavenging and trapping of water molecules by complex aggregates in solutions of quaternary ammonium fluorides and hydroxides are investigated by FAB to complement previous NMR-studies.

Strong hydrogen bonding in organic synthesis

The preparation of phenacyl and para-phenylphenacyl esters, the reactions of carboxylic acids, phenols, 2-nitropropane and alcohols with alkyl halides in the presence of fluoride anion are described. The reactions are thought to be accelerated by the formation of hydrogen bonds between the fluoride anion and the organic electron acceptor. The fluoride ,carboxylic acids, fluoride-phenols and fluoride-2-nitropropane are better reaction systems than the fluoride-alcohol. The source of the fluoride anion and the choice of solvents are also discussed.

The importance of hydrogen bonding in the alkylation of phenols

Hydrogen bond assisted alkylation of phenols is compared with the classical base assisted reactions. The influence of solvents on the fluoride assisted reactions is discussed,· with emphasis on the localization of hydrogen bond charge density. Polar aprotic solvents such as DMF favour a-alkylation, and nonpolar aprotic solvents such as toluene favourC-alkylation of phenol. For more reactive and soluble fluorides, such as tetrabu~ylammoniumfluoride, the polar aprotic solvent favours a-alkylation and nonpolar aprotic solvent favours fluorination. Freeze-dried potassium fluoride is a better catalytic agent in hydrogen bond assisted alkylation reactions of phenol than the oven-dried fluoride. The presence of water in the alkylation reactions reduces the expected yield drastically. The tolerance of the reaction to water has also been studied. The use ofa phase transfer catalyst such as tetrabutylammonium bromide in the alkylation reactions of phenol in the presence of potassium fluoride is very effective under anhydrous conditions. Sterically hindered phenols such as 2,6-ditertiarybutyl-4-methyl phenol could not be alkylated even by using the more reactive fluorides, such as tetrabutylammonium fluoride in either polar or nonpolar aprotic solvents. Attempts were also made to alkylate phenols in the presence of triphenylphosphine oxide.

Mirroring Enzymes: The Role of H-Bonding In HQuin-BAM Catalyzed Asymmetric Aza-Henry Reactions: A DFT Study into the Reactivity, Mechanism, and Origins of Selectivity

The exact mechanistic understanding of various organocatalytic systems in asymmetric reactions such as Henry and aza-Henry transformations is important for developing and designing new synthetic organocatalysts. The focus of this dissertation will be on the use of density functional theory (DFT) for studying the asymmetric aza-Henry reaction. The first part of the thesis is a detailed mechanistic investigation of a poorly understood chiral bis(amidine) (BAM) Brønsted acid catalyzed aza-Henry reaction between nitromethane and N-Boc phenylaldimine. The catalyst, in addition to acting as a Brønsted base, serves to simultaneously activate both the electrophile and the nucleophile through dual H-bonding during C-C bond formation and is thus essential for both reaction rate and selectivity. Analysis of the H-bonding interactions revealed that there was a strong preference for the formation of a homonuclear positive charge-assisted H-bond, which in turn governed the relative orientation of substrate binding. Attracted by this well-defined mechanistic investigation, the other important aspect of my PhD research addressed a detailed theoretical analysis accounting for the observed selectivity in diastereoselective versions of this reaction. A detailed inspection of the stereodetermining C-C bond forming transition states for monoalkylated nitronate addition to a range of electronically different aldimines, revealed that the origins of stereoselectivity were controlled by a delicate balance of different factors such as steric, orbital interactions, and the extent of distortion in the catalyst and substrates. The structural analysis of different substituted transition states established an interesting dependency on matching the shape and size of the catalyst (host molecule) and substrates (guest molecules) upon binding, both being key factors governing selectivity, in essence, offering an analogy to positive cooperative binding effect of catalytic enzymes and substrates in Nature. In addition, both intra-molecular (intra-host) and inter-molecular (host-guest, guest-guest) stabilizing interactions play a key role to the high π-facial selectivity. The application of dispersion-corrected functionals (i.e., ωB97X-D and B3LYP-D3) was essential for accurately modeling these stabilizing interactions, indicating the importance of dispersion effects in enantioselectivity. As a brief prelude to more extensive future studies, the influence of a triflate counterion on both reactivity and selectivity in this reaction was also addressed.

Configuration et exploitation d'une machine émotionnelle

Dans ce travail, nous explorons la faisabilité de doter les machines de la capacité de prédire, dans un contexte d'interaction homme-machine (IHM), l'émotion d'un utilisateur, ainsi que son intensité, de manière instantanée pour une grande variété de situations. Plus spécifiquement, une application a été développée, appelée machine émotionnelle, capable de «comprendre» la signification d'une situation en se basant sur le modèle théorique d'évaluation de l'émotion Ortony, Clore et Collins (OCC). Cette machine est apte, également, à prédire les réactions émotionnelles des utilisateurs, en combinant des versions améliorées des k plus proches voisins et des réseaux de neurones. Une procédure empirique a été réalisée pour l'acquisition des données. Ces dernières ont fourni une connaissance consistante aux algorithmes d'apprentissage choisis et ont permis de tester la performance de la machine. Les résultats obtenus montrent que la machine émotionnelle proposée est capable de produire de bonnes prédictions. Une telle réalisation pourrait encourager son utilisation future dans des domaines exploitant la reconnaissance automatique de l'émotion.

La configuration spatiale et la hiérarchie urbaine en Colombie

Ce mémoire présente les résultats d'une recherche portant sur cinq villes colombiennes sélectionnées selon leur taille et le nombre d’emplois par secteur économique, d’après les données des recensements du Département Administratif National des Statistiques (DANE) qui s’étalent entre 1985 et 2005. La méthode d'analyse adoptée est de type descriptif et met l'accent sur des facteurs de taille et de nombre d’habitants afin d’évaluer la hiérarchie entre les villes entre 1985 et 2005, et de comprendre, pour la même période, la spécialisation des cinq villes à travers leurs données d’emplois respectives. Cette méthode est complétée par l’utilisation des outils d’évaluation régionale et urbaine pour comprendre le processus de polarisation en Colombie. L’économie colombienne est dominée par les villes de grande taille ou les régions métropolitaines qui constituent les plus grands marchés (économies d'agglomération). Cependant, ces zones (Bogota, Medellin, Cali, Barranquilla et Bucaramanga) ne sont pas intégrées. Par conséquent, la prédominance de ces centres s’est considérablement réduite.

Indirect Bonding Method: in vitro Comparison of the Shear Bond Strength between Metallic Orthodontic Brackets and Different Porcelain Surface Preparations

Introduction : La force d’adhésion à l'interface métal-céramique avec les résines auto-polymérisantes destinées au collage indirect des boîtiers orthodontiques n'a pas été évaluée à ce jour et un protocole clinique basé sur la littérature scientifique est inexistant. Objectifs : 1) Comparer la force de cisaillement maximale entre des boîtiers métalliques et des surfaces en porcelaine préparées selon différentes méthodes; 2) Suggérer un protocole clinique efficace et prévisible. Matériel et méthodes : Quatre-vingt-dix disques en leucite (6 groupes; n = 15/groupe) ont été préparés selon 6 combinaisons de traitements de surface : mécaniques (+ / - fraisage pour créer les rugosités) et chimiques (acide fluorhydrique, apprêt, silane). Des bases en résine composite Transbond XT (3M Unitek, Monrovia, California) faites sur mesure ont été collées avec le système de résine adhésive auto-polymérisante Sondhi A + B Rapid Set (3M Unitek, Monrovia, California). Les échantillons ont été préservés (H2O/24hrs), thermocyclés (500 cycles) et testés en cisaillement (Instron, Norwood, Massachusetts). Des mesures d’Index d’adhésif résiduel (IAR) ont été compilées. Des tests ANOVAs ont été réalisés sur les rangs étant donné que les données suivaient une distribution anormale et ont été ajustés selon Tukey. Un Kruskall-Wallis, U-Mann Whitney par comparaison pairée et une analyse de Weibull ont aussi été réalisés. Résultats : Les médianes des groupes varient entre 17.0 MPa (- fraisage + acide fluorhydrique) à 26.7 MPa (- fraisage + acide fluorhydrique + silane). Le fraisage en surface ne semble pas affecter l’adhésion. La combinaison chimique (- fraisage + silane + apprêt) a démontré des forces de cisaillement significativement plus élevées que le traitement avec (- fraisage + acide fluorhydrique), p<0,05, tout en possédant des forces similaires au protocole typiquement suggéré à l’acide fluorhydrique suivi d’une application de silane, l’équivalence de (- fraisage + acide fluorhydrique + silane). Les mesures d’IAR sont significativement plus basses dans le groupe (- fraisage + acide fluorhydrique) en comparaison avec celles des 5 autres groupes, avec p<0,05. Malheureusement, ces 5 groupes ont des taux de fracture élévés de 80 à 100% suite à la décimentation des boîtiers. Conclusion : Toutes les combinaisons de traitement de surface testées offrent une force d’adhésion cliniquement suffisante pour accomplir les mouvements dentaires en orthodontie. Une application de silane suivie d’un apprêt est forte intéressante, car elle est simple à appliquer cliniquement tout en permettant une excellente adhésion. Il faut cependant avertir les patients qu’il y a un risque de fracture des restorations en céramique lorsque vient le moment d’enlever les broches. Si la priorité est de diminuer le risque d’endommager la porcelaine, un mordançage seul à l’acide hydrofluorique sera suffisant.

Effect of curing temperature , fibre loading and bonding agent on the equilibrium swelling of isora-natural rubbercomposites

Isora fibre-reinforced natural rubber (NR) composites were cured at 80, 100, 120 and 150°C using a low temperature curing accelerator system. Composites were also prepared using a conventional accelerator system and cured at 150°C. The swelling behavior of these composites at varying fibre loadings was studied in toluene and hexane. Results show that the uptake of solvent and volume fraction of rubber due to swelling was lower for the low temperature cured vulcanizates which is an indication of the better fibre/rubber adhesion. The uptake of aromatic solvent was higher than that of aliphatic solvent, for all the composites. As the fibre content increased, the solvent uptake decreased, due to the superior solvent resistance of the fibre and good fibre-rubber interactions. The bonding agent improved the swelling resistance of the composites due to the strong interfacial adhesion. Due to the improved adhesion between the fibre and rubber, the ratio of the change in volume fraction of rubber due to swelling to the volume fraction of rubber in the dry sample (V,) was found to decrease in the presence of bonding agent. At a fixed fibre loading, the alkali treated fibre composite showed a lower percentage swelling than untreated one for both systems showing superior rubber-fibre interactions.

Mechanical Properties of Short-Isora-Fiber-Reinforced Natural Rubber Composites: Effects of Fiber Length, Orientation, and Loading; Alkali Treatment;and Bonding Agent

A series of short-isora-fiber-reinforced natural rubber composites were prepared by the incorporation of fibers of different lengths (6, 10, and 14 mm) at 15 phr loading and at different concentrations (10, 20, 30, and 40 phr) with a 10 mm fiber length. Mixes were also prepared with 10 mm long fibers treated with a 5% NaOH solution. The vulcanization parameters, processability, and stress-strain properties of these composites were analyzed. Properties such as tensile strength, tear strength, and tensile modulus were found to be at maximum for composites containing longitudinally oriented fibers 10 mm in length. Mixes containing fiber loadings of 30 phr with bonding agent (resorcinol-formaldehyde [RF] resin) showed mechanical properties superior to all other composites. Scanning electron microscopy (SEM) studies were carried out to investigate the fiber surface morphology, fiber pullout, and fiber-rubber interface. SEM studies showed that the bonding between the fiber and rubber was improved with treated fibers and with the use of bonding agent.

The Use of Carboxy Terminated Liquid Natural Rubber (CTNR) as an Adhesive in Bonding Rubber to Rigid and Non-rigid Substrates

Carboxy Terminated Liquid Natural Rubber (CTNR) was prepared by photochemical reaction using maleic anhydride and masticated natural rubber (NR). The use of CTNR as an adhesive in bonding rubber to rubber and rubber to metal was studied. The peel strengths and lap shear strengths of the adherends which were bonded using CTNR were determined. The effect of using a tri isocyanate with CTNR in rubber to metal bonding was also studied. It is found that CTNR can effectively be used in bonding rubber to rubber and rubber to mild steel.

Studies on Rubber-to-Nylon Tire Cord Bonding

ABSTRACT: Nylon tire cord (1680/2) was dipped in different adhesives based on resorcinol formaldehyde resin and latex (RFL) and was bonded to natural rubber-based compounds. The resin-rubber ratio in the RFL adhesive was optimized. The variation of pull-through load was studied by varying the drying and curing temperature of the dipped nylon tire cord. RFL adhesive based on vinylpyridine latex was found to have better rubber-to-nylon tire cord bonding, compared with the one based on natural rubber latex. Addition of a formaldehyde donor into the RFL adhesive/rubber compound improves adhesion.

Rubber Solution Adhesives for Wood-to-Wood Bonding

Rubber solutions were prepared and used for bonding wood pieces. The effect of the variation of chlorinated natural rubber (CNR) and phenolformaldehyde (PF) resin in the adhesive solutions on lap shear strength was determined. Natural rubber and neoprene-based adhesive solutions were compared for their lap shear strength. The storage stability of the adhesive prepared was determined. The change in lap shear strength before and after being placed in cold water, hot water, acid, and alkali was tested. The bonding character of these adhesives was compared with different commercially available solution adhesives. The room-temperature aging resistance of wood joints was also determined. In all the studies, the adhesive prepared in the laboratory was found to be superior compared to the commercial adhesives.