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CHAPTER ONE reviews the literature, discussing aspects of transition metal mediated asymmetric catalysis in the presence of enantiomerically pure ligands. CHAPTER TWO discusses the asymmetric addition of dialkyl-zinc reagents to aromatic aldehydes. The work presented is particularly concerned with the design and construction of enantiomerically pure oxazoline ligands tethered to alcohols These ligands have proved effective in the acceleration of the alkylation reaction and are able to influence good levels of asymmetric induction in the resultant secondary alcohol products CHAPTER THREE examines the electronic (and steric) effects of enantiomerically pure oxazoline ligands for the palladium catalysed allylic substitution reaction. Using ligands possessing two electronically different donor atoms, it is possible to create electronic distortion upon the intermediate allyl complex. In doing so it is possible to direct nucleophilic addition to one carbon centre preferentially to the other, resulting in asymmetric induction. Manipulation of these ligands enables control in the extent of electron distortion inflicted upon the allyl complex and consequently influences the levels of enantioselectivity observed. CHAPTER FOUR investigates the ability of hydrolytic enzymes to kinetically resolve a series of allylic acetates, under varying conditions. Lipases appeared superior to esterases for the substrates employed. In particular cis-3-acetoxy-5-carbomethoxycyclohexene was smoothly resolved m high yield and enantioselectivity. CHAPTER FIVE reports on the potentiality of a dynamic resolution of allylic acetates, using hydrolytic enzymes in the presence of a palladium catalyst. A proposed mechanism is discussed. Initial results are promising, however, the sensitivity of the reaction is realised and optimisation of conditions still needs to be addressed.

O processamento de celulose em moinho de bolas de porcelana produz um pó no qual se detecta a presença de radicais livres, por Ressonância Paramagnética Eletrônica. O espectro obtido e complexo e pode ser decomposto em pelo menos dois sinais: uma linha instável, eliminada apôs alguns dias de exposição ao ar, e outra estável, que decai lentamente por aquecimento a 400-500ºC, e desaparece a 900ºC. A moagem simultânea de celulose com amido, acrilamida, azul de dextrana e sacarose produz celulose quimicamente modificada. Por exemplo, no caso da acrilamida, dosagem pelo método kjeldahl revela a presença de ate 0,65% de nitrogênio no material obtido apôs lavagens exaustivas. A celulose moída na presença de vários reagentes foi submetida a ensaios de hidrólise e em alguns casos, mostrou-se significativamente mais suscetível ao ataque enzimático do que a celulose simplesmente moída. Os resultados deste trabalho mostram que a conversão enzimática de celulose em açucares redutores e sensível a uma pequena modificação no pré-tratamento mecanoquímico. / Cellulose processing on porcelain ball mill produce a powder in which one can detect free radicals by means of Electronic Paramagnetic Ressonance. The spectrum attained is complex and it may be decomposed in at least two signals: an unstable line that is eliminated after some days of exposition tc air and another stable line, which slowly decays due to heating at 400-500ºC and disapears at 900ºC. Cellulose simultaneously milled with starch, acrylamide, dextran blue and saccharose produce chemically modified cellulose. For instance, when using acrylamide one can detect, by means of the Kjeldhal method, the existence of nitrogen up to 0.65% in the material obtained after exhaustive washings. In some cases cellulose milled with several reactants seemed to be significantly more susceptible to the enzymatic attack during hydrolysis than cellulose milled without any reactant. The results of this work show that the enzymatic conversion of cellulose into reducible sugars is sensible to a little change in the mechano-chemical pre-treatment.

2-(2-mandelyl)thiamin (MTh), the adduct of benzoylformate and thiamin, is an accurate model of 2-(2-mandelyl)thiamin diphosphate, the initial covalent intermediate in the decarboxylation of benzoylformate by benzoylformate decarboxylase (BFDC). The first order rate constant for spontaneous decarboxylation of MTh is about 106 times smaller than the enzymic rate (kcat) for the BFDC reaction. Based on the similarities of MTh and the corresponding enzymic intermediate, as well as the inherent nature of the intermediate, it is not obvious why the enzyme-catalyzed reaction is so much faster. However, earlier studies showed that the decarboxylation of MTh is catalyzed by protonated pyridines and this was proposed to occur through a preassociation mechanism. If this explanation is correct, then the observed 12C/13C kinetic isotope effect (CKIE) will increase in the presence of the catalyst as a more favorable forward commitment is made possible. This provides a specific model for the enzyme-catalyzed process. We developed a technique using headspace analysis and compound specific isotope analysis (CSIA) to determine the CKIE for the decarboxylation of MTh in the presence and absence of pyridinium. We found that the CKIE increases in the presence of the catalyst, as predicted for the preassociation mechanism. In a related study, we investigated the kinetics of decarboxylation of pyrrole-2-carboxylic acid, which was known to be subject to acid catalysis in highly acidic solutions. In the expected mechanism, protonation of the pyrrole ring at C2 destroys the aromaticity of the ring. C-C bond cleavage in the process of decarboxylation will re-establish the aromatic pyrrole. However, the overall reaction rate would not increase as it is counteracted by a larger concentration of the undissociated carboxyl group compared to carboxylate ion necessary for decarboxylation. Since the reaction occurs readily, there must be an alternative pathway for the acid-catalyzed reaction. This can be achieved in an associative mechanism that is initiated by addition of water to the carboxyl group of the carboxyl-protonated reactant. C-C bond cleavage results in formation of pyrrole and protonated carbonic acid, a species that has been recognized as a viable intermediate in related processes. Protonated carbonic acid is spontaneously converted to H3O+ and carbon dioxide. The associative mechanism is consistent with solvent-deuterium kinetic isotope effects and 12C/13C kinetic isotope effects.

2-(2-mandelyl)thiamin (MTh), the adduct of benzoylformate and thiamin, is an accurate model of 2-(2-mandelyl)thiamin diphosphate, the initial covalent intermediate in the decarboxylation of benzoylformate by benzoylformate decarboxylase (BFDC). The first order rate constant for spontaneous decarboxylation of MTh is about 106 times smaller than the enzymic rate (kcat) for the BFDC reaction. Based on the similarities of MTh and the corresponding enzymic intermediate, as well as the inherent nature of the intermediate, it is not obvious why the enzyme-catalyzed reaction is so much faster. However, earlier studies showed that the decarboxylation of MTh is catalyzed by protonated pyridines and this was proposed to occur through a preassociation mechanism. If this explanation is correct, then the observed 12C/13C kinetic isotope effect (CKIE) will increase in the presence of the catalyst as a more favorable forward commitment is made possible. This provides a specific model for the enzyme-catalyzed process. We developed a technique using headspace analysis and compound specific isotope analysis (CSIA) to determine the CKIE for the decarboxylation of MTh in the presence and absence of pyridinium. We found that the CKIE increases in the presence of the catalyst, as predicted for the preassociation mechanism. In a related study, we investigated the kinetics of decarboxylation of pyrrole-2-carboxylic acid, which was known to be subject to acid catalysis in highly acidic solutions. In the expected mechanism, protonation of the pyrrole ring at C2 destroys the aromaticity of the ring. C-C bond cleavage in the process of decarboxylation will re-establish the aromatic pyrrole. However, the overall reaction rate would not increase as it is counteracted by a larger concentration of the undissociated carboxyl group compared to carboxylate ion necessary for decarboxylation. Since the reaction occurs readily, there must be an alternative pathway for the acid-catalyzed reaction. This can be achieved in an associative mechanism that is initiated by addition of water to the carboxyl group of the carboxyl-protonated reactant. C-C bond cleavage results in formation of pyrrole and protonated carbonic acid, a species that has been recognized as a viable intermediate in related processes. Protonated carbonic acid is spontaneously converted to H3O+ and carbon dioxide. The associative mechanism is consistent with solvent-deuterium kinetic isotope effects and 12C/13C kinetic isotope effects.

The first project is focused on kinetic analysis of two enzymes: Rsc1362 (Ralstonia solanacearum GMI1000) and PA0810 (Pseudomonas aeruginosa PA01). Rsc1362 had a kcat of 504±66 min-1 and a KM of 0.06±0.02 mM, PA0810 had a kcat of 2.6±0.6 min-1 and a KM of 0.44±0.2 mM. A lack of environmental context for a chloroacetate dehalogenase was noted in Pseudomonas aeruginosa PA01. The second project focuses on kinetic and stable isotope fractionation of 1,2- dichloroethane by DhlA (Xanthobacter autotrophicus GJ10), and Jann2620 (Jannaschia CCS1). Although both enzymes had different kinetics (DhlA: KM = 4.8±0.6 mM and kcat = 133±8 min-1, Jann2620: KM = 25.9±2.3 mM and kcat = ~1.7 min-1), they fractionated similarly (ε values of -33.9‰ and -32.9‰ for DhlA and Jann2620, respectively). As calculated AKIE values were similar to the expected values of an abiotic reaction, it was determined that neither enzyme masks the intrinsic fractionation.

The first project is focused on kinetic analysis of two enzymes: Rsc1362 (Ralstonia solanacearum GMI1000) and PA0810 (Pseudomonas aeruginosa PA01). Rsc1362 had a kcat of 504±66 min-1 and a KM of 0.06±0.02 mM, PA0810 had a kcat of 2.6±0.6 min-1 and a KM of 0.44±0.2 mM. A lack of environmental context for a chloroacetate dehalogenase was noted in Pseudomonas aeruginosa PA01. The second project focuses on kinetic and stable isotope fractionation of 1,2- dichloroethane by DhlA (Xanthobacter autotrophicus GJ10), and Jann2620 (Jannaschia CCS1). Although both enzymes had different kinetics (DhlA: KM = 4.8±0.6 mM and kcat = 133±8 min-1, Jann2620: KM = 25.9±2.3 mM and kcat = ~1.7 min-1), they fractionated similarly (ε values of -33.9‰ and -32.9‰ for DhlA and Jann2620, respectively). As calculated AKIE values were similar to the expected values of an abiotic reaction, it was determined that neither enzyme masks the intrinsic fractionation.