The hydrogenation of β-ketoesters over chirally modified Ni catalysts is a celebrated and thoroughly researched example of an enantioselective heterogeneous catalytic reaction. Enantioselective heterogeneous processes, although extremely attractive in terms of fewer complications in the separation of products from the catalyst, are hindered in their viability as industrial applications due to the lack of detailed knowledge on how chirality is conferred to the metal surface. Surface science techniques have afforded substantial progress into determining mechanisms between modifier, reactant and catalyst to explain the source of enantioselectivity of the system. In this study, a combination of solution and ultra-high vacuum (UHV)-based experiments allow a more realistic interpretation of the surface chemistry underpinning the catalytic reaction as the key step in achieving enantioselective performance is the adsorption of chiral modifiers from solution. The behaviour of (S)-aspartic acid and (S)-lysine on Ni{111} and their interaction with the prochiral β-ketoester methylacetoacetate is investigated in this study to understand their potential as chiral modifiers for the system. In UHV, scanning tunnelling microscopy (STM), reflection absorption infrared spectroscopy (RAIRS), and temperature programmed desorption (TPD) are used to analyse the conformation and order of the amino acids on the metal, and their thermal stability. Additionally, liquid-solid interface RAIRS and X-ray photoelectron spectroscopy (XPS) are used to examine the modified Ni surface, prepared under aqueous conditions, to give an accurate representation of the catalytic studies. It has been found highly likely that, for (S)-aspartic acid modified Ni{111}, enantioselective sites exist at step or step/kink defects, formed by corrosive leaching of the Ni substrate. Conversely, lysine appears to bind with a high sticking probability to Ni, in the form of lysine islands, and does not appear to etch the Ni chirally. Finally, similar experiments have been carried out on Au{111}, where lysine was found to chiral restructure the surface and form nanofingers, and 2D Ni clusters grown on Au{111} in order to investigate the formation of possible metal-organic frameworks.

Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically: Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal-oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example. In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants.

Epóxido hidrolases (EHs) são enzimas que catalisam a hidrólise de epóxidos a seus correspondentes dióis, apresentam potencial aplicação biotecnológica (separação de enantiômeros na produção de fármacos), estão envolvidas no metabolismo de ácidos graxos poliinsaturados e inibidores de EHs estão sendo estudados para possível utilização no tratamento de doenças. Uma enzima epóxido hidrolase (TrEH) do fungo filamentoso Trichoderma reesei QM9414 foi clonada, expressa, purificada e caracterizada funcionalmente e estruturalmente. A atividade de TrEH foi determinada com o substrato óxido de estireno (racêmico), demonstrando maior atividade nas temperaturas de 23 a 50 °C, no pH 7,2 a 37 °C, e as constantes cataliticas Km= 4,6 mM e kcat= 336 s-1. A enzima recombinante mostrou ser enantiosseletiva, pois hidrolisa preferencialmente (S)-(-)-óxido de estireno, (R)-(-)- epicloridrina e (S)-(-)-1,2-epoxibutano. Moléculas inibidoras da atividade de TrEH foram identificadas e algumas delas inibem até 60% o crescimento de T. reesei. A estrutura terciária de TrEH (1,7 Å) foi determinada por cristalografia, apresenta dobramento α/β-hidrolase e não tem alta homologia com nenhuma outra estrutura de EH. TrEH é uma nova enzima epóxido hidrolase solúvel cujas propriedades mostram seu potencial de utilização em aplicações biotecnológicas. / Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to their corresponding diols, present a potential biotechnological application (separation of enantiomers for the production of drugs), they are involved in the metabolism of polyunsaturated fatty acids and EH inhibitors are being studied for possible use in the treatment of diseases. An epoxide hydrolase enzyme(TrEH) from the filamentous fungus Trichoderma reesei QM9414 was cloned, expressed, purified and functionally and structurally characterized. The activity of TrEH was determined with the substrate styrene oxide (racemic), showing higher activity at temperatures of 23 to 50 °C, at pH 7.2 at 37 °C, and the catalytic constants Km= 4.6 mM and kcat= 336 s-1. The recombinant enzyme has been shown to be enantioselective, because it preferably hydrolyzes (S)-(-)-styrene oxide, (R)-(-)-epichlorohydrin and (S)-(-)-1,2- epoxybutane. TrEH inhibitors have been identified and some of them inhibit up to 60% growth of T. reesei . The tertiary structure of TrEH (1.7 Å) was determined by crystallography, showing α/ β-hydrolase folding and low homology with any other EH structure. TrEH is a new soluble epoxide hydrolase enzyme whose properties show its potential for use in biotechnological applications.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-02-12T14:11:12Z No. of bitstreams: 2 Dissertação - Fernanda de Sousa Fernandes Pimentel - 2015.pdf: 3552044 bytes, checksum: 6506fc310b497a929ec9acf4d4db5942 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-02-16T06:47:11Z (GMT) No. of bitstreams: 2 Dissertação - Fernanda de Sousa Fernandes Pimentel - 2015.pdf: 3552044 bytes, checksum: 6506fc310b497a929ec9acf4d4db5942 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-02-16T06:47:11Z (GMT). No. of bitstreams: 2 Dissertação - Fernanda de Sousa Fernandes Pimentel - 2015.pdf: 3552044 bytes, checksum: 6506fc310b497a929ec9acf4d4db5942 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-06-12 / The allylation reaction of allyl organometallic compounds with aldehydes promoted by Lewis acids in the presence of chiral auxiliaries are a kind of nucleophilic addition reaction to control the stereochemistry of the product. The (R) -BINOL and (S) -4-benzylthiazolidine-2-thione compounds are used as chiral auxiliaries in asymmetric synthesis, although the literature describing the application of the first allylation reaction in the presence of various Lewis acids, it was not employed in the presence of niobium pentachloride. For the second compound, its application is limited to the aldol reactions and their study on enantioselective allylation reactions of aldehydes in the presence of Lewis acids was not described in the main databases used in this research. Therefore, this study investigated the application of these two compounds as chiral inductors in addition reactions of allyl tri-n-butilestanana to aldehydes in the presence of Lewis acid such as NbCl5. To optimize the reaction conditions used the p-nitrobenzaldehyde and was found to influence the proportion of the reactants in enantioselectivity and reaction yields. It was observed that the best yields (between 63-65% using (R)-BINOL and 41-50% using (S)-4-benzylthiazolidine-2-thione) were obtained when ethyl ether is used as solvent one temperature of -15 °C. However, the best enantioselectivity using a chiral auxiliary (R)-BINOL was obtained when the reaction rose while stirring for 1 hour the reaction between (R)-BINOL and NbCl5, then adding the aldehyde and stirred for 30 minuitos using diethyl ether and methylene chloride at -15 °C and -75 °C temperature conditions. On the other hand, the best enantioselectivity using the chiral auxiliary (S)-4-benzylthiazolidine-2-thione was obtained when it was used NbCl5 Lewis acid followed by stirring the reaction for 10 minutes and addition of diethyl ether as solvent under the conditions -15 °C temperature. / As reações de alilação de aldeídos com compostos organometálicos alílicos promovidas por ácidos de Lewis na presença de auxiliares quirais são um tipo de reação de adição nucleofílica para o controle da estereoquímica do produto. O (R)-BINOL e a (S)-4-benziltiazolidino-2-tiona são compostos utilizados como auxiliares quirais em síntese assimétrica e embora a literatura descreva a aplicação do primeiro em reações de alilação na presença de vários ácidos de Lewis, o mesmo não foi empregado na presença do pentacloreto de nióbio. No caso do segundo composto, a sua aplicação se limita as reações aldólicas e o seu estudo em reações de alilação enantiosseletiva de aldeídos na presença de ácidos de Lewis não foi descrito nas principais bases de dados utilizados nesta pesquisa. Portanto, nesse trabalho investigou-se a aplicação desses dois compostos como indutores quirais em reações de adição de alil-tri-n-butilestanana a aldeídos na presença de NbCl5 como ácido de Lewis. Para otimizar as condições reacionais utilizou-se o p-nitrobenzaldeído e verificou-se a influência da proporção dos reagentes na enantiosseletividade e nos rendimentos da reação. Percebe-se que os melhores rendimentos (entre 63-65% utilizando o (R)-BINOL e 41-50% utilizando a (S)-4-benziltiazolidino-2-tiona) foram obtidos quando o éter etílico foi utilizado como solvente a uma temperatura de -15ºC. Porém, a melhor enantiosseletividade utilizando o auxiliar quiral (R)-BINOL foi obtida quando elevou-se o tempo reacional para 1 hora de agitação na reação entre (R)-BINOL e o NbCl5, seguida da adição do aldeído e agitação por 30 minuitos, utilizando éter etílico e cloreto de metileno nas condições de temperatura de -15ºC e -75ºC. Por outro lado, a melhor enantiosseletividade utilizando o auxiliar quiral (S)-4-benziltiazolidino-2-tiona foi obtida quando utilizou-se o ácido de Lewis NbCl5 seguido pela agitação da reação por 10 minutos e adição de éter etílico como solvente nas condições de temperatura de -15ºC.

Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R)- and 2-(S)-hydroxypropyl-CoM (HPC) dehydrogenases (DH)) of the epoxide carboxylation pathway in Xanthobacter autotrophicus strain Py2. The main goal of this dissertation is to kinetically, mechanistically and structurally characterize S-HPCDH and through the comparison studies with R-HPCDH reveal the basis for high degree of stereoselectivity exhibited by both enzymes. Analysis of the molecular structure of R-HPCDH and the homology model of S-HPCDH suggests a mechanism of substrate specificity in which the binding of the substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for the hydride abstraction. The positively charged residues responsible for binding the CoM moiety of the substrate were identified in R-HPCDH (Arg152 and Arg196), and in S-HPCDH (Arg211 and Lys214). Site-directed mutagenesis confirmed their importance in binding and orienting physiological substrates, but not the substrates lacking the CoM moiety. Extensive kinetic and mechanistic characterization of S-HPCDH reveals its key catalytic features similar to those of R-HPCDH, but also points out a few important differences. Furthermore, the role of the methionine residues flanking the substrate in the active site of both dehydrogenases was investigated. Substitution of these residues to alanine resulted in enzymes with significantly altered catalytic parameters and suggested their importance in binding and catalysis. Additionally, the X-ray crystal structures of the Met187Ala and Met192Ala mutants of R-HPCDH have revealed their role as "gate keepers," protecting the active site from the surrounding solvent. Kinetic analysis of Met187Leu and Met192Leu mutants implied a structural, rather than catalytic function of the methionines. It is proposed that steric clashes of the terminal methyl group of the HPC substrates with the nicotinamide ring of NAD+ are a major determinant of the enantioselectivity in S-HPCDH. This research provides the first side-by-side characterization of a pair of short-chain dehydrogenase/reductase (SDR) enzymes expressed simultaneously to act on two enantiomers of the same alcohol produced in a metabolic pathway. The R-HPCDH and S-HPCDH enzymes are distinguished from all other known members of the SDR family in using the novel sulfonate functional group of coenzyme M as a handle for chiral discrimination. These results provide a standard for examining the molecular basis of stereoselectivity in other such enzyme pairs.

A series of five linked β-cyclodextrin (βCD) dimers N,N-bis(6 [superscript]A-deoxy-6[superscript]A-β-cyclodextrinyl)-succinamide, 66βCD₂su, N-((2[superscript]A S,3 [superscript]A S)-3 [superscript]A-deoxy-3 [superscript]A-β-cyclodextrinyl)-N’-(6 [superscript]A-deoxy-6 [superscript]A -β-cyclodextrinyl)-urea, 36βCD₂su, N,N-bis((2 [superscript]A S,3 [superscript]A S)-3 [superscript]A -deoxy-3 [superscript]A-β-cyclodextrinyl)-succinamide, 33βCD₂su, N,N-bis(6[superscript]A-deoxy-6[superscript]A-β-cyclodextrinyl)-urea, 66βCD₂ur, and N-((2 [superscript]A S,3 [superscript]A S)-3 [superscript]A-deoxy-3 [superscript]A-β-cyclodextrinyl)-N’-(6 [superscript]A -deoxy-6 [superscript]A -β-cyclodextrinyl)urea, 36βCD₂ur, has been prepared. The complexation of 6-(4’-(toluidinyl)naphthalene-2-sulphonate, TNS⁻, by βCD and the five linked βCD dimers was characterized by UV, fluorescence and 2D ¹H ROESY NMR spectroscopy. In aqueous phosphate buffer at pH 7.0, I = 0.10 mol dm⁻³ and 298.2 K, TNS⁻ forms host-guest complexes with βCD of stoichiometry βCD.TNS⁻ (K₁ = 3020 and 3320 dm³ mol⁻¹) and βCD₂.TNS⁻ (K₂ = 57 and 11 dm³ mol⁻¹) where the first and second values were determined in UV and fluorescence studies, respectively. For 66βCD₂su, 36βCD₂su, 33βCD₂su, 66βCD₂ur and 36βCD₂ur, the analogous K₁ = 16100, 10900, 10700, 55100 and 18300 dm³ mol⁻¹ and K₁ = 12500, 8700, 9600, 38000 and 9800 dm³ mol⁻¹(fluorimetric studies), respectively. ¹H 2D ROESY NMR studies provided evidence for variation of the mode of complexation of the TNS⁻ guest as the βCD host is changed. The factors affecting complexation are discussed. UV and ¹H NMR studies showed that 6-(4’-(t-butyl)-phenyl)naphthalene-2-sulphonate, BNS⁻, and its dimer, (BNS⁻)₂, form host-guest complexes with βCD of the stoichiometry βCD.BNS⁻ (K₁ = 5.54 × 10⁴ dm³ mol⁻¹ ) and βCD.BNS₂ ²⁻(K₂ = 3.07 × 10² dm³ mol⁻¹ ) where the complexation constant K₁ = [βCD.BNS⁻]/([βCD][BNS⁻] and K₂ = [βCD.(BNS⁻)₂]/([βCD.BNS⁻][BNS⁻]) in aqueous phosphate buffer at pH 7.0, I = 0.10 mol dm⁻³ and 298.2 K. For 66βCD₂su, 36βCD₂su, 33βCD₂su, 66βCD₂ur and 36βCD₂ur the analogous K₁ = 125, 74, 10.2, 364 and 16.1 (× 10⁴ dm³ mol⁻¹ ) and K₂ = 25.7, 2.30, 2.57, 17.6 and 17.2 (× 10² dm³ mol⁻¹ ), respectively. For the dimerisation of BNS⁻ K[subscript]d = 2.63 × 10² dm³ mol⁻¹ . Fluorimetric studies showed that the complexation stability for βCD.BNS⁻, forms βCD. BNS⁻, 66βCD₂su.BNS⁻, 36βCD₂su.BNS⁻, 33βCD₂su. BNS⁻, 66βCD₂ur.BNS⁻ and 36βCD₂ur. BNS⁻ characterized by K₁ = 4.67, 330, 101, 11.0, 435 and 29.6 (× 10⁴ dm³ mol⁻¹ ), respectively. The factors affecting the variations in these data are discussed. The enantioselectivity of substituted βCDs 6 [superscript]A -[bis (carboxylatomethyl)amino]-6 [superscript]A -deoxy-β-cyclodextrin (6βCDidaH₂) and (2 [superscript]A S,3 [superscript]A S)-3 [superscript]A -[bis(carboxylatomethyl)amino]-3[superscript]A -deoxy-β-cyclodextrin (3βCDidaH₂) and 6 [superscript]A -[tris(carboxylatomethyl)(2- aminoethyl)amino]-6 [superscript]A -deoxy-β-cyclodextrin (6βCDedtaH₃) and their Eu³ ⁺ complexes in forming host-guest complexes with six enantiomeric guests in D₂O was studied by 1D and 2D ¹H NMR (600 MHz) spectroscopy. The guests are D/L-tryptophanate (Trp⁻), 4-hydroxyl-D/L-phenylglycinate (4HOPhg⁻), D/L-histidinate (His⁻), D/L-pheniramine (Phm), D/L-phenylglycinate (Phg⁻) and (D/L)-β-phenylserinate (βPhs⁻). Enantioselective host-guest complexation was observed between the [Eu(6βCDida)]⁺ , [Eu(3βCDida)]⁺ and [Eu(6βCDedta)] complexes and Trp⁻, [Eu(6βCDida)]⁺ and [Eu(3βCDida)]⁺ and 4HOPhg⁻, and βCD, 6βCDida²⁻, 3βCDida²⁻, 6βCDedta³⁻ and the Eu³⁺complexes of the three substituted βCDs and Phm. The His⁻, Phg⁻ and βPhs⁻ enantiomers showed no evidence for selective host-guest complexation. The preparation of 3βCDidaH₂ and 6βCDedtaH₃ and the determination of their pK[subscript]a s are also reported. In collaboration with the research group of Prof. Matthew A. Tarr, (University of New Orleans, USA), the 6βCDida²⁻ and the 6βCDedta³⁻ has been utilized to improve Fenton oxidation of aromatic pollutants. To further support to this work, the binary complexation of Fe² ⁺ by 6βCDida²⁻ has been studied by potentiometric titrations. A series of six modified poly(acrylic acid)s 3% substituted with either βCD or the adamantyl moiety with different length of substituent chain was synthesised. To advance the understanding and control of aqueous supramolecular assembly, the host-guest interactions between the βCD substituted poly(acrylic acid)s and adamantane-1-carboxylic; adamantyl substituted poly(acrylic acid)s with βCD and linked βCD dimers; and between both βCD and adamantyl substituted poly(acrylic acid)s have been studied. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311237 / Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 2008

This PhD thesis deals with the activation of diorganozinc compounds by asymmetric catalysts in the framework of their enantioselective addition on carbon oxygen and carbon nitrogen double bonds. The properties of several new catalysts systems were investigated. Several chiral sulfonamido alcohols derived from (S)-leucinol and (1S) 3 aminoisoborneol were synthesized to cover a range of electronic and steric properties. These were evaluated in the addition of Et2Zn to N phosphinoyl and N sulfonyl-imines, but they did not provide any acceptable yields or enantioselectivities while used in stoichiometric quantities. Their catalytic properties were also investigated for the addition of Et2Zn to benzaldehyde. Unfortunately, only modest enantiocontrol (max. 47% ee) could be obtained with 10mol% of chiral ligand. We investigated another approach based on the possible activation of the nucleophilicity of dialkylzinc compounds towards benzaldehyde by fluoride anion. The combination of stoichiometric amount of an inorganic fluoride salt and a substoichiometric amount of a phase transfer catalyst (10mol% of crown ether 18 C 6) provided non-negligible rate acceleration. However, an asymmetric version of this catalytic system was not considered. Following our discovery of the activation provided by the fluoride anion, metal fluoride complexes were tapped as potential catalysts. In particular, copper(I) fluoride complex, (Ph3P)3CuF, was shown to accelerate the reaction on benzaldehyde. After screening a range of ligands, the combination of (Ph3P)3CuF and chiral BINAP in catalytic amounts (1mol%) was found to catalyze efficiently the Et2Zn addition on aromatic aldehydes. Yields were good but enantiocontrol was modest (49-63% ee). An improved copper fluoride-based catalyst was developed by combining a copper salt, a fluoride source and a chiral diphosphine ligand. Optimization of this second generation catalyst was carried out (copper source, fluoride source, chiral ligand, relative stoichiometries of catalyst's precursors, catalyst's loading, solvent, temperature) and applied to a range of aldehydes (upto 87% ee). A mechanism was proposed based on literature data and experimental observations. The influence of the counterion was then thoroughly explored by assessing the catalytic potential of various copper complexes in combination with chiral BINAP in the Et2Zn addition to benzaldehyde. Copper(II) diketonates provided good enantiocontrol (upto 88% ee) which could be improved even further by using optically active copper diketonates in a matched combination with chiral BINAP (upto 91% ee). This new system was applied to control the addition of Et2Zn to a range of aldehydes with a low catalyst loading (0.5mol%). A mechanism was proposed to explain a series of experimental observations. / Cette dissertation traite de l'activation de composés diorganozinciques par des catalyseurs asymétriques dans le cadre de leur addition énantiosélective sur des doubles liaisons carbone-oxygène et carbone-azote. Les propriétes de divers catalyseurs originaux ont été investiguées. Plusieurs sulfonamidoalcools chiraux dérivés du (S)-leucinol et du (1S) 3 aminoisoborneol ont été synthétisés pour couvrir un domaine de propriétés électroniques et stériques. Ceux-ci ont été testés lors l'addition du Et2Zn sur les N phosphinoyl et N-sulfonyl-imines, mais ils n'ont pas fourni de rendements et excès énantiomériques intéressants malgré leur utilisation en quantité stoechiométrique. Leurs propriétés catalytiques ont également été examinées dans le cadre de l'addition de Et2Zn au benzaldéhyde. Malheureusement, un faible contrôle de l'énantiosélectivité (max. 47% ee) est obtenu en présence de 10mol% de ligand chiral. Une autre approche abordée concerne l'influence de l'anion fluorure sur la nucléophilie des composés dialkylzincs envers le benzaldehyde. La combinaison de quantités stoechiométriques d'un sel fluoré inorganique et d'une quantités sub stoechiométrique d'un agent de transfert de phase (10mol% éther couronne 18 C 6) conduit à un accroissement non négligeable de la vitesse de réaction. Toutefois, le développement d'une version asymétrique de ce système n'a pas été considéré. A la suite de notre découverte de l'activation due à l'anion fluorure, les complexes métalliques fluorés ont été examinés en tant que catalyseurs potentiels. En particulier, le complexe du cuivre, (Ph3P)3CuF, accélère la réaction d'addition du Et2Zn sur le benzaldéhyde. Après un criblage de divers ligands, la combinaison de (Ph3P)3CuF et de la diphosphine chirale BINAP en quantité catalytique (1mol%) conduit à une catalyse efficace de l'addition du Et2Zn sur des aldéhydes aromatiques. Les rendements obtenus sont bons, par contre l'énantiosélectivité est modeste (49-63% ee). Une version améliorée de catalyseurs cuivre-fluorure a été obtenue en combinant un sel de cuivre, une source de fluorure et une diphosphine chirale. L'optimisation de divers paramètres de réaction a été effectuée (source de cuivre, origine de l'anion fluorure, ligand chiral, stoechiométries relatives des précurseurs du catalyseur, charge de catalyseur, solvant et température). Le catalyseur optimisé a été appliqué à divers aldéhydes (jusqu'à 87% ee). Un mécanisme réactionnel a été proposé sur la base d'arguments de la littérature et d'observations expérimentales. Le rôle du contre-ion a été exploré par un examen attentif de divers complexes du cuivre en combinaison avec la BINAP chirale en tant que catalyseurs de l'addition du Et2Zn sur le benzaldehyde. Les dicétonates du cuivre(II) ont conduit à une bonne énantiosélectivité (jusqu'à 88% ee) qui a pu être améliorée en utilisant des dicétonates du cuivre(II) optiquement actifs assortis avec le bon énantiomère de la BINAP (jusqu'à 91% ee). Ce nouveau système a été appliqué pour l'addition du Et2Zn sur divers aldéhydes avec une charge réduite en catalyseur (0.5mol%). Un mécanisme a été proposé afin de supporter les observations expérimentales.

The haemolymph of the adult Colorado potato beetle, Lepinotarsa decemlineata Say, contains a high molecular weight (MW > 200,000) JH-III specific binding protein. The Kd value of the protein for racemic JH-III is 1.3 ± 0.2 × 10−7 M. It has a lower affinity for racemic JH-I and it does not bind JH-III-diol or JH-III-acid. The binding protein does discriminate between the enantiomers of synthetic, racemic JH-III as was determined by stereochemical anaysis of the bound and the free JH-III. Incubation of racemic JH-III with crude haemolymph results in preferential formation of (10S)-JH-III-acid, the unnatural configuration. The JH-esterase present in L. decemlineata haemolymph is not enantioselective. It is concluded that the most important function of the binding protein is that of a specific carrier, protecting the natural hormone against degradation by esterases. The carrier does not protect JH-I as efficiently as the lower homologue.

The work leading to this thesis has provided additional information and novel knowledge concerning structure-function relationship in the potato epoxide hydrolase. Epoxide hydrolases are enzymes catalyzing the hydrolysis of epoxides to yield the corresponding vicinal diols. The reaction mechanism proceeds via a nucleophilic attack resulting in a covalent alkylenzyme intermediate, which in turn is attacked by a base-activated water molecule, followed by product release. Epoxides and diols are precursors in the production of chiral compounds and the use of epoxide hydrolases as biocatalysts is growing. The promising biocatalyst StEH1, a plant epoxide hydrolase from potato, has been investigated in this thesis. In paper I the active site residue Glu35, was established to be important for the formation of the alkylenzyme intermediate, activating the nucleophile for attack by facilitated proton release through a hydrogen bond network. Glu35 is also important during the hydrolytic half reaction by optimally orienting the hydrolytic water molecule, aiding in the important dual function of the histidine base. Glu35 makes it possible for the histidine to work as both an acid and a base. In paper II a putative proton wire composed of five water molecules lining a protein tunnel was proposed to facilitate effective proton transfer from the exterior to the active site, aiding in protonation of the alkylenzyme intermediate. The protein tunnel is also proposed to stabilize plant epoxide hydrolases via hydrogen bonds between water molecules and protein. Enzyme variants with modified enantiospecificity for the substrate (2,3-epoxypropyl)benzene have been constructed by in vitro evolution using the CASTing approach. Residues lining the active site pocket were targeted for mutagenesis. From the second generation libraries a quadruple enzyme variant, W106L/L109Y/V141K/I155V, displayed a radical shift in enantioselectivity. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the quadruple variant showed a 15:1 preference for the R-enantiomer.

Biocatalysis has become a popular technique in organic synthesis due to high activity and selectivity of enzyme catalyzed reactions. Enantioselectivity is a particularly attractive enzyme property, which is utilized for the production of enantiopure substances. Determination of the temperature dependence of enzyme enantioselectivity allows for thermodynamic analyses that reveal the contribution of differential activation enthalpy, ΔR-SΔH‡, and entropy, ΔR-SΔS‡. In the present investigation the influence of substrate structure, variations on enzyme structure and of reaction media on the enantioselectivity of Candida Antarctica lipase B has been studied. The contribution of enthalpy, ΔR-SΔH‡, and entropy, TΔR-SΔS‡, to the differential free energy, ΔR-SΔG‡, of kinetic resolutions of sec-alcohols were of similar magnitude. Generally the two terms were counteracting, meaning that the enantiomer favored by enthalpy was disfavored by entropy. 3-Hexanol was an exception where the preferred enantiomer was favored both by enthalpy and by entropy. Resolution of 1-bromo-2-butanol revealed non-steric interactions to influence both ΔR-SΔH‡ and ΔR-SΔS‡. Molecular modeling of the spatial freedom of the enzyme-substrate transition state indicated correlation tothe transition state entropy. The acyl chain length was shown to affect enantioselectivity in transesterifications of a sec-alcohol. Point mutations in the active site were found to decrease or increase enantioselectivity. The changes were caused by partly compensatory changes in both ΔR-SΔH‡ and ΔR-SΔS‡. Studies on single and double mutation variants showed that the observed changes were not additive. Enantioselectivity was strongly affected by the reaction media. Transesterifications of a sec-alcohol catalyzed by Candida Antarctica lipase B was studied in eight liquidorganic solvents and supercritical carbon dioxide. A correlation of enantioselectivity and the molecular volume of the solvent was found. Differential activation enthalpy, ΔR-SΔH‡, and entropy, ΔR-SΔS‡, display a compensatory nature. However this compensation is not perfect, which allows for modifications of enantioselectivity. The components of the thermodynamic parameters are highly complex and interdependent but if their roles are elucidated rational design of enantioselective enzymatic processes may be possible. / QC 20100616