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Stereochemistry and chirality are arguably two of the most important subjects pertaining to the development of new pharmaceutical drugs. Since enantiomers have the potential to encompass different pharmacological effects in biological systems, both enantiomers have to be tested for pharmacological activity. Not only has obtaining these single enantiomers become crucial, but formulation of the pure enantiomer of a drug also has the potential to contain advantages for both pharmaceutical formulation and therapeutic effect. Epoxide hydrolase is an enzyme commonly found in nature that catalyses the hydrolysis of epoxides, resulting in the formation of the corresponding vicinal diol. Over the last few years a large amount of research has been completed on these enzymes from sources such as mammals, insects, bacteria and fungi. Micro-organisms especially have enjoyed ample attention because of their abundant supply. Recently it was found that certain yeasts contain this enzyme and have the ability to enantioselectively catalyse certain hydrolysis reactions. Styrene oxides are terminal epoxides that are, due to the reactivity of the epoxide ring, useful synthons in the organic synthesis of pharmaceutical products. The first objective of this project was to synthesize three nitro derivatives of styrene oxide namely para-, meta-, and ortho-nitrostyrene oxide. Al three products were obtained from the corresponding nitrophenacyl bromide in yields of 52%, 90% and 57% respectively. The second objective was lo find a suitable yeast slrain containing the epoxide hydrolase enzyme to enantioselectively hydrolyse the synthesised products and unsubstituted styrene oxide. A screening was completed during which 410 yeast strains from more than 44 genera were tested. Epoxide hydrolase activity was found to be widespread throughout the screened yeast domain, while the genera Candida, Debaryomyces, Pichia, Rhodosporidium, Rhodotorula and Trichosporon specifically were very successful in catalysing the hydrolysis of the substrates. Rhodosporidium toruloides UOFS Y-0471 and Rhodotorula glutinis UOFS Y-0653 were chosen for further studies because of their superior enantioselectivity. The final objective was to optimise these reactions in terms of pH, temperature and substrate concentration. It was found that a pH value of 7.2 and a temperature of 45’C yielded optimal enzyme activity. Increased temperatures (45’C), however, lead to a decrease in enantioselectivity and, in the case of R. toruloides together with the substrate puranitrostyrene oxide, reversed enantioselectivity. Lower temperatures (15’C) increased enantioselectivity, resulting in a remarkable improvement from a 10% yield of the single enantiomer (45’C) to a 35% yield. Surprisingly this temperature decrease had a very small affect upon the reaction time. / Thesis (M.Sc. (Pharmaceutical Chemistry)--Potchefstroom University for Christian Higher Education, 2002.

O primeiro capítulo desta tese é dedicado aos estudos que visaram à aplicação da biocatálise na síntese de γ-butirolactonas quirais. Inicialmente investigou-se a preparação de fenilseleno-γ-butirolactonas quirais, através da resolução cinética enzimática dos fenilseleno-γ-hidróxi ésteres correspondentes. Através desta estratégia, ambos os enantiômeros da fenilseleno-γ-butirolactona foram preparados em excessos enantioméricos razoáveis. Ainda no primeiro capítulo é demonstrado o potencial sintético do (R)-3-(5-oxotetraidro-2-furanil)-propanoato de benzila, obtido através de uma enantiolactonização catalisada por PPL na etapa chave. Este intermediário quiral foi utilizado com sucesso na síntese enantiosseletiva da (R)- e da (S)-y-jasmolactona, aromatizantes de interesse industrial, além de ambos enantiômeros da (7Z)-7,15-hexadecadien-4-oIida, feromônio sexual da \"Yellowish Elongate Chafer, Heptophy/a picea\" . A segunda parte deste trabalho foi efetuada na Universidade da Califórnia, EUA, sob a orientação do professor Bruce H. Lipshutz, durante o período de novembro de 2003 a outubro de 2004. Dois novos reagentes foram desenvolvidos e aplicados com sucesso na preparação estereosseletiva de polienos conjugados com estrutura análoga a da vitamina A . / The first chapter of this thesis is dedicated to the studies on the application of biocatalysis in the synthesis of chiral γ-butyrolactones. We started investigating the preparation of phenylselanyl-γ-butyrolactones through the kinetic enzymatic resolution of the corresponding phenylselanyl-γ-hydroxyesters. By using this strategy both enantiomers of phenylselanyl-γ-butyrolactone were prepared in reasonable enantiomeric excesses . In the first chapter is also demonstrated the synthetic potential of benzyl 3[(R)-tetrahydro-5-oxofuran-2-yl] propanoate, obtained through a PPL catalyzed enantiolactonization in the key step. This chiral intermediate was successfully applied in the enantioselective synthesis of (R)- and (S)-γ-jasmolactone, flavors that find industrial use, and both enantiomers of (7Z)-7,15-hexadecadien-4-olide, the sex pheromone of Yellowish Elongate Chafer, Heptophy/a picea. The second part of this work was carried out at University of California, USA, between November 2003 and October 2004, under supervision of Professor Bruce H. Lipshutz. Two new reagents were developed and successfully applied in the stereoselective synthesis of conjugated polienes analogs to vitamin A .

Natural products represent a large and diverse array of molecules. Natural products and their derivatives play important roles in the human sphere, serving as pharmaceuticals, biofuels, and more. However, the structural complexity of many promising natural products prohibits industrial production sufficient to make full use of their capabilities. The challenge posed by natural products has spurred many advances in multiple fields. Despite these achievements, ignorance of the native metabolic pathways and inefficiencies in manipulating the genes involved has slowed the ability of science to capitalize on the enormous potential of natural products. In Chapter 1, we begin by surveying the fields concerned with the production or variation of natural products. This begins with organic synthesis, continues with in vivo and in vitro biocatalytic methods, and concludes with the “combination” techniques that seek to unite the strengths of biocatalysis and organic chemistry: precursor-driven biosynthesis, mutasynthesis and semi-synthesis. After examining the advantages and disadvantages of the extant technologies, in Chapter 2 we describe a novel strategy to develop semi-synthetic routes to underexplored classes of natural products. While it employs features of existing techniques, our strategy originates from a fundamentally different conception of natural product production, which looks away from the native precursors of a single target, and towards versatile precursors amenable to multiple forms of chemical modification. We then carry out a demonstration of this strategy by first biosynthetically producing 2Z,7E-farnesol from heterologously expressed Mycobacterium tuberculosis synthetases, and subsequently derivatizing this unnatural precursor into a set of novel Ambrox© analogs. Complex biocatalytic applications rely on DNA manipulation technologies to rapidly construct and diversify metabolic pathways. When components of the targeted pathway are unknown or poorly understood, the creation of large libraries of variant pathways can be employed to circumvent these limitations and rapidly develop the desired phenotype. In Chapter 3, we harness our existing library building technology, Reiterative Recombination, to the yeast sexual reproduction cycle for the purpose of combining separately constructed library strains via simple mating and chromosome segregation into an exponentially larger combinatorial library. This chapter describes the design, construction, and initial validation of this system, termed Reiterative Segregation. Finally, in Chapter 4, we explore possible elaborations of the Reiterative Segregation design, and work towards combining libraries of alternative sugar metabolic pathways as an application relevant to biofuel production.

Thesis (M.Sc. (Pharmaceutical Chemistry)--Potchefstroom University for Christian Higher Education, 2002.

Sustainable technologies are vital to reducing the environmental impact of chemical enterprises. Solvents are often seen as just a medium in which a reaction takes place; however they can also play a dominant role in the overall toxicity of a typical pharmaceutical/fine chemicals batch chemical operation. Further, careful solvent selection for a reaction may also lead to more facile separation and purification of products, thus reducing the overall cost of a chemical process. This thesis presents an environmentally benign processing technique for sustainable biocatalytic reactions coupled with facile built-in separation. An organic aqueous tunable solvent (OATS) system allows access to a hydrophobic substrate which is transformed with a homogeneous enzymatic catalyst in a single liquid phase. Subsequent CO2 addition produces a biphasic mixture where the hydrophobic product partitions preferentially into the organic rich phase for separation while the hydrophilic enzyme catalyst partitions into the aqueous rich phase, where it is recyclable. Processing parameters in OATS systems are discussed and an overall product recovery of 80% is observed after six reaction cycles. Additionally, greater than 99% enantiomeric excess (ee) is shown for catalyzed hydrolysis of rac-1-phenylethyl acetate with Candida antarctica lipase B (CAL B) both before and after CO2-induced separation.

Due to recent legislation requiring the determining of the pharmacokinetic effect of both enantiomers separately, of any new racemic drug before commercialisation, much research is done to improve and optimise methods for obtaining chirally pure compounds important for the pharmaceutical industry, for example epoxide precursors. To date most experiments regarding the biocatalytic chiral separation of 1,2-epoxyoctane has been done in batch processes. The aim of this study was to optimise the enantioselective hydrolysis of 1,2-epoxyoctane by Rhodosporidiurn tondoides in both a batch and continuous process. The batch process was optimised in terms of stir speed, biomass (cell) concentration and reaction time, while the flow-through reactor (continuous process) was optimised with regards to the flow rate as a function of the pressure and the amount of chitosan and biomass in the reactor. Initial inconsistencies of epoxide concentrations in preliminary studies were found to be due to adsorption by reaction and sampling vessels, and the lower than expected solubility of 1,2- epoxyoctane (3.85 mM instead of 6 mM as reported by previous investigators). The results from the batch process suggest that as the reaction time increases, the % ee-epox increases initially, but decreases after 40 minutes. Optimum yield in terms of % ee-epox were obtained at medium stir speed (400 rpm) and biomass (cell) concentration (13 %). Below these values the % ee-epox increases with an increase in stir speed and/or biomass concentration. Above these values however, the increased stir speed and/or biomass concentration causes abrasion between cells, which negatively affects the % ee-epox. The % ee-diol reached a steady state after 10 minutes, and the effect of the different operating conditions on % ee-diol was negligible. In the flow-through reactor chitosan was used as a spacer material (antifouling agent) to help decrease the fouling due to biomass deposition. The use of chitosan as a spacer ensured higher and stabilised flow rates for extended periods of time. In initial studies 0.5 g chitosan increased the flow rate by 34 % with a resistance removal of 25 %. For 1 g chitosan these values were 130 % flow increase and 57 % resistance removal. The flow rate was optimised in relation to the chitosan amount, biomass (cell) amount and pressure. The maximum flow rate was obtained at a pressure of 40 kPa, using the minimum amount of cells (0.4 g) and a maximum amount of chitosan (1.6 g) / Thesis (M.Sc.)--North-West University, Potchefstroom Campus, 2004.

Due to recent legislation requiring the determining of the pharmacokinetic effect of both enantiomers separately, of any new racemic drug before commercialisation, much research is done to improve and optimise methods for obtaining chirally pure compounds important for the pharmaceutical industry, for example epoxide precursors. To date most experiments regarding the biocatalytic chiral separation of 1,2-epoxyoctane has been done in batch processes. The aim of this study was to optimise the enantioselective hydrolysis of 1,2-epoxyoctane by Rhodosporidiurn tondoides in both a batch and continuous process. The batch process was optimised in terms of stir speed, biomass (cell) concentration and reaction time, while the flow-through reactor (continuous process) was optimised with regards to the flow rate as a function of the pressure and the amount of chitosan and biomass in the reactor. Initial inconsistencies of epoxide concentrations in preliminary studies were found to be due to adsorption by reaction and sampling vessels, and the lower than expected solubility of 1,2- epoxyoctane (3.85 mM instead of 6 mM as reported by previous investigators). The results from the batch process suggest that as the reaction time increases, the % ee-epox increases initially, but decreases after 40 minutes. Optimum yield in terms of % ee-epox were obtained at medium stir speed (400 rpm) and biomass (cell) concentration (13 %). Below these values the % ee-epox increases with an increase in stir speed and/or biomass concentration. Above these values however, the increased stir speed and/or biomass concentration causes abrasion between cells, which negatively affects the % ee-epox. The % ee-diol reached a steady state after 10 minutes, and the effect of the different operating conditions on % ee-diol was negligible. In the flow-through reactor chitosan was used as a spacer material (antifouling agent) to help decrease the fouling due to biomass deposition. The use of chitosan as a spacer ensured higher and stabilised flow rates for extended periods of time. In initial studies 0.5 g chitosan increased the flow rate by 34 % with a resistance removal of 25 %. For 1 g chitosan these values were 130 % flow increase and 57 % resistance removal. The flow rate was optimised in relation to the chitosan amount, biomass (cell) amount and pressure. The maximum flow rate was obtained at a pressure of 40 kPa, using the minimum amount of cells (0.4 g) and a maximum amount of chitosan (1.6 g) / Thesis (M.Sc.)--North-West University, Potchefstroom Campus, 2004.

Stereochemistry and chirality are arguably two of the most important subjects pertaining to the development of new pharmaceutical drugs. Since enantiomers have the potential to encompass different pharmacological effects in biological systems, both enantiomers have to be tested for pharmacological activity. Not only has obtaining these single enantiomers become crucial, but formulation of the pure enantiomer of a drug also has the potential to contain advantages for both pharmaceutical formulation and therapeutic effect. Epoxide hydrolase is an enzyme commonly found in nature that catalyses the hydrolysis of epoxides, resulting in the formation of the corresponding vicinal diol. Over the last few years a large amount of research has been completed on these enzymes from sources such as mammals, insects, bacteria and fungi. Micro-organisms especially have enjoyed ample attention because of their abundant supply. Recently it was found that certain yeasts contain this enzyme and have the ability to enantioselectively catalyse certain hydrolysis reactions. Styrene oxides are terminal epoxides that are, due to the reactivity of the epoxide ring, useful synthons in the organic synthesis of pharmaceutical products. The first objective of this project was to synthesize three nitro derivatives of styrene oxide namely para-, meta-, and ortho-nitrostyrene oxide. Al three products were obtained from the corresponding nitrophenacyl bromide in yields of 52%, 90% and 57% respectively. The second objective was lo find a suitable yeast slrain containing the epoxide hydrolase enzyme to enantioselectively hydrolyse the synthesised products and unsubstituted styrene oxide. A screening was completed during which 410 yeast strains from more than 44 genera were tested. Epoxide hydrolase activity was found to be widespread throughout the screened yeast domain, while the genera Candida, Debaryomyces, Pichia, Rhodosporidium, Rhodotorula and Trichosporon specifically were very successful in catalysing the hydrolysis of the substrates. Rhodosporidium toruloides UOFS Y-0471 and Rhodotorula glutinis UOFS Y-0653 were chosen for further studies because of their superior enantioselectivity. The final objective was to optimise these reactions in terms of pH, temperature and substrate concentration. It was found that a pH value of 7.2 and a temperature of 45’C yielded optimal enzyme activity. Increased temperatures (45’C), however, lead to a decrease in enantioselectivity and, in the case of R. toruloides together with the substrate puranitrostyrene oxide, reversed enantioselectivity. Lower temperatures (15’C) increased enantioselectivity, resulting in a remarkable improvement from a 10% yield of the single enantiomer (45’C) to a 35% yield. Surprisingly this temperature decrease had a very small affect upon the reaction time. / Thesis (M.Sc. (Pharmaceutical Chemistry)--Potchefstroom University for Christian Higher Education, 2002.

This thesis investigated polymer properties for their application as extractants in two-phase partitioning bioreactors (TPPBs), which are intended to remove inhibitory fermentation products as they are produced. Three applications of polymer TPPB extractants were studied, followed by an investigation into poly(ether)-based polymers’ affinity toward representative target molecules, to identify properties which confer improved extraction performance. The first investigation aimed to replace a liquid extractant (silicone oil) using a block copolymer, Hytrel® 8206, in the biotransformation of indene to cis-(1S,2R)-indandiol, a chiral pharmaceutical intermediate, by Pseudomonas putida ATCC55687. The polymer simultaneously delivered substrate and removed the product and by-products to alleviate inhibition, improving operability and productivity relative to silicone oil, which could only deliver substrate. Subsequently, soft segment composition and proportion were varied in different block copolymers to selectively extract product or by-product(s) from the same biotransformation, altering the cells’ production profile. This demonstrated selective polymer extraction to help direct substrate utilization toward the product rather than by-product(s) in complex biotransformations. The next study was on absorptive extraction of a hydrophilic target molecule, 4-valerolactone, produced by recombinant Pseudomonas putida KT2440, featuring an equilibrium-limited final step. The aim was to demonstrate the first application of equilibrium-pulling using selective product absorption, improving production by 30%. Furthermore, this study showed that limited polymer water absorption is helpful to aid in extraction of hydrophilic target molecules, but high polymer water content compromises selectivity, diminishing the equilibrium-pulling effect. Finally, the effects of soft block proportion, molecular weight, and chain-end composition on affinity toward representative target molecules, carveol and carvone, were studied using commercial block copolymers and their representative homopolymer components. Target molecule affinity improved at low molecular weights in the absence of polar homopolymer end-groups. End-group polarity had an effect whose direction depended on the polarity of the target molecule, improving affinity toward a third, polar target molecule, 4-valerolactone, thereby providing a means to tailor selectivity. Crystallinity and hard segment proportion were both found to reduce uptake. This work has provided insights into the selection of polymeric TPPB absorbents by identifying polymer properties which improve affinity and selectivity toward different fermentation target molecules, especially relatively hydrophilic ones. The future design of purpose-built polymer extractants will benefit from considering these findings. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2014-01-17 13:43:54.818