“The Thiopyran Route to Polypropionates” is a synthetic strategy that involves the stepwise aldol reactions of 6 and 7a to rapidly access stereochemically complex tetrapropionate 8 and hexapropionate synthons 72 or 73. Coupling racemic 7a with any of the four enantioenriched diastereomers 8 with kinetic resolution (KR) is possible with rational design of the reaction using the 'multiplicativity' rule. Thus, any of two of the eight possible aldol adducts, 72 or 73, are selectively available from the same reactants.

Dynamic kinetic resolution (DKR) is a powerful method for obtaining compounds with high optical purity. The process relies on the combination of a kinetic resolution with an in situ racemization. In this thesis, a combination of an immobilized hydrolase and a transition metal-based racemization catalyst was employed in DKR to transform racemic alcohols and amines into enantioenriched esters and amides, respectively. In the first part the DKR of 1,2-amino alcohols with different rings sizes and N-protecting groups is described. We showed that the immobilization method used to support the lipase strongly influenced the stereoselectivity of the reaction. The second part deals with the DKR of C3-functionalized cyclic allylic alcohols affording the corresponding allylic esters in high yields and high ee’s. The protocol was also extended to include carbohydrate derivatives, leading to inversion of a hydroxyl substituted chiral center on the carbohydrate. The third part focuses on an improved method for obtaining benzylic primary amines. By using a novel, recyclable catalyst composed of Pd nanoparticles on amino-functionalized mesocellular foam, DKR could be performed at 50 °C. Moreover, Lipase PS was for the first time employed in the DKR of amines. In the fourth part DKR was applied in the total synthesis of Duloxetine, a compound used in the treatment of major depressive disorder. By performing a six-step synthesis, utilizing DKR in the enantiodetermining step, Duloxetine could be isolated in an overall yield of 37% and an ee &gt;96%. In the final part we investigated how the enantioselectivty of reactions catalyzed by Candida Antarctica lipase B for δ-substituted alkan-2-ols are influenced by water. The results showed that the enzyme displays much higher enantioselectivity in water than in anhydrous toluene. The effect was rationalized by the creation of a water mediated hydrogen bond in the active site that helps the enzyme form enantiodiscriminating binding modes. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Thesis advisor: Marc L. Snapper / Chapter 1: Brief overview of the catalytic enantioselective functionalization and kinetic resolution of alcohols Chapter 2: Kinetic resolution of syn-diols by catalytic enantioselective sulfonylation Chapter 3: Significant improvement on catalytic enantioselective silylation of syn-diols and triols through the use of a tetrazole additive / Thesis (MS) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Enantiopure epoxides are versatile compounds in the production of single enantiomer drugs, and are of high value as building blocks and intermediates in the preparation of more complex single enantiomer pharmaceuticals and agrochemicals. Epoxide hydrolases, ubiquitous enzymes in nature, can be versatile tools in the biocatalytic production of these single enantiomer epoxides due to their capability of selectively hydrolysing one enantiomer of a wide range of these compounds, and thus rendering an enantiopure epoxide and diol. The value of epoxide hydrolases for the kinetic resolution of epoxide compounds are dependant on factors such as availability, ease of production, long term stability, activity and the displayed enantioselectivity. The first objective of this study was to investigate and optimise the growth media and time for the production of two red yeasts, Rhodotorula glutinis and Rhodospondium toruloides, and their epoxide hydrolysing enzymes. Maximum and minimum epoxide hydrolase (EH) activity for R. glutinis was respectively observed with the YMvit (0,26 mM.min"1) and malt (0,17 mM.min"1) media, while peak biomass production was observed from the YM medium (64,9 mg.mL"1). For R. toruloides, the highest biomass was produced in the YM (130,8 mg.mL"1) medium, with similar epoxide hydrolase activities (average c = 0,75 ± 0,01) displayed for the YM, YMvit and malt grown biocatalysts. With varying the YM medium glucose concentration (0,5 - 2,0 %) the most biomass was produced for R. glutinis with the addition of 1,5 % glucose (60,0 + 0,9 mg.mL"1), with a slight drop in the biomass observed with the addition of 2,0% glucose (56,0 + 1,7 mg.mL"1). No significant differences in epoxide hydrolase activity was observed for the lower glucose additive concentrations (0,5 - 1,5 %), while 2,0 % (m/v) rendered a biocatalyst with almost 20 % higher activity (0,29 mM.min"1). For R. toruloides an increase in the glucose concentration lead to a significantly higher biomass production while the time needed to attain the stationary phase increased progressively from 40 to 96 hours. Almost equal activity was observed for the top three glucose concentrations (average c = 0,82 ± 0,01) at 36 hours growth time, but in all cases a decrease in the EH activity was observed during the stationary phase, with the most pronounced decrease for the 2,0 % (m/v) glucose concentration, that showed a drop in conversion of almost 62 % at 144 hours growth time. The second objective was to synthesise meta and para nitro-, methyl- and methoxystyrene oxides and the successive production of their single enantiopure epoxides through R. glutinis EH mediated kinetic resolution, and the determination of the absolute configuration of the pure residual enantiomers through VCD analysis. R. glutinis selectively hydrolysed the whole range of styrene oxide derivatives, with the highest activity displayed towards the meta substituted derivatives in the order of methyl > methoxy > nitro. m-Methylstyrene oxide reached a % e.e. of >98 within 60 minutes, with an exceptionally high yield of 42,5 %. The absolute configuration of the residual epoxide enantiomers of /n-nitro, m-methyl and m-methoxystyrene oxides were determined to be of the (S)-configuration, indicating that R. glutinis EH preferentially hydrolyses the (R)-epoxides. Thirdly, we attempted to increase the R. glutinis EH activity through the addition of hydroxypropyl-p-cyclodextrin (HPB) and to correlate the rate of chemical and R. glutinis EH mediated enzymatic hydrolysis, and the enzyme's enantioselectivity to the electronic properties of their substituents and the spatial arrangement of the substrates in relation to the EH catalytic triad of the EH active site. An increase in the HPB concentration (0 - 20 % w/v) lead to a substantial increase in both the solubility as well as enzyme activity for p-N02 (para-nitrostyrene oxide) with a significant increase in the solubility of between 2,89 and 6,28 times for the substrate range with the addition of 5 % HPB in comparison to the buffer solution. The acid induced chemical and R. glutinis EH mediated enzymatic reaction rate was correlated to both the Hammett constant as well as the Mulliken charge distributions. The Mulliken charge distribution over the protonated epoxides was correlated to the acid induced chemical hydrolysis rates, while the Mulliken charge distribution over the neutral epoxides could be correlated to the enzymatic reaction rates. An increase in the electron-donating properties of the styrene oxide substituent groups was correlated to an increase in both the chemical as well as the R. glutinis EH mediated hydrolysis reaction rates of the styrene oxide derivatives. Docking of the possible conformers of the (R)- and (S)-enantiomers of these meta and para substituted styrene oxides into the EH binding site of the closely related Aspergillus niger displayed a closer and more preferential fit of the (R)-epoxides which is the faster reacting enantiomerfor both A. niger and R. glutinis EHs. The proven relationship between R. glutinis EH activity and selectivity and the electronic properties of substituent groups, as well as the relationship between spatial arrangement of the epoxide hydrolase binding site and the enantioselectivity of the enzyme, could open up the possibility to correctly predict both the enantioselectivity as well as the activity of R. glutinis EH, and possibly other red yeasts, towards more complex epoxide substrates without the need of time consuming screenings. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

Enantiopure epoxides are versatile compounds in the production of single enantiomer drugs, and are of high value as building blocks and intermediates in the preparation of more complex single enantiomer pharmaceuticals and agrochemicals. Epoxide hydrolases, ubiquitous enzymes in nature, can be versatile tools in the biocatalytic production of these single enantiomer epoxides due to their capability of selectively hydrolysing one enantiomer of a wide range of these compounds, and thus rendering an enantiopure epoxide and diol. The value of epoxide hydrolases for the kinetic resolution of epoxide compounds are dependant on factors such as availability, ease of production, long term stability, activity and the displayed enantioselectivity. The first objective of this study was to investigate and optimise the growth media and time for the production of two red yeasts, Rhodotorula glutinis and Rhodospondium toruloides, and their epoxide hydrolysing enzymes. Maximum and minimum epoxide hydrolase (EH) activity for R. glutinis was respectively observed with the YMvit (0,26 mM.min"1) and malt (0,17 mM.min"1) media, while peak biomass production was observed from the YM medium (64,9 mg.mL"1). For R. toruloides, the highest biomass was produced in the YM (130,8 mg.mL"1) medium, with similar epoxide hydrolase activities (average c = 0,75 ± 0,01) displayed for the YM, YMvit and malt grown biocatalysts. With varying the YM medium glucose concentration (0,5 - 2,0 %) the most biomass was produced for R. glutinis with the addition of 1,5 % glucose (60,0 + 0,9 mg.mL"1), with a slight drop in the biomass observed with the addition of 2,0% glucose (56,0 + 1,7 mg.mL"1). No significant differences in epoxide hydrolase activity was observed for the lower glucose additive concentrations (0,5 - 1,5 %), while 2,0 % (m/v) rendered a biocatalyst with almost 20 % higher activity (0,29 mM.min"1). For R. toruloides an increase in the glucose concentration lead to a significantly higher biomass production while the time needed to attain the stationary phase increased progressively from 40 to 96 hours. Almost equal activity was observed for the top three glucose concentrations (average c = 0,82 ± 0,01) at 36 hours growth time, but in all cases a decrease in the EH activity was observed during the stationary phase, with the most pronounced decrease for the 2,0 % (m/v) glucose concentration, that showed a drop in conversion of almost 62 % at 144 hours growth time. The second objective was to synthesise meta and para nitro-, methyl- and methoxystyrene oxides and the successive production of their single enantiopure epoxides through R. glutinis EH mediated kinetic resolution, and the determination of the absolute configuration of the pure residual enantiomers through VCD analysis. R. glutinis selectively hydrolysed the whole range of styrene oxide derivatives, with the highest activity displayed towards the meta substituted derivatives in the order of methyl > methoxy > nitro. m-Methylstyrene oxide reached a % e.e. of >98 within 60 minutes, with an exceptionally high yield of 42,5 %. The absolute configuration of the residual epoxide enantiomers of /n-nitro, m-methyl and m-methoxystyrene oxides were determined to be of the (S)-configuration, indicating that R. glutinis EH preferentially hydrolyses the (R)-epoxides. Thirdly, we attempted to increase the R. glutinis EH activity through the addition of hydroxypropyl-p-cyclodextrin (HPB) and to correlate the rate of chemical and R. glutinis EH mediated enzymatic hydrolysis, and the enzyme's enantioselectivity to the electronic properties of their substituents and the spatial arrangement of the substrates in relation to the EH catalytic triad of the EH active site. An increase in the HPB concentration (0 - 20 % w/v) lead to a substantial increase in both the solubility as well as enzyme activity for p-N02 (para-nitrostyrene oxide) with a significant increase in the solubility of between 2,89 and 6,28 times for the substrate range with the addition of 5 % HPB in comparison to the buffer solution. The acid induced chemical and R. glutinis EH mediated enzymatic reaction rate was correlated to both the Hammett constant as well as the Mulliken charge distributions. The Mulliken charge distribution over the protonated epoxides was correlated to the acid induced chemical hydrolysis rates, while the Mulliken charge distribution over the neutral epoxides could be correlated to the enzymatic reaction rates. An increase in the electron-donating properties of the styrene oxide substituent groups was correlated to an increase in both the chemical as well as the R. glutinis EH mediated hydrolysis reaction rates of the styrene oxide derivatives. Docking of the possible conformers of the (R)- and (S)-enantiomers of these meta and para substituted styrene oxides into the EH binding site of the closely related Aspergillus niger displayed a closer and more preferential fit of the (R)-epoxides which is the faster reacting enantiomerfor both A. niger and R. glutinis EHs. The proven relationship between R. glutinis EH activity and selectivity and the electronic properties of substituent groups, as well as the relationship between spatial arrangement of the epoxide hydrolase binding site and the enantioselectivity of the enzyme, could open up the possibility to correctly predict both the enantioselectivity as well as the activity of R. glutinis EH, and possibly other red yeasts, towards more complex epoxide substrates without the need of time consuming screenings. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

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.

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

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.

No description available.

Enzymes are attractive catalysts in organic synthesis since they are efficient, selective and environmentally friendly. A large number of enzyme-catalyzed transformations have been described in the literature. If no natural enzyme can carry out a desirable reaction, one possibility is to modify an existing enzyme by protein engineering and thereby obtain a catalyst with the desired properties. In this thesis, the development of enantioselective enzymes and their use in synthetic applications is described.  In the first part of this thesis, enantioselective variants of Candida antarctica lipase A (CALA) towards α-substituted p-nitrophenyl esters were developed by directed evolution. A highly selective variant of CALA towards p-nitrophenyl 2-phenylpropanoate was developed by pairwise randomization of amino acid residues close to the active site. The E value of this variant was 276 compared to 3 for the wild type. An approach where nine residues were altered simultaneously was used to discover another highly enantioselective CALA variant (E = 100) towards an ibuprofen ester. The sterical demands of this substrate made it necessary to vary several residues at the same time in order to reach a variant with improved properties. In the second part of the thesis, a designed variant of Candida antarctica lipase B (CALB) was employed in kinetic resolution (KR) and dynamic kinetic resolution (DKR) of secondary alcohols. The designed CALB variant (W104A) accepts larger substrates compared to the wild type, and by the application of CALB W104A, the scope of these resolutions was extended. First, a DKR of phenylalkanols was developed using CALB W104A. An enzymatic resolution was combined with in situ racemization of the substrate, to yield the products in up to 97% ee. Secondly, the KR of diarylmethanols with CALB W104A was developed. By the use of diarylmethanols with two different aryl groups, highly enantioselective transformations were achieved. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows:<strong>  </strong>Paper 5: Submitted.