CP
C.E. Paul
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1
Enzymes offer a transformative solution to traditional chemical catalysts, providing both highly selective and highly pure compounds for the fine chemical, pharmaceutical, and insecticide industries. Despite significant progress, the enzymatic toolbox remains somewhat limited, spurring scientists in the biocatalysis field to strive for the expansion of chemical reactivities. The aim of this thesis was to study the Old Yellow Enzymes (OYEs) chemical versatility, in an attempt to broaden their reaction portfolio. This work is organized into five research chapters: four focusing on the chemical reactivities of OYE, and the fifth on a scaled-up reaction. We begin with an introduction outlining the state-of-the-art of OYEs and conclude with a conclusion and outlook. A detailed overview of each chapter is provided in the following paragraphs.
Chapter one provides an overview of our current understanding of OYEs, covering their history, physiological roles, classification among enzymes, structural characteristics, coenzymes, chemical reactivities, and their potential applications within industry.
In chapter two, we reveal how versatile OYEs are, by exploring an unknown reactivity, the stereoselective monoreduction of α,β-dicarbonyls towards chiral α-hydroxycarbonyls. We investigated ten aromatic, cyclic, aliphatic α,β-dicarbonyl compounds and evaluated their reduction using five OYEs and one flavin-independent double bond reductase (DBR). The most effective substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, which was converted to phenylacetylcarbinol with 91% conversion using OYE3 (R-selectivity >99.9% ee).
In chapter three, we continue to highlight OYEs’ versatility by yet another reactivity, the semireduction of allenes. Six activated allene substrates were screened against eighteen enzymes, including sixteen OYEs and two DBRs. The best results occurred using a class I OYE, PETNR, with 99% conversion of 10 mM ethyl-2,3-pentadienoate to ethyl-pent-3-enoate (E:Z ratio, 49:51). High selectivity was observed with class II OYE3 using methyl 2-methyl-2,3-pentadienoate as a substrate (81% conversion, E:Z ratio, 11:89), as well as with another class II OYE, EBP1 and ethyl 2-methyl-2,3-butadienoate (87% conversion, 97% ee).
Chapter four covers our re-examination of OYEs’ oxidative reaction, developing a new method for selective desaturation without requiring high temperatures. We show that by a simple pH adjustment, the active site tyrosine is deprotonated and serves as a catalytic base. Several OYEs and substrates were screened to demonstrate this desaturation method. This study expands the range of biocatalytic applications for OYEs, introducing an elegant approach to synthesizing chiral α,β-unsaturated carbonyl compounds.
In chapter five we further examined the intricacies of oxidation, by assessing whether redox potential influences desaturation. We measured the redox midpoint potential of eleven OYEs and their mutants from various classes, focusing on specific active site mutations that might shed light on desaturase activity. Our findings revealed a range of redox potentials across the different OYE classes, but no clear correlation between desaturation activity and redox potential. We examined the active site’s threonine/cysteine near the flavin N5 position and the proton-donating tyrosine with mutant enzymes to understand their role in desaturation.
In chapter six we demonstrate that OYEs are well suited for industrial use, by carrying out a 150 g/L scale-up for monoterpene asymmetric reduction. Until now, OYEs have rarely been applied in scale-up reactions, with limited turnover numbers of 102-104. We present a preparative scale using the thermostable OYE from Thermus scotoductus (TsOYE) for the asymmetric reduction of activated alkenes achieving a record turnover number of 123,000 with 1 M of (S)-carvone (98% conversion, 90% isolated yield) towards product (2R,5S)-dihydrocarvone with a diastereomeric excess of 92% (>99% ee).
In general this work advances the understanding of the biocatalytic reactivity of the OYE family, demonstrating their capacity to catalyze diverse and novel chemical reactions towards industrial applications.
...
Chapter one provides an overview of our current understanding of OYEs, covering their history, physiological roles, classification among enzymes, structural characteristics, coenzymes, chemical reactivities, and their potential applications within industry.
In chapter two, we reveal how versatile OYEs are, by exploring an unknown reactivity, the stereoselective monoreduction of α,β-dicarbonyls towards chiral α-hydroxycarbonyls. We investigated ten aromatic, cyclic, aliphatic α,β-dicarbonyl compounds and evaluated their reduction using five OYEs and one flavin-independent double bond reductase (DBR). The most effective substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, which was converted to phenylacetylcarbinol with 91% conversion using OYE3 (R-selectivity >99.9% ee).
In chapter three, we continue to highlight OYEs’ versatility by yet another reactivity, the semireduction of allenes. Six activated allene substrates were screened against eighteen enzymes, including sixteen OYEs and two DBRs. The best results occurred using a class I OYE, PETNR, with 99% conversion of 10 mM ethyl-2,3-pentadienoate to ethyl-pent-3-enoate (E:Z ratio, 49:51). High selectivity was observed with class II OYE3 using methyl 2-methyl-2,3-pentadienoate as a substrate (81% conversion, E:Z ratio, 11:89), as well as with another class II OYE, EBP1 and ethyl 2-methyl-2,3-butadienoate (87% conversion, 97% ee).
Chapter four covers our re-examination of OYEs’ oxidative reaction, developing a new method for selective desaturation without requiring high temperatures. We show that by a simple pH adjustment, the active site tyrosine is deprotonated and serves as a catalytic base. Several OYEs and substrates were screened to demonstrate this desaturation method. This study expands the range of biocatalytic applications for OYEs, introducing an elegant approach to synthesizing chiral α,β-unsaturated carbonyl compounds.
In chapter five we further examined the intricacies of oxidation, by assessing whether redox potential influences desaturation. We measured the redox midpoint potential of eleven OYEs and their mutants from various classes, focusing on specific active site mutations that might shed light on desaturase activity. Our findings revealed a range of redox potentials across the different OYE classes, but no clear correlation between desaturation activity and redox potential. We examined the active site’s threonine/cysteine near the flavin N5 position and the proton-donating tyrosine with mutant enzymes to understand their role in desaturation.
In chapter six we demonstrate that OYEs are well suited for industrial use, by carrying out a 150 g/L scale-up for monoterpene asymmetric reduction. Until now, OYEs have rarely been applied in scale-up reactions, with limited turnover numbers of 102-104. We present a preparative scale using the thermostable OYE from Thermus scotoductus (TsOYE) for the asymmetric reduction of activated alkenes achieving a record turnover number of 123,000 with 1 M of (S)-carvone (98% conversion, 90% isolated yield) towards product (2R,5S)-dihydrocarvone with a diastereomeric excess of 92% (>99% ee).
In general this work advances the understanding of the biocatalytic reactivity of the OYE family, demonstrating their capacity to catalyze diverse and novel chemical reactions towards industrial applications.
...
Enzymes offer a transformative solution to traditional chemical catalysts, providing both highly selective and highly pure compounds for the fine chemical, pharmaceutical, and insecticide industries. Despite significant progress, the enzymatic toolbox remains somewhat limited, spurring scientists in the biocatalysis field to strive for the expansion of chemical reactivities. The aim of this thesis was to study the Old Yellow Enzymes (OYEs) chemical versatility, in an attempt to broaden their reaction portfolio. This work is organized into five research chapters: four focusing on the chemical reactivities of OYE, and the fifth on a scaled-up reaction. We begin with an introduction outlining the state-of-the-art of OYEs and conclude with a conclusion and outlook. A detailed overview of each chapter is provided in the following paragraphs.
Chapter one provides an overview of our current understanding of OYEs, covering their history, physiological roles, classification among enzymes, structural characteristics, coenzymes, chemical reactivities, and their potential applications within industry.
In chapter two, we reveal how versatile OYEs are, by exploring an unknown reactivity, the stereoselective monoreduction of α,β-dicarbonyls towards chiral α-hydroxycarbonyls. We investigated ten aromatic, cyclic, aliphatic α,β-dicarbonyl compounds and evaluated their reduction using five OYEs and one flavin-independent double bond reductase (DBR). The most effective substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, which was converted to phenylacetylcarbinol with 91% conversion using OYE3 (R-selectivity >99.9% ee).
In chapter three, we continue to highlight OYEs’ versatility by yet another reactivity, the semireduction of allenes. Six activated allene substrates were screened against eighteen enzymes, including sixteen OYEs and two DBRs. The best results occurred using a class I OYE, PETNR, with 99% conversion of 10 mM ethyl-2,3-pentadienoate to ethyl-pent-3-enoate (E:Z ratio, 49:51). High selectivity was observed with class II OYE3 using methyl 2-methyl-2,3-pentadienoate as a substrate (81% conversion, E:Z ratio, 11:89), as well as with another class II OYE, EBP1 and ethyl 2-methyl-2,3-butadienoate (87% conversion, 97% ee).
Chapter four covers our re-examination of OYEs’ oxidative reaction, developing a new method for selective desaturation without requiring high temperatures. We show that by a simple pH adjustment, the active site tyrosine is deprotonated and serves as a catalytic base. Several OYEs and substrates were screened to demonstrate this desaturation method. This study expands the range of biocatalytic applications for OYEs, introducing an elegant approach to synthesizing chiral α,β-unsaturated carbonyl compounds.
In chapter five we further examined the intricacies of oxidation, by assessing whether redox potential influences desaturation. We measured the redox midpoint potential of eleven OYEs and their mutants from various classes, focusing on specific active site mutations that might shed light on desaturase activity. Our findings revealed a range of redox potentials across the different OYE classes, but no clear correlation between desaturation activity and redox potential. We examined the active site’s threonine/cysteine near the flavin N5 position and the proton-donating tyrosine with mutant enzymes to understand their role in desaturation.
In chapter six we demonstrate that OYEs are well suited for industrial use, by carrying out a 150 g/L scale-up for monoterpene asymmetric reduction. Until now, OYEs have rarely been applied in scale-up reactions, with limited turnover numbers of 102-104. We present a preparative scale using the thermostable OYE from Thermus scotoductus (TsOYE) for the asymmetric reduction of activated alkenes achieving a record turnover number of 123,000 with 1 M of (S)-carvone (98% conversion, 90% isolated yield) towards product (2R,5S)-dihydrocarvone with a diastereomeric excess of 92% (>99% ee).
In general this work advances the understanding of the biocatalytic reactivity of the OYE family, demonstrating their capacity to catalyze diverse and novel chemical reactions towards industrial applications.
Chapter one provides an overview of our current understanding of OYEs, covering their history, physiological roles, classification among enzymes, structural characteristics, coenzymes, chemical reactivities, and their potential applications within industry.
In chapter two, we reveal how versatile OYEs are, by exploring an unknown reactivity, the stereoselective monoreduction of α,β-dicarbonyls towards chiral α-hydroxycarbonyls. We investigated ten aromatic, cyclic, aliphatic α,β-dicarbonyl compounds and evaluated their reduction using five OYEs and one flavin-independent double bond reductase (DBR). The most effective substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, which was converted to phenylacetylcarbinol with 91% conversion using OYE3 (R-selectivity >99.9% ee).
In chapter three, we continue to highlight OYEs’ versatility by yet another reactivity, the semireduction of allenes. Six activated allene substrates were screened against eighteen enzymes, including sixteen OYEs and two DBRs. The best results occurred using a class I OYE, PETNR, with 99% conversion of 10 mM ethyl-2,3-pentadienoate to ethyl-pent-3-enoate (E:Z ratio, 49:51). High selectivity was observed with class II OYE3 using methyl 2-methyl-2,3-pentadienoate as a substrate (81% conversion, E:Z ratio, 11:89), as well as with another class II OYE, EBP1 and ethyl 2-methyl-2,3-butadienoate (87% conversion, 97% ee).
Chapter four covers our re-examination of OYEs’ oxidative reaction, developing a new method for selective desaturation without requiring high temperatures. We show that by a simple pH adjustment, the active site tyrosine is deprotonated and serves as a catalytic base. Several OYEs and substrates were screened to demonstrate this desaturation method. This study expands the range of biocatalytic applications for OYEs, introducing an elegant approach to synthesizing chiral α,β-unsaturated carbonyl compounds.
In chapter five we further examined the intricacies of oxidation, by assessing whether redox potential influences desaturation. We measured the redox midpoint potential of eleven OYEs and their mutants from various classes, focusing on specific active site mutations that might shed light on desaturase activity. Our findings revealed a range of redox potentials across the different OYE classes, but no clear correlation between desaturation activity and redox potential. We examined the active site’s threonine/cysteine near the flavin N5 position and the proton-donating tyrosine with mutant enzymes to understand their role in desaturation.
In chapter six we demonstrate that OYEs are well suited for industrial use, by carrying out a 150 g/L scale-up for monoterpene asymmetric reduction. Until now, OYEs have rarely been applied in scale-up reactions, with limited turnover numbers of 102-104. We present a preparative scale using the thermostable OYE from Thermus scotoductus (TsOYE) for the asymmetric reduction of activated alkenes achieving a record turnover number of 123,000 with 1 M of (S)-carvone (98% conversion, 90% isolated yield) towards product (2R,5S)-dihydrocarvone with a diastereomeric excess of 92% (>99% ee).
In general this work advances the understanding of the biocatalytic reactivity of the OYE family, demonstrating their capacity to catalyze diverse and novel chemical reactions towards industrial applications.
Chiral amines are valuable compounds in the pharmaceutical industry. These valuable compounds have been made via organocatalysis, metallocatalysis but in the last two decades also via biocatalysis. Enzymes are highly selective, making them attractive catalysts to target chiral amine synthesis. The aim of this dissertation was to apply different biocatalytic pathways with oxidoreductases to synthesize chiral amines.
...
Chiral amines are valuable compounds in the pharmaceutical industry. These valuable compounds have been made via organocatalysis, metallocatalysis but in the last two decades also via biocatalysis. Enzymes are highly selective, making them attractive catalysts to target chiral amine synthesis. The aim of this dissertation was to apply different biocatalytic pathways with oxidoreductases to synthesize chiral amines.
Producing chiral amines
A bi-enzymatic cascade involving Old Yellow Enzymes and amine dehydrogenases
Chiral amines are valuable compounds for their use as building blocks for pharmaceutical and fine chemical industries. Previously, chiral amines were made with metals as catalysts, but these are unsustainable and difficult to remove from the product. Organocatalysis is a sustainable alternative, but requires chiral ligands, which are costly. Over the last decade, biocatalytic methods have been developed as well. Our goal was to produce chiral amines with our designed bi-enzymatic cascades, containing an ene reductase and amine dehydrogenase. We produced and purified ene reductases and assayed the asymmetric reduction of our proposed substrate scope, consisting of unsaturated ketones and aldehydes. We also investigated the impact of multiple reaction conditions on these performances that are optimal for amine dehydrogenases. Starting with 10 mM of substrate, we obtained concentrations up to 9.7 mM amine. Also, we obtained 3-methylcyclohexylamine with an enantiomeric and diastereomeric excess up to 99%. Therefore, we conclude that this bi-enzymatic cascade is capable of producing chiral amines with both high enantiomeric and diastereomeric excess. Further research to perform cascades on a larger scale is recommended.
...
Chiral amines are valuable compounds for their use as building blocks for pharmaceutical and fine chemical industries. Previously, chiral amines were made with metals as catalysts, but these are unsustainable and difficult to remove from the product. Organocatalysis is a sustainable alternative, but requires chiral ligands, which are costly. Over the last decade, biocatalytic methods have been developed as well. Our goal was to produce chiral amines with our designed bi-enzymatic cascades, containing an ene reductase and amine dehydrogenase. We produced and purified ene reductases and assayed the asymmetric reduction of our proposed substrate scope, consisting of unsaturated ketones and aldehydes. We also investigated the impact of multiple reaction conditions on these performances that are optimal for amine dehydrogenases. Starting with 10 mM of substrate, we obtained concentrations up to 9.7 mM amine. Also, we obtained 3-methylcyclohexylamine with an enantiomeric and diastereomeric excess up to 99%. Therefore, we conclude that this bi-enzymatic cascade is capable of producing chiral amines with both high enantiomeric and diastereomeric excess. Further research to perform cascades on a larger scale is recommended.