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Chiral amine synthesis using ω-transaminases: an amine donor that displaces equilibria and enables high-throughput screening.

Green AP, Turner NJ, O'Reilly E - Angew. Chem. Int. Ed. Engl. (2014)

Bottom Line: The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition.This operationally simple method is compatible with the most widely used (R)- and (S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone).Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN (UK). anthony.green@manchester.ac.uk.

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Selected examples of widely utilized conditions for small/medium-scale (a) and large-scale (b) processes that employ ω-TAs.
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fig01: Selected examples of widely utilized conditions for small/medium-scale (a) and large-scale (b) processes that employ ω-TAs.

Mentions: The structural simplicity and broad availability of prochiral ketones make these structures attractive starting materials for the synthesis of chiral building blocks. For example, the application of ketoreductases is currently one of the most common strategies employed for chiral alcohol synthesis.[11] The analogous asymmetric reductive amination of ketones represents a significant challenge in organic synthesis and has been highlighted as an extremely desirable transformation for use in the pharmaceutical industry.[1] ω-TAs are a family of pyridoxal-5′-phosphate (PLP)-dependent enzymes that require a sacrificial amine donor to mediate the reversible conversion of prochiral ketones into the corresponding optically pure amines.[4] The broad substrate scope and high levels of regio- and stereoselectivity associated with these enzymes make them ideal biocatalysts for chiral amine synthesis in research laboratories and in manufacturing processes. This point is highlighted by the recent application of an engineered (R)-selective ω-TA in a biocatalytic process for the large-scale production of the anti-diabetic API sitagliptin.[10a] Despite the enormous potential of ω-TAs, fundamental challenges associated with severe by-product inhibition and with displacing unfavorable equilibrium positions towards product formation have prevented the widespread application of these biocatalysts.[12a] The use of amine donors in large excess combined with the in situ removal of ketone by-products is generally required to achieve high yields of the desired amines (Figure 1). The most widely employed strategy uses alanine as the amine donor and relies on combinations of expensive, cofactor-dependent enzymes for pyruvate removal (Figure 1 a).[12] An elegant alternative strategy was recently described in which the ketone by-product is effectively removed by spontaneous conversion into the more stable phenol tautomer.[13] Unfortunately, the application of these approaches for scalable amine synthesis is limited by the high costs associated with their use. The preferred method for large-scale production currently involves the use of a significant excess of isopropyl amine donor in combination with the technically challenging removal of the acetone by-product by evaporation (Figure 1 bii).[10a, 14] A further challenge that complicates the development of efficient ω-TA-mediated processes is the limited availability of simple high-throughput screening methods to identify new enzymes and to evaluate large libraries of engineered variants for enhanced activity, selectivity, and stability under the required reaction conditions.[15] Herein, we describe the application of a non-chiral, low-cost amine donor that serves the dual function of efficiently displacing unfavorable reaction equilibria towards product formation whilst providing a substrate-independent high-throughput colorimetric screening method to detect desired ω-TA activity.


Chiral amine synthesis using ω-transaminases: an amine donor that displaces equilibria and enables high-throughput screening.

Green AP, Turner NJ, O'Reilly E - Angew. Chem. Int. Ed. Engl. (2014)

Selected examples of widely utilized conditions for small/medium-scale (a) and large-scale (b) processes that employ ω-TAs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4497610&req=5

fig01: Selected examples of widely utilized conditions for small/medium-scale (a) and large-scale (b) processes that employ ω-TAs.
Mentions: The structural simplicity and broad availability of prochiral ketones make these structures attractive starting materials for the synthesis of chiral building blocks. For example, the application of ketoreductases is currently one of the most common strategies employed for chiral alcohol synthesis.[11] The analogous asymmetric reductive amination of ketones represents a significant challenge in organic synthesis and has been highlighted as an extremely desirable transformation for use in the pharmaceutical industry.[1] ω-TAs are a family of pyridoxal-5′-phosphate (PLP)-dependent enzymes that require a sacrificial amine donor to mediate the reversible conversion of prochiral ketones into the corresponding optically pure amines.[4] The broad substrate scope and high levels of regio- and stereoselectivity associated with these enzymes make them ideal biocatalysts for chiral amine synthesis in research laboratories and in manufacturing processes. This point is highlighted by the recent application of an engineered (R)-selective ω-TA in a biocatalytic process for the large-scale production of the anti-diabetic API sitagliptin.[10a] Despite the enormous potential of ω-TAs, fundamental challenges associated with severe by-product inhibition and with displacing unfavorable equilibrium positions towards product formation have prevented the widespread application of these biocatalysts.[12a] The use of amine donors in large excess combined with the in situ removal of ketone by-products is generally required to achieve high yields of the desired amines (Figure 1). The most widely employed strategy uses alanine as the amine donor and relies on combinations of expensive, cofactor-dependent enzymes for pyruvate removal (Figure 1 a).[12] An elegant alternative strategy was recently described in which the ketone by-product is effectively removed by spontaneous conversion into the more stable phenol tautomer.[13] Unfortunately, the application of these approaches for scalable amine synthesis is limited by the high costs associated with their use. The preferred method for large-scale production currently involves the use of a significant excess of isopropyl amine donor in combination with the technically challenging removal of the acetone by-product by evaporation (Figure 1 bii).[10a, 14] A further challenge that complicates the development of efficient ω-TA-mediated processes is the limited availability of simple high-throughput screening methods to identify new enzymes and to evaluate large libraries of engineered variants for enhanced activity, selectivity, and stability under the required reaction conditions.[15] Herein, we describe the application of a non-chiral, low-cost amine donor that serves the dual function of efficiently displacing unfavorable reaction equilibria towards product formation whilst providing a substrate-independent high-throughput colorimetric screening method to detect desired ω-TA activity.

Bottom Line: The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition.This operationally simple method is compatible with the most widely used (R)- and (S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone).Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN (UK). anthony.green@manchester.ac.uk.

Show MeSH
Related in: MedlinePlus