Multi‐faceted Set‐up of a Diverse Ketoreductase Library Enables the Synthesis of Pharmaceutically‐relevant Secondary Alcohols

Abstract

Enzymes are valuable tools to introduce chirality into small molecules. Especially, ketoreductase (KRED)-catalyzed transformations of ketones to yield chiral secondary alcohols have become an established biocatalytic process step in the pharmaceutical and fine chemical industry. Development time, however, remains a critical factor in chemical process development and thus, the competitiveness of a biocatalytic reaction step is often governed by the availability of off-the-shelf enzyme libraries. To expand the biocatalytic toolbox with additional ketoreductases, we established a multi-faceted screening procedure to capture KRED diversity from different sources, such as literature, available genome data, and uncharacterized microbial strains. Overall, we built a library consisting of 51 KRED enzymes, 29 of which have never been described in literature before. Notably, 18 of the newly described enzymes exhibited anti-Prelog preference complementing the majority of ketoreductases which generally follow Prelog's rule. Analysis of the library's catalytic activity toward a chemically diverse ketone substrate set of pharmaceutical interest further highlighted the broad substrate scope and the complementing enantio-preference of the individual KREDs. Using the generated sequence-function data of the included short chain dehydrogenases in a bioinformatic analysis led to the identification of possible sequence determinants of the stereospecificity exhibited by these enzymes.

Expanding the Reactivity of Flavin-Dependent Halogenases toward Olefins via Enantioselective Intramolecular Haloetherification and Chemoenzymatic Oxidative Rearrangements

“…19 We envisioned that such alcohols could be accessed via reduction of the corresponding ketones using a ketoreductase (KRED). 34 These enzymes are NADdependent and require a reduced cofactor supplied by a cofactor regeneration system like the glucose/glucose dehydrogenase (GDH) system that we use to drive FAD reduction or by oxidation of a sacrificial alcohol like isopropanol (IPA). We therefore anticipated that KREDs could be used in a onepot cascade reaction with FDHs to enable sequential ketone reduction/bromoetherification with the respective enzymes controlling the enantioselectivity of the former and the diastereoselectivity of the latter.…”

Biocatalytic Alkylation Chemistry: Building Molecular Complexity with High Selectivity

“…Like natural enzyme families that can be rapidly tested and profiled on a broad set of substrates, [152,153] the activity profile of artificial enzyme families could be determined. This might not only eliminate the need for multiple directed evolution campaigns but also provide necessary data for machine learning models [154–156] …”

Expanding the Reactivity of Flavin Dependent Halogenases Toward Olefins via Enantioselective Intramolecular Haloetherification and Chemoenzymatic Oxidative Rearrangements

“…19 We envisioned that such alcohols could be accessed via reduction of the corresponding ketones using a ketoreductase (KRED). 34 These enzymes are NAD-dependent and require reduced cofactor supplied by a cofactor regeneration system like the glucose/glucose dehydrogenase (GDH) system that we use to drive FAD reduction or by oxidation of a sacrificial alcohol like isopropanol (IPA). We therefore anticipated that KREDs could be used in a one-pot cascade reaction with FDHs to enable sequential ketone reduction/bromoetherification with the respective enzymes controlling the enantioselectivity of the former and the diastereoselectivity of the latter.…”