Efficient synthesis of an apremilast precursor and chiral ß-hydroxy sulfones via ketoreductase-catalyzed asymmetric reduction.

Ketoreductase (KRED)-catalyzed asymmetric reduction of prochiral ketones is an attractive method to synthesize chiral alcohols. Herein, two KREDs LfSDR1-V186A/E141I and CgKR1-F92I with complementary stereopreference were identified towards reduction of apremilast prochiral ketone intermediate 1a. LfSDR1-V186A/E141I exhibited >99% conversion and 99.2% ee yielding an apremilast chiral alcohol intermediate ((R)-2a) at 50 g L-1 substrate loading. Furthermore, we investigated the substrate scope of ß-keto sulfones by using LfSDR1-V186A/E141I and CgKR1-F92I to produce both enantiomers of the corresponding ß-hydroxy sulfones, with good-to-excellent conversion (up to >99%) and enantioselectivity (up to 99.9% ee) being obtained in most cases. Finally, the gram-scale synthesis of (R)-2a was performed by employing the crude enzyme of LfSDR1-V186A/E141I and BsGDH to afford the desired enantiomer with >99% conversion, 85.9% isolated yield and 99.2% ee. This study presents a biocatalytic strategy to synthesize chiral ß-hydroxy sulfones.

Identification of four ene reductases and their preliminary exploration in the asymmetric synthesis of (R)-dihydrocarvone and (R)-profen derivatives.

The ene reductases (ERs) from the old yellow enzymes (OYEs) family have the ability to reduce activated alkenes to generate up to two stereocenters, therefore they have been received extensive attention as powerful biocatalysts. In this study, through gene mining, four ERs were identified from the genomes of Ensifer adhaerens, Pseudomonas fluorescens, and Pseudomonas veronil. The biocatalytic properties of these four ERs were identified, and their applications in the synthesis process of dihydrocarvone and profen derivatives were further evaluated. Among them, three ERs (EaER2, PvER1, and PvER2) belonging to the classic OYEs showed the best catalytic activity at 30 °C and pH 7.0 (100 mM potassium phosphate buffer) and the PfER2, which belongs to the thermophilic-like OYEs exhibited the best catalytic at 40 °C and pH 7.0 (100 mM potassium phosphate buffer). When exploring the influence of organic solvents on the catalytic efficiency, it was found that the four ERs were more sensitive to toluene and had tolerance to several other selected organic solvents. In addition, EaER2, PfER2, PvER1 and PvER2 showed excellent catalytic activity toward carvone, and the stereoselectivity of PvER2 toward carvone could reach up to 88.7 % de. EaER2 and PfER2 can catalyze the synthesis of a variety of profen derivatives with a stereoselectivity over 99 % ee. Moreover, through homology modeling and molecular docking, we preliminarily explained the mechanism of catalytic activity and stereoselectivity of the four ERs, which provided a solid base on the rational design of their stereo-preference in the future. The discovery of EaER2, PfER2, PvER1, and PvER2 provides four new enzyme sources for the study of the OYEs family and enriches the biocatalytic toolbox of ERs. Our exploration of the enzymatic properties of these four ERs will provide the sufficient data basis for future research and industrialization progress.

Old yellow enzymes: structures and structure-guided engineering for stereocomplementary bioreduction.

Since the first discovery of old yellow enzyme 1 (OYE1) from Saccharomyces pastorianus in 1932, biocatalytic asymmetric reduction of activated alkenes by OYEs has become a valuable reaction in organic synthesis. To access stereocomplementary C=C-bond bioreduction, the mining of novel OYEs and especially the protein engineering of existing OYEs have been performed, which successfully achieved the stereocomplementary reduction in several cases and further raise the potential of applications. In this review, we analyzed the structures, active sites, and substrate recognition of OYEs, which are the bases for their substrate specificity and stereospecificity. Sequence similarity network of OYEs superfamily was also constructed to investigate the scope of characterized OYEs. The structure-guided engineering to switch the stereoselectivity of OYEs and thus access stereocomplementary bioreduction over the last decade (2009-2020) was then reviewed and discussed, which might give new insights into the mining and engineering of related biocatalysts. KEY POINTS: • The sequence similarity network of OYEs superfamily was constructed and annotated. • The structures and active sites of OYEs from different classes were compared. • "Left/right" binding mode was used to explain the stereopreferences of OYEs. • Structure-guided engineering of OYEs to switch their stereoselectivity was reviewed.