"A Study in Yellow": Investigations in the Stereoselectivity of Ene-Reductases

Abstract

Ene-reductases from the Old Yellow Enzyme (OYE) superfamily are a well-known and efficient biocatalytic alternative for the asymmetric reduction of C=C bonds. Considering the broad variety of substituents that can be tolerated, and the excellent stereoselectivities achieved, it is apparent why these enzymes are so appealing for preparative and industrial applications. Different classes of C=C bonds activated by at least one electron-withdrawing group have been shown to be accepted by these versatile biocatalysts in the last decades, affording a vast range of chiral intermediates employed in the synthesis of pharmaceuticals, agrochemicals, flavours, fragrances and fine chemicals. In order to access both enantiomers of reduced products, stereodivergent pairs of OYEs are desirable, but their natural occurrence is limited. The detailed knowledge of the stereochemical course of the reaction can uncover alternative strategies to orient the selectivity via mutagenesis, evolution, and substrate engineering. An overview of the ongoing studies on OYE-mediated bioreductions will be provided, with particular focus on stereochemical investigations by deuterium labelling.

Keywords: biocatalysis; enzymes; oxidoreductases; reduction; stereoselectivity.

Figure 1

Famous detectives at work to analyse stereochemical clues.

Scheme 1

General mechanism of OYE‐mediated bioreductions of activated C=C bonds (residues numbered according to the sequence of OYE1).

Scheme 2

Representations of the classical and flipped binding modes (adapted from Ref. [4]).

Figure 2

Eight possible paths for the bioreduction of diethyl citraconate. In all cases H^– is delivered to the β‐position from below the plane (and H⁺ to the α‐position from above the plane for anti addition, and from below the plane for syn addition). The experimentally proven option is shown as the first, indicated by black arrows.

Scheme 3

Isotopic labelling experiments to identify the activating EWG and the addition mode for diethyl citraconate as a representative example (NMR spectra adapted from Ref. [9]).

Figure 3

General model for the analysis of C=C bioreductions mediated by canonical OYEs (in all cases H⁻ is delivered from below the plane to the β‐position and H⁺ from above the plane to the α‐position).

Figure 4

Alternative strategies for enantiodivergent reductions with OYEs (reproduced with permission from Ref. [4]).