Structure‐Based Insight into the Asymmetric Bioreduction of the CC Double Bond of α,β‐Unsaturated Nitroalkenes by Pentaerythritol Tetranitrate Reductase

Abstract

Biocatalytic reduction of α-or β-alkyl-β-arylnitroalkenes provides a convenient and efficient method to prepare chiral substituted nitroalkanes. Pentaerythritol tetranitrate reductase (PETN reductase) from Enterobacter cloacae st. PB2 catalyses the reduction of nitroolefins such as 1-nitrocyclohexene (1) with steady state and rapid reaction kinetics comparable to other old yellow enzyme homologues. Furthermore, it reduces 2-aryl-1-nitropropenes (4a-d) to their equivalent (S)-nitropropanes 9a-d. The enzyme shows a preference for the (Z)-isomer of substrates 4a-d, providing almost pure enantiomeric products 9a-d (ees up to > 99%) in quantitative yield, whereas the respective (E)-isomers are reduced with lower enantioselectivity (63-89% ee) and lower product yields. 1-Aryl-2-nitropropenes (5a, b) are also reduced efficiently, but the products (R)-10 have lower optical purities. The structure of the enzyme complex with 1-nitrocyclohexene (1) was determined by X-ray crystallography, revealing two substrate-binding modes, with only one compatible with hydride transfer. Models of nitropropenes 4 and 5 in the active site of PETN reductase predicted that the enantioselectivity of the reaction was dependent on the orientation of binding of the (E)-and (Z)-substrates. This work provides a structural basis for understanding the mechanism of asymmetric bioreduction of nitroalkenes by PETN reductase.

Activated α,β‐Unsaturated Aldehydes as Substrate of Dihydroxyacetone Phosphate (DHAP)‐Dependent Aldolases in the Context of a Multienzyme System

“…Similar trends were previously observed for nitroalkenes. [16] In contrast, the activity with 2-methylcyclohexenone 5a was lower than with the respective parent ketone (4a; Table 1). The presence of a methyl substituent at Cβ (3-methylcyclohexanone 15a and 3-methylcyclopentanone 16a) leads to a significant reduction in enzyme activity (data not shown).…”

“…Moreover, for (E)-nitroolefins 18-19a the yields were not quantitative and the formation of by-products was observed, as described previously. [16] The respective 1-aryl-2-nitropropanes were obtained under the reaction conditions in almost racemic form (data not shown), compared with up to 54% ee reported for the biphasic reductions. [16] As the configuration of the newly formed stereogenic centre at Cβ depends on highly stereoselective hydride transfer from the flavin (Scheme 1d-e), the moderate ees obtained for 11b and 18-19b are likely to be the result of an enantiodivergent course for the reduction of (E) and (Z)-isomers of the substrates.…”

“…[16] The respective 1-aryl-2-nitropropanes were obtained under the reaction conditions in almost racemic form (data not shown), compared with up to 54% ee reported for the biphasic reductions. [16] As the configuration of the newly formed stereogenic centre at Cβ depends on highly stereoselective hydride transfer from the flavin (Scheme 1d-e), the moderate ees obtained for 11b and 18-19b are likely to be the result of an enantiodivergent course for the reduction of (E) and (Z)-isomers of the substrates. [23] The kinetic data for nitroalkenes showed a clear preference for the (Z)-isomers of β,β-disubstituted nitroalkenes 18a and 19a compared with their respective (E)-counterparts.…”

“…[23] The kinetic data for nitroalkenes showed a clear preference for the (Z)-isomers of β,β-disubstituted nitroalkenes 18a and 19a compared with their respective (E)-counterparts. [16] Since the reduction of both (E) and (Z)-isomers of nitroalkene 18a lead to the formation of (S)-enantiomer ( Table 2, entries 12 and 13), one cannot exclude an enzyme-driven shift in equilibrium between the (E) and (Z)-isomers towards the isomer that binds more strongly to the active site, leading to an apparent enantioconvergent course of the reduction of both isomers. The kinetic data obtained for nitroolefins [16] …”

“…[16] To obtain a like-for-like comparison with other substrates reported in this paper, we have also repeated the reductions of 2-aryl-1-nitropropenes 18-19a, using DMF (2%) to solubilise the substrates in a single phase aqueous system (Table 2, entries 12-14). Similarly to our previous study, [16] under monophasic reaction conditions, the (Z)-isomer of 18a gave the respective (S)-product quantitatively in high optical purity (95% ee, compared to 96% in the biphasic system). The reduction of (E)-isomers of nitroalkenes 18a and 19a, furnished the same (S)-enantiomer of the products in moderate enantioselectivity (69 and 64% ee, compared with 89 and 72% in the biphasic system).…”