Asymmetric Reduction of Activated Alkenes by Pentaerythritol Tetranitrate Reductase: Specificity and Control of Stereochemical Outcome by Reaction Optimisation

Abstract

We show that pentaerythritol tetranitrate reductase (PETNR), a member of the 'ene' reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated alpha,beta-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49-99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cbeta (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18-19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Calpha is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.

Figure 1

Models of the active site of PETNR containing (a) ketoisophorone 3a and (b) 2-methylmaleimide 2a overlaid with the known position of 2-cyclohexenone 4a. The position of 4a is derived from a superimposition of the 2CH-PETNR structure onto the model (pdb code 1GVQ).[14] All residues are shown as atom-coloured sticks with green, yellow, magenta and blue carbons for amino acids, FMN, substrate 3a or 2a and 4a, respectively. Predicted hydrogen bonds are indicated by red dotted lines. The figures were generated in Pymol.[29]

Figure 2

¹H NMR (400 MHz) analysis of N-phenyl-2-methylsuccinamide (1b): a) reduction in the presence of (R)-[4-²H]-NADPH (NADPD) in phosphate buffer; b) reduction in the presence of NADPH in deuterated phosphate buffer; c) a parent non-deuterated compound.

Scheme 1

Model of the postulated binding mode and mechanism of alkene reduction by PETNR. Panel a: substrates 3–6a and 15a; panel b: substrates 16–17a; panel c: substrates 1–2a; panel d: substrates 10–11a; panel e: substrates 18–19a.

Scheme 2

Stereochemical course of the reduction of maleimides 1–2a by PETNR (R=Ph or H). Two possible binding modes of 1–2a give the same stereochemical outcome: a) hydrogen at new stereogenic centre (C2) is derived from protonation step; b) from hydride addition step.