Asymmetric Monoreduction\nof α,β-Dicarbonyls\nto α‑Hydroxy Carbonyls by Ene Reductases

Ene reductases (EREDs)\ncatalyze asymmetric reduction\nwith exquisite\nchemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking\nother types of reactivities toward oxime reduction and reductive C–C\nbond formation. Exploring nontypical reactions can further expand\nthe biocatalytic knowledgebase, and evidence alludes to yet another\nvariant reaction where flavin mononucleotide (FMN)-bound ERs from\nthe old yellow enzyme family (OYE) have unconventional activity with\nα,β-dicarbonyl substrates. In this study, we demonstrate\nthe nonconventional stereoselective monoreduction of α,β-dicarbonyl\nto the corresponding chiral hydroxycarbonyl, which are valuable building\nblocks for asymmetric synthesis. We explored ten α,β-dicarbonyl\naliphatic, cyclic, or aromatic compounds and tested their reduction\nwith five OYEs and one nonflavin-dependent double bond reductase (DBR).\nOnly GluER reduced aliphatic α,β-dicarbonyls, with up\nto 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with\nan <i>R</i>-selectivity of 83% <i>ee</i>. The\nbest substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione,\nwith 91% conversion to phenylacetylcarbinol using OYE3 with <i>R</i>-selectivity >99.9% <i>ee</i>. Michaelis–Menten\nkinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover <i>k</i><sub>cat</sub> of 0.71 ± 0.03 s<sup>–1</sup> and a <i>K</i><sub>m</sub> of 2.46 ± 0.25 mM. Twenty-four\nEREDs from multiple classes of OYEs and DBRs were further screened\non 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like)\nhave the best overall selectivity and conversion. EPR studies detected\nno radical signal, whereas NMR studies with deuterium labeling indicate\nproton incorporation at the benzylic carbonyl carbon from the solvent\nand not the FMN hydride. A crystal structure of OYE2 with 1.5 Å\nresolution was obtained, and docking studies showed a productive pose\nwith the substrate.