Asymmetric Monoreduction of α,β-Dicarbonyls to α-Hydroxy Carbonyls by Ene Reductases

Abstract

Ene reductases (EREDs) catalyze asymmetric reduction with exquisite chemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking other types of reactivities toward oxime reduction and reductive C-C bond formation. Exploring nontypical reactions can further expand the biocatalytic knowledgebase, and evidence alludes to yet another variant reaction where flavin mononucleotide (FMN)-bound ERs from the old yellow enzyme family (OYE) have unconventional activity with α,β-dicarbonyl substrates. In this study, we demonstrate the nonconventional stereoselective monoreduction of α,β-dicarbonyl to the corresponding chiral hydroxycarbonyl, which are valuable building blocks for asymmetric synthesis. We explored ten α,β-dicarbonyl aliphatic, cyclic, or aromatic compounds and tested their reduction with five OYEs and one nonflavin-dependent double bond reductase (DBR). Only GluER reduced aliphatic α,β-dicarbonyls, with up to 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with an R-selectivity of 83% ee. The best substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, with 91% conversion to phenylacetylcarbinol using OYE3 with R-selectivity >99.9% ee. Michaelis-Menten kinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover k _cat of 0.71 ± 0.03 s^-1 and a K _m of 2.46 ± 0.25 mM. Twenty-four EREDs from multiple classes of OYEs and DBRs were further screened on 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like) have the best overall selectivity and conversion. EPR studies detected no radical signal, whereas NMR studies with deuterium labeling indicate proton incorporation at the benzylic carbonyl carbon from the solvent and not the FMN hydride. A crystal structure of OYE2 with 1.5 Å resolution was obtained, and docking studies showed a productive pose with the substrate.

Figure 1

Simplified schematic representation of ERED-catalyzed reductions (left) via hydride transfer from reduced FMN and protonation via a tyrosine (right): (a) the native alkene reduction, (b) the previously observed oxime reduction, and (c) the currently examined vicinal dicarbonyl monoreduction.

Figure 2

Biocatalytic approaches to produce α-hydroxy carbonyl compounds. DKR (dynamic kinetic resolution) with lipases; reduction by dehydrogenase such as BDH (2,3-butanediol dehydrogenase or acetoin reductase such as BudC) and other ADHs; aldol condensation via lyases including transketolase, MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase), and an oxidase-lyase cascade. Here, we show the state of art for biocatalytic pathways (black) and our added ERED approach. References (−32).

Figure 3

Products 1–10b–c of ERED-catalyzed selective monoreduction of α,β-dicarbonyl substrates. Conditions: 1.1 eq. NADPH, 10 mM 1a–10a, 2% v/v DMSO, 5 μM ERED, 50 mM MOPS-NaOH pH 7.0, 6 h, 30 °C, 900 rpm, 0.5 mL, average of duplicates. EREDS screened: GluER, TsOYE, OYE3, OYE2, YqjM and NtDBR. Conversions and ee values were measured on (chiral) GC. 7b and 8c were additionally measured on (chiral) HPLC. Full details are in Table S3. Substrate and product names: 1a 3,4-hexanedione, 1b 4-hydroxyhexan-3-one, 2a 2,3-hexanedione, 2b 2-hydroxyhexan-3-one, 3a 2,3-heptanedione, 3b 2-hydroxyheptan-3-one, 4a 2,3-pentanedione, 4b 2-hydroxypentan-3-one, 5a 2,3-butanedione, 5b 3-hydroxybutan-2-one, 6a 1,2-cyclohexanedione, 6b 2-hydroxycyclohexanone, 7a 1-phenyl-1,2-propanedione, 7b phenylacetylcarbinol, 7c 2-hydroxy-1-phenylpropan-1-one, 8a phenylglyoxal, 8b 2-hydroxy-2-phenyl-acetaldehyde, 8c 2-hydroxyacetophenone, 9a ethylbenzoylformate, 9b ethyl 2-hydroxy-2-phenylacetate, 10a benzoyl formic acid, and 10b 2-hydroxy-2-phenylacetic acid.

Figure 4

ERED screening of 1-phenyl-1,2-propanedione 7a. Conditions: 5 μM ERED or 2 mg/mL for the JM kit, 11 mM NADPH, 10 mM 1-phenyl-1,2-propanedione, 6 h, 30 °C, 900 rpm, 2% v/v DMSO, 0.5 mL, 50 mM MOPS-NaOH pH 7.0, average of duplicate experiments measured on HPLC at 210 nm. The OYE classes listed are Ia, Ib, Ic, II, and III. JM: Johnson Matthey ERED kit EZK002. A scientific color map was used to ensure accurate data representation and inclusivity for readers with color-vision deficiencies.

Figure 5

OYE3-catalyzed monoreduction of 7a to 7b with (a) dideuterated cofactor [4-²H]-BNAH in buffer (50 mM MOPS-NaOH pH 7.0); (b) BNAH in deuterated buffer (50 mM MOPS-NaOD pD 7.0); (c, d) control reactions without enzyme; (e) reaction with OYE3 Y197F. Conditions: 60 μM OYE3, 30 mM 1-phenyl-1,2-propanedione, 30 mM cofactor, 4.5 h at 30 °C, anaerobic.

Figure 6

Docking study of 7a (green) in OYE2 (PDB ID 9FH7). Distances are shown as dashed lines in Å.