Sequential Enzymatic Conversion of α-Angelica Lactone to γ-Valerolactone through Hydride-Independent C=C Bond Isomerization

Abstract

A case of hydride-independent reaction catalyzed by flavin-dependent ene-reductases from the Old Yellow Enzyme (OYE) family was identified. α-Angelica lactone was isomerized to the conjugated β-isomer in a nicotinamide-free and hydride-independent process. The catalytic cycle of C=C bond isomerization appears to be flavin-independent and to rely solely on a deprotonation-reprotonation sequence through acid-base catalysis. Key residues in the enzyme active site were mutated and provided insight on important mechanistic features. The isomerization of α-angelica lactone by OYE2 in aqueous buffer furnished 6.3 mm β-isomer in 15 min at 30 °C. In presence of nicotinamide adenine dinucleotide (NADH), the latter could be further reduced to γ-valerolactone. This enzymatic tool was successfully applied on semi-preparative scale and constitutes a sustainable process for the valorization of platform chemicals from renewable resources.

Keywords: biobased chemicals; biocatalysis; ene-reductases; isomerization; α-angelica lactone.

Scheme 1

Versatile catalytic behavior of ene‐reductases (ER) on α,β‐unsaturated cycloalkenones and lactones. A) NADH‐independent intermolecular hydrogen transfer; B) NADH‐catalyzed intermolecular hydrogen transfer; C) stoichiometric NADH‐dependent C=C reduction. E‐value: enantiomeric ratio.

Scheme 2

Sequential biotransformation of unactivated substrates 1 a and 2 a by OYE2 from Saccharomyces cerevisiae to 1 c and 2 c, respectively.

Scheme 3

Initial proposed mechanism for the isomerization of α‐angelica lactone by OYE2. Return to histidine (His) and tyrosine (Tyr) initial states is solvent mediated (buffer pH 7.5). Asn=asparagine.