Stereoselective conversion of α-angelica lactone to (R)-γ-valerolactone in a one-pot two-step enzymatic cascade

Ene-reductases from the Old Yellow Enzyme (OYE) family are versatile flavin-dependent catalysts that have been applied to the asymmetric trans-reduction of a broad range of activated C=C-bonds at the expense of NAD(P)H cofactor. Alternative catalytic activities of OYE homologues were also observed2 such as the hydrideindependent isomerization of non-activated C=C-bonds. In this work, we focused on the design of a two-step enzymatic cascade combining ene-reductase catalyzed stereoselective isomerization of α-angelica lactone to β-angelica lactone, with subsequent reduction by a suitable OYE homologue of the enantioenriched β-isomer to γ-valerolactone. We here report the first case of stereoselective hydride-independent isomerization of α-angelica lactone to (R)-β-angelica lactone catalyzed by a new ene-reductase from a photosynthetic organism (ER1). The cascade toward formation of (R)-γ-valerolactone included a second ene-reductase (ER2) for the following reductive step (Scheme 1). Key parameters were identified that allowed the sequential reaction to run in one-pot, while avoiding substrate/product spontaneous degradation (e.g, pH, presence of co-solvents, reaction time). In the final design, a one-pot three-enzyme system was set-up, including glucose dehydrogenase for cofactor regeneration, which led to the formation of 3.5 mM (R)-γ-valerolactone in ~92% ee. Preliminary results from co-crystallization of ER1 in the presence of α-angelica lactone will also be presented. In summary, the combination of two OYE homologues in a one-pot cascade allowed the formal asymmetric reduction of non-activated C=C-bonds. The isomerization-reduction sequence was applied to α-angelica lactone and furnished (R)-γ-valerolactone in high enantiopurity.