Cofactor-Free, Direct Photoactivation of Enoate Reductases for the Asymmetric Reduction of C=C Bonds

Abstract

Enoate reductases from the family of old yellow enzymes (OYEs) can catalyze stereoselective trans-hydrogenation of activated C=C bonds. Their application is limited by the necessity for a continuous supply of redox equivalents such as nicotinamide cofactors [NAD(P)H]. Visible light-driven activation of OYEs through NAD(P)H-free, direct transfer of photoexcited electrons from xanthene dyes to the prosthetic flavin moiety is reported. Spectroscopic and electrochemical analyses verified spontaneous association of rose bengal and its derivatives with OYEs. Illumination of a white light-emitting-diode triggered photoreduction of OYEs by xanthene dyes, which facilitated the enantioselective reduction of C=C bonds in the absence of NADH. The photoenzymatic conversion of 2-methylcyclohexenone resulted in enantiopure (ee>99 %) (R)-2-methylcyclohexanone with conversion yields as high as 80-90 %. The turnover frequency was significantly affected by the substitution of halogen atoms in xanthene dyes.

Keywords: asymmetric reduction; enoate reductases; green chemistry; photocatalysis; redox enzymes.

Scheme 1

Illustration of the light‐driven activation of flavin‐containing TsOYE using rose bengal (RB) as a photosensitizer. Photoexcitation of the molecular photosensitizer reduces the active species of TsOYE to catalyze an enantioselective reduction of 2‐methylcyclohexenone to 2‐methylcyclohexanone.

Figure 1

A) Possible steps of enzyme‐bound FMN reduction. B) UV/Vis absorbance spectra of TsOYE‐RB mixture under visible light irradiation. C) The change in the absorbance of TsOYE at 464 nm with or without RB or TEOA. An aqueous solution containing 60 μm TsOYE, 2 μm RB, 5 mm CaCl₂, and 200 mm TEOA was irradiated by a 450 W Xenon lamp equipped with 420 nm cut‐off filter.

Figure 2

A) Spectrophotometric changes in the absorbance of RB upon addition of TsOYE. B) Cyclic voltammograms of TsOYE modified electrode in the presence or absence of RB. C) Photocurrent response of TsOYE, RB, and TsOYE with RB at an applied potential of −0.6 V (vs. Ag/AgCl). D) Proposed mechanism of electron transfer from RB to TsOYE upon visible‐light irradiation in the presence of sacrificial electron donor (TEOA).