Selective Activation of C-H Bonds in a Cascade Process Combining Photochemistry and Biocatalysis

Abstract

Selective oxyfunctionalizations of inert C-H bonds can be achieved under mild conditions by using peroxygenases. This approach, however, suffers from the poor robustness of these enzymes in the presence of hydrogen peroxide as the stoichiometric oxidant. Herein, we demonstrate that inorganic photocatalysts such as gold-titanium dioxide efficiently provide H₂ O₂ through the methanol-driven reductive activation of ambient oxygen in amounts that ensure that the enzyme remains highly active and stable. Using this approach, the stereoselective hydroxylation of ethylbenzene to (R)-1-phenylethanol was achieved with high enantioselectivity (>98 % ee) and excellent turnover numbers for the biocatalyst (>71 000).

Keywords: Biocatalysis; TiO2; oxyfunctionalization; peroxygenases; photocatalysis.

Scheme 1

Comparison with the previously reported in situ H₂O₂ generation method to promote peroxygenase‐catalyzed hydroxylations of alkanes using the recombinant peroxygenase from Agrocybe aegerita (rAaeUPO). Top: The previously reported multienzyme cascade comprising alcohol oxidase (AOx), formaldehyde dismutase (FDM), formate dehydrogenase (FDH), 3‐hydroxybenzoate‐6‐hydroxylase (3HB6H), as well as the nicotinamide cofactor (NADH/NAD⁺).3 Bottom: Photochemical oxidation of methanol using Au‐loaded TiO₂ (Au‐TiO₂).

Figure 1

Photochemoenzymatic hydroxylation of ethylbenzene to (R)‐1‐phenylethanol with Au‐TiO₂ as the photocatalyst for in situ H₂O₂ generation and rAaeUPO for the stereospecific hydroxylation reaction (•). Negative controls without enzyme (▪), light (▴), methanol (⧫), or rutile Au‐TiO₂ (○). Reaction conditions: [methanol]=250 mm, [Au‐TiO₂]=5 mg mL⁻¹, [rAaeUPO]=150 nm, and [ethylbenzene]=15 mm in 60 mm phosphate buffer (pH 7.0) under illumination.

Figure 2

Qualitative and quantitative determination of radicals formed during the photocatalytic process. A) EPR spectra recorded during the illumination of rutile Au‐TiO₂ in water with methanol for 20 min. Signals marked with an asterisk (★) belong to the oxidation product of DMPO, 5,5‐dimethyl‐2‐oxopyrroline‐1‐oxyl (DMPOX).12 Signals marked with triangles (▾) belong to the spin adduct ^.DMPO–OH, and signals marked with circles (•) belong to the spin adduct ^.DMPO–CH₂OH from methanol.13 Reaction conditions: [Au‐TiO₂]=5 g L⁻¹, [DMPO]=30 mm, [methanol]=100 mm, RT, under illumination. B) Time course of the photocatalytic umbelliferone generation from coumarin as a specific detection method for ^.OH radicals. Reaction conditions: 60 mm phosphate buffer (pH 7), [Au‐TiO₂]=5 g L⁻¹, [coumarin]=0.1 mm, [methanol]=0 (⧫) or 250 mm (▪), T=30 °C, under illumination.