Photoenzymatic Hydroxylation of Ethylbenzene Catalyzed by Unspecific Peroxygenase: Origin of Enzyme Inactivation and the Impact of Light Intensity and Temperature

Abstract

Photoenzymatic cascades can be used for selective oxygenation of C−H‐Bonds under mild conditions circumventing the hydrogen peroxide mediated peroxygenase inactivation via in situ H2O2 generation. Here, we report the “on demand” production of hydrogen peroxide via methanol assisted reduction of molecular oxygen using UV‐illuminated titanium dioxide (Aeroxide P25) combined with the enantioselective hydroxylation of ethylbenzene to (R)‐1‐phenylethanole catalyzed by the Unspecific Peroxygenase from Agrocybe Aegerita. For the application of the system it is important to understand the influence of the reaction parameters to be able to optimize the system. Therefore, we systematically investigated product formation and enzyme inactivation as well as ROS formation (H2O2, .OH and .O2−) applying different light intensities and temperatures. As a result, Turnover Numbers up to 220 000, photonic efficiencies up to 13.6 % and production rates up to 0.9 mM h−1 were achieved.

A Glimpse into Green Chemistry Practices in the Pharmaceutical Industry

“…Besides, it does not require any protection/deprotection steps, and it is generally highly enantioselective and can be performed under mild conditions . Enzymes, a key component in biocatalysis, have been employed successfully in many key transformations, such as reduction, hydroxylation, oxidation, hydrolysis, and some other useful transformations . Biocatalysis has been reviewed extensively by others, and we will only discuss a few important results herein.…”

A chemo-enzymatic oxidation cascade to activate C–H bonds with in situ generated H2O2

“…The complex photobiocatalytic system Churakova et al used with native Aae UPO for the hydroxylation of several substrates including ethylbenzene, cyclohexane and octane also still required flavin mononucleotide as a cofactor (FMN) and co-substrate (ethylenediaminetetraacetic acid) to supply reducing equivalents 17 . Further recent papers by Hollmann and coworkers describe in situ H 2 O 2 production by photocatalytic oxidation of methanol and water using various gold loaded TiO 2 photocatalysts 18–20 . When using recombinant Aae UPO (r Aae UPO) and methanol as electron donor they obtained TTNs in the order of 80,000 with ethylbenzene and 61,500 with cyclohexane.…”

“…However, the scale up of photocatalytic transformations is challenging and costly. Exposure of the enzyme to the photocatalyst was reported to cause significant loss in enzyme activity due to oxidative damage by H 2 O 2 decomposition and reactive oxygen species being generated under light irradiation 18–20 . This necessitated immobilization of the enzyme onto a solid carrier to minimize exposure to the photocatalytic TiO 2 surface and increase lifetime.…”

Solar‐Assisted eBiorefinery: Photoelectrochemical Pairing of Oxyfunctionalization and Hydrogenation Reactions

“…In particular, OH . radicals can oxidize the heme iron in the active site of peroxygenases, leading to their inactivation . We expect that these issues associated with OH .…”