A Peroxygenase‐Alcohol Dehydrogenase Cascade Reaction to Transform Ethylbenzene Derivatives into Enantioenriched Phenylethanols

Abstract In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)‐ or (S)‐phenylethanols using the (R)‐selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)‐selective ADH from Rhodococcus ruber (ADH‐A). In a one‐pot two‐step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity.


General information
Unless otherwise mentioned, all chemicals were purchased from Sigma-Aldrich, TCI-Europe or abcr GmbH, and used without further purification. Columns and column material for enzyme purification were purchased from GE Healthcare

Synthesis of acetophenone derivatives
Scheme S1. Synthesis of acetophenone derivatives.
The reactions were performed in 20 mL glass vials at room temperature, under ambient atmosphere. The ethylbenzene derivatives (0.325 mmol) were diluted in CH3CN (650 µL), and mixed with 4850 µL 50 mM KPi buffer, pH 7.0 and 2 µM rAaeUPO (83.88 µM stock, 160 µL). H2O2 (1 M in MilliQ) was added in the vial via a tube connected to a syringe pump (20 mM/h, 130 µL/h). The system was closed to avoid evaporation as much as possible. A ThermoMixer C from Eppendorf ( Figure S2) was used for the reactions at a speed of 600 rpm.

Acetophenone reduction by ADHs
The reactions were performed in GC glass vials of 1.5 mL ( Figure S2). Containing 2 mM MgCl2, 0.1 mM NAD(P)H, ADH-A/LkADH: 50 µL cell free extract/20 mg dry cell, 10% v/v 2-propanol and 15 mM acetophenone (total volume: 700 µL). A ThermoMixer C from Eppendorf was used to control the temperature at 30 °C and speed at 600 rpm.

One-pot one-step system
The reactions were performed in a 1.5 GC glass vials a solution (700 µL total volume) containing 2 µM rAaeUPO, 50 mM ethylbenzene, 2 mM MgCl2, 0.1 mM NAD(P)H, 10% v/v 2-propanol, 50 µL of ADH-A/LkADH cell free extract in 50 mM KPi buffer (pH 7.0). H2O2 (1 M in MilliQ) was added in the vial via a tube connected to a syringe pump (10 mM/h, 7 µL/h, 10 h). The reaction was running 24 h. A ThermoMixer C from Eppendorf was used to control the temperature at 30 °C and speed at 600 rpm.

One-pot-two-steps system
The reactions were performed in 20 mL glass vials starting at room temperature. The ethylbenzene derivatives (0.325 mmol) were diluted in CH3CN (650 µL), and mixed with 4850 µL 50 mM KPi buffer, pH 7.0 and 2 µM rAaeUPO (83.88 µM stock, 160 µL). H2O2 (1 M in MilliQ) was added in the vial via a tube connected to a syringe pump (130 µL/h). After 6 h, 365 µL of the reaction mixture was taken into a 1.5 mL glass vial, 2 mM MgCl2, 0.1 mM NAD(P)H, 10% v/v 2-propanol, 100 µL of ADH-A/LkADH cell free extract was added. The reaction was running overnight. A ThermoMixer C from Eppendorf was used to control the temperature at 30 °C and speed at 600 rpm.

Determination of rAaeUPO activity
The volumetric activity was determined via ABTS-assay at 25 °C. ABTS-assay was performed in sodium citrate buffer (50 mM, pH 4.4) containing H2O2 (2.0 mM), ABTS (0.5 mM) and a diluted enzyme solution. The oxidation of ABTS was monitored by absorption change at 420 nm (ɛ420 = 36.0 mM -1 cm -1 ). Reactions were performed in triplicates.

SDS-PAGE
10 μL Protein samples were diluted with distilled water and mixed with 9.5 μL staining agent (Laemmli Sample buffer 4x) and 0.5 μL reducing agent (DDT 20x) to a final volume of 20 μL. After heat incubation the samples, 10 μL samples were loaded onto the gel. A precast gel was used (Criterion TM TGX Stain-Free) with 1x TGS running buffer. 10 μL of marker solution (Precision Plus ProteinTM Unstained Standard) was applied for every run. As running conditions, a constant voltage of 200 V and a starting current of 165 mA per gel with a running time of 45 min were applied.

GC measurements
GC measurements were performed on a Shimadzu GC-14A/FID or Shimadzu GC-2010 plus/FID equipped with different columns (Table S1 and S2). The reactions were stopped at different time points by addition of ethyl acetate containing n-octanol (5 mM) as internal standard. After extraction and centrifugation, the organic phase was dried with magnesium sulfate and analysed via gas chromatography. All concentrations reported, are based on calibration curves obtained from authentic and synthesised standards.