Deracemisation of profenol core by combining a laccase / TEMPO-mediated oxidation and an alcohol dehydrogenase-catalysed dynamic kinetic resolution

A mild one-pot methodology has been developed to deracemise rac-2-phenyl-1-propanol by combining the use of non-selective laccase/TEMPO-mediated oxidation with enantioselective bioreduction of the racemic aldehyde intermediate under dynamic conditions. The process was easily scalable and stereocontrollable by selecting the suitable biocatalyst.


I. General
Materials.Racemic 2-phenyl-1-propanol, 2-phenylpropionaldehyde, and 2phenylpropionic acid are commercially available and were obtained from Sigma Aldrich.Laccase from Trametes versicolor was also purchased from Sigma Aldrich.
(RasADH) were overexpressed following the methodology previously described. 1ADH was obtained from Prof. Martina Pohl (Forschungszentrum Jülich GmbH, Germany).All other reagents were obtained from commercial sources and used as received unless otherwise indicated.

General experimental information. Oxidation reactions using the laccase from
Trametes versicolor/TEMPO catalytic system were performed in a Erlenmeyer flask, open to air and in an orbital shaker.
NMR spectra were recorded on a Bruker DPX 300 MHz or AV400 MHz spectrometer.
All chemical shifts (δ) are given in parts per million (ppm) and referenced to the residual solvent signal as internal.Gas chromatography (GC) analyses were performed on a Agilent 7820 A GC chromatograph equipped with a FID detector.High performance liquid chromatography (HPLC) analyses were carried out in a Hewlett Packard 1100 chromatograph UV detector at 210 nm.Thin-layer chromatography (TLC) was conducted with Merck Silica Gel 60 F 254 precoated plates and visualised with UV and potassium permanganate stain.Column chromatography was performed using Merck Silica Gel 60 (230-400 mesh).

II. Oxidation of 2-phenyl-1-propanol (1) using different laccases
In a 25 mL erlenmeyer flask, to a solution of alcohol 1 (20 mg, 0.14 mmol) in acetate buffer 50 mM pH 5.5 (4.5 mL), was added TEMPO (4.0 mg, 0.025 mmol), and this mixture was stirred for a few minutes to dissolve the reagents.Then, the corresponding laccase was added (12 U/mL) and the reaction was shaken in an orbital shaker at 250 rpm and 30 ºC for 2.5 h.The reaction mixture was acidified using HCl (3 M) and extracted with DCM (2 x 5 mL).The combined organic layers were washed with brine and dried over Na 2 SO 4 .The residue was analysed by GC and NMR (Table S1).Control reaction without the laccase showed no detectable conversion.

Optimising parameters
Our previously reported conditions for the oxidation of 1 using Trametes versicolor/TEMPO as catalytic system are shown in Table S2 (entry 1). 2 In this work, the reaction was performed under more concentrated conditions and monitored within the time (entries 2-4, Table S2).Using these conditions, the pH and concentration of substrate for the subsequent bioreduction could be fixed easily by dilution or adjustment of the pH by the addition of a base.The general protocol is given below: In a 25 mL erlenmeyer flask, rac-alcohol 1 (150 mg, 1.13 mmol) was dissolved in citrate buffer (10 mL; 50 mM, pH 5.5).Then, TEMPO (30 mg, 0.19 mmol) and laccase from Trametes versicolor (55 mg) were added.The reaction mixture was shaken at 30 °C and 250 rpm in an orbital shaker open to air for 3.5 h.The reaction mixture was acidified using HCl (3 M) and extracted with DCM (2 x 5 mL).The residue was analysed by GC (Table S2).
The solvent was evaporated and conversions were determined by GC and ee by chiral HPLC (see Table 1 in Manuscript).

Optimising parameters
In order to improve the enantioselectivities for the preparation of alcohol 1, additional experiments were performed changing parameters such as pH and temperature.The general protocol is described below: In a 1.5 mL vial, 2-phenylpropionaldehyde 2 (3 mg, 0.022 mmol) was dissolved in phosphate buffer at different pHs as indicated in Table S3 (700 μL, 50 mM) containing 1 mM NAD(P)H and the cosubstrate (EtOH or 2-PrOH, 5% v v -1 ).MgCl 2 was added for Evo-1.1.200(1 mM).Finally, the ADH was added.Reactions were shaken at the temperature indicated in Table S3 and 250 rpm for 22 h and extracted with EtOAc (2 x 0.5 mL).The organic layer was separated by centrifugation (2 min, 13000 rpm) and dried over Na 2 SO 4 .Conversions and enantiomeric excess were determined by GC and HPLC, respectively.
Note: Under more drastic conditions, the ee slightly increases in some cases but more acetophenone was detected.

IV. Bioreduction of 2-phenylpropionaldehyde (2) at 500 mg-scale
In a 25 mL erlenmeyer flask, rac-2 (0.5 g, 3.6 mmol) was dissolved in phosphate buffer (115 mL, pH 8 for HLADH and pH 9 for Evo-1.1.200)containing 5% v/v of cosubstrate (EtOH for HLADH and 2-PrOH for Evo-1.1.200)and 1 mM of NADH.In the case of Evo-1.1.200, 1 mM of MgCl 2 was added.Finally, the ADH (10 mg) was added to the reaction mixture and after 3 h, additional 10 mg were added.The reaction was monitored by GC and stopped when no aldehyde was remaining (7 h).

Table S3 .
Bioreductions of 2 at different reaction conditions.
a Conversions were determined by GC and the amount of acetophenone is indicated in brackets.b Enantiomeric excess were determined by chiral HPLC.