Metals in Biotechnology: Cr‐Driven Stereoselective Reduction of Conjugated C=C Double Bonds

Abstract Elemental metals are shown to be suitable sacrificial electron donors to drive the stereoselective reduction of conjugated C=C double bonds using Old Yellow Enzymes as catalysts. Both direct electron transfer from the metal to the enzyme as well as mediated electron transfer is feasible, although the latter excels by higher reaction rates. The general applicability of this new chemoenzymatic reduction method is demonstrated, and current limitations are outlined.


Determination of YqjM activity
The YqjM activity assay was established with 2-methylcyclohex-2-en-1-one as standard substrate. The consumption of NADPH during the enzymatic reaction was directly followed at 340 nm for 120 s. The 2-methylcyclohex-2-en-1-one concentration in the assay was 1 mM and the initial concentration of NADPH was 150 µM. Because of background activity with oxygen, glucose and glucose oxidase was also added at a concentration of 20 mM and 10 U/mL respectively. The buffer used was KPi buffer 100 mM pH 6.5.

Biocatalytic reduction of 2-methylcyclohex-2-en-1-one by YqjM regenerated by metals
Each reaction was carried out in the glovebox in a 1.5 mL glass vial with a working volume of 400 µL.
To ensure the absence of oxygen in the reaction, each component was placed in the glovebox at least few hours before use. Stirring bars were used for getting a good suspension of metals in the reaction. Reactions with mediators were protected from the light with an aluminium foil. Duplicates were performed. 50 mg/mL of chromium (or 65 mg/mL of zinc) correspond to a concentration of 1 M in the reaction.
For analysis, as each time-point corresponds to one reaction glass vial, 400 µL of ethyl acetate (containing 5 mM of dodecane as internal standard) was added for performing the extraction. The separation of the two phases was obtained via centrifugation. The combined organic phases were dried over anhydrous MgSO 4 and transferred into a GC vials for analysis. All concentrations reported on this paper are based on calibration curves obtained from authentic standards and treated in the same manner as described here. For 2-methylcyclohex-2-en-1-one, the ee value during this study was >90%, knowing that the substrate is only pure at 90% (the 10% of impurity is a racemic mixture of 6-methylcyclohex-2-en-1-one which can also be reduced by YqjM).

Spectrophotometer
The instrument used is a Cary 60 UV-Vis spectrophotometer (equipped with a single cell Peltier accessory) from Agilent technologies.

XPS analysis
The used XPS was a ThermoFisher K-Alpha. The X-ray gun uses an Al Kα source with an energy of 1486 eV. The (nominal) spot size was set to 400 µm. During the measurements a flood gun was used for charge compensation, setting the pressure to about 5.10 -7 mbar.

Stability assay of YqjM toward chromium
Stability assays of YqjM in presence of chromium species have been performed. Table S2 demonstrate that chromium species do not have an influence on YqjM's residual activity after 24 hours.

Control reactions of substrate in presence of chromium and FMN
Control reactions have been performed for determining the chemical background of the system. When only chromium was in presence of substrates, no reduction was observed. However when FMN and chromium were present, ketoisophorone and citral were reduced in a racemic mixture.

XPS analysis
XPS measurements have been performed on a sample of chromium powder (sample 1) and on a sample of powder chromium used for the regeneration of YqjM (Sample 2). On each samples, two measurements have been performed and these duplicates are reproducible. In Table S5, the content of each sample is summarised. Phosphate and potassium are present in the sample 2 due to the use of KPi buffer for the reaction. Sample 2 has been washed but small quantities of phosphate and potassium are still present at the surface of the particles.   Figure S3. Chromatogram of the chemoenzymatic reduction of C=C-double bonds of 2-methylcyclohexenone by YqjM regenerated with zinc. The reaction is not enantioselective and 2-methylcyclohexenol is also produced. Figure S4. Chromatogram of the chemical reduction of 2-methylcyclohexenone by zinc. Zinc reduced the C=C-double bond and the ketone. Figure S5. Chromatogram of the chemical reduction of carvone by reduced mediators. The peak at 28.9 min corresponds to the reduction of the non-conjugated C=C-double bond.