A Photochemoenzymatic Hunsdiecker‐Borodin‐Type Halodecarboxylation of Ferulic Acid

Abstract A photochemoenzymatic halodecarboxylation of ferulic acid was achieved using vanadate‐dependent chloroperoxidase as (bio)catalyst and oxygen and organic solvent as sole stoichiometric reagents in a biphasic system. Performance and selectivity were improved through a phase transfer catalyst, reaching a turnover number of 660.000 for the enzyme.


Partition coefficient of ferulic acid in the biphasic system under different TBAB
concentrations  8. 1 H-NMR of product 2 isolated from the semi-preparative photochemoenzymatic reaction.

Materials
All reagents, solvents and standards were purchased form Sigma-Aldrich in the highest purity available and used without further purification except for 2-methyltetraidrofuran that was freshly distilled, degassed and stored under Argon prior to use it. All stock solutions used in this study were prepared with freshly distilled and degassed 2-methyltetrahydrofuran, stored under argon atmosphere in amber glass bottles to avoid any autoxidation processes.

Production of CiVCPO
CiVCPO was produced in recombinant E. coli TOP10 pBADgIIIB VCPO following previously described procedures. 1 CiVCPO was purified via heat treatment according to a modified protocol from literature. 1 33 g of E. coli TOP10 pBADgIIIB VCPO were resuspended in around 100 mL 20 mM Tris-H2SO4 buffer (pH 8.1) containing 0.1 mM sodium-orthovanadate. One protease inhibitor tablet (cOmplete ULTRA Tablets, Mini, EDTA-free, Roche) and bovine DNAse I (Merck) with a spatula tip of MgSO4 were further added prior to cell disruption. Cells were disrupted by use of a Multi Shot Cell Disruption System (3 cycles) at 1.5 kBar. The soluble fraction (crude cell extract) was separated from the cell debris by centrifugation (47.850 x g for 30 min at 4°C) and subsequently exposed at 70°C for 30 min. Then, heat precipitated proteins were removed by centrifugation (47.850 x g for 30 min, at 4 °C). The supernatant containing CiVCPO was concentrated to around 20 mL (Amicon filters with 30 kDa cut-off membrane) and washed four times with fresh 20 mM Tris-H2SO4 buffer (pH 8.1) containing 0.1 mM sodium-orthovanadate. The final VCPO fraction (20 mL) was flash frozen in a roundbottom flask with liquid nitrogen and lyophilized at 0.1 mbar and -58 °C. Around 200 mg lyophilized CiVCPO with a purity of 87% (according to SDS-PAGE analysis, see Figure   S1) and an enzyme concentration of 5 nmol per mg lyophilized powder was obtained.

Figure S1
: SDS-PAGE analysis following the purification of CiVCPO. CE: crude extract, HE: soluble fraction after heat treatment, FT: flow through during concentration with Amicon filter (30kDa cutoff), W: Wash fraction with buffer during concentration with the Amicon, fin: final fraction used for lyophilisation. 2, 5 or 10 µg protein of each sample was applied (indicated by the indices). A purity of 87% VCPO was determined using the band intensities (5 µg protein load). S: Precision Plus Protein All Blue Standard (Biorad).

Analytical procedure.
HPLC measurements were performed using an Ultimate 3000 Rapid Resolution UHPLC system (ThermoFisher scientific) equipped with Alltima C18 (250 mm×4.6 mm, 5 m) column and a multiwavelength detector using the following method: Conversions and yields are always based on calibration curves with authentic substrate standards and with chemically synthetized product standards. singlet. The purity of product standards has been quantified by NMR using ethylene carbonate as internal standard and then used as standard for HPLC calibration curve of products.

Procedure
The selected ,-unsaturated carboxylic acid (1.0 mmol, 1.0 eq.) and triethylamine (TEA) (5.0 mol%) were dissolved in 3.0 mL of dry N,N-dimethylformamide (DMF) under inert atmosphere (argon). The resulting solution was stirred for 5 minutes at room temperature then, N-Bromosuccinimide (1.05 mmol, 1.05 eq.) was added, and the reaction was further stirred for 2 hrs at room temperature. After this time, 20.0 mL of ethyl acetate (AcOEt) were added to the reaction and the resulting organic layer was washed with LiCl 3% aqueous solution (3 x 30 mL), distilled water (1x 30 mL) and BRINE (1x30 mL), dried over sodium sulfate and evaporated under vacuum.
The obtained crude was purified by flash column chromatography using the indicated mobile phase.