Thiols Act as Methyl Traps in the Biocatalytic Demethylation of Guaiacol Derivatives

Abstract Demethylating methyl phenyl ethers is challenging, especially when the products are catechol derivatives prone to follow‐up reactions. For biocatalytic demethylation, monooxygenases have previously been described requiring molecular oxygen which may cause oxidative side reactions. Here we show that such compounds can be demethylated anaerobically by using cobalamin‐dependent methyltransferases exploiting thiols like ethyl 3‐mercaptopropionate as a methyl trap. Using just two equivalents of this reagent, a broad spectrum of substituted guaiacol derivatives were demethylated, with conversions mostly above 90 %. This strategy was used to prepare the highly valuable antioxidant hydroxytyrosol on a one‐gram scale in 97 % isolated yield.


Experimental Procedures
General Information 1 H-and 13 C-NMR spectra were recorded at 20°C on a 300 MHz Bruker NMR.The conversion of all biotransformations was measured by reversed phase HPLC with an Agilent 1260 Infinity HPLC system at 25°C, using a Phenomenex Luna® 5 mm, C18, 100 A (250 × 4.6 mm) column.Detection was performed with a diode array detector (G4212B) and quantification was achieved by calibration with standards.Alternatively, an Agilent 7890 A GC system together with a mass-selective Agilent 5975 detector (electron impact ionization, 70 eV, mass scan= 33-400 m/z) was used for detection.For separation, an Agilent J&W HP-5ms column (30 m, 250 μm, 0.25 μm) and helium (0.7 mL/min) as carrier gas were employed.TLC was carried out with pre-coated aluminum sheets (TLC Silica gel 60 F254, Merck) with detection by UV (254 nm) and/or by staining with cerium molybdate solution.All chemicals and solvents were obtained from commercial suppliers (TCI, Sigma Aldrich/Fluka, VWR International/Merck, Roth) and used as received unless stated otherwise.Enzymes were recombinantly expressed in E. coli and used as lyophilized cell-free extract (CFE) or purified preparation.The corrinoid protein (CP) was reconstituted with methylcobalamin prior to use to yield active holo-CP.Biocatalytic reactions were performed in triplicates in degassed buffers under inert atmosphere, using a Labstar MB10-compact glovebox (MBraun, Germany) equipped with a special O2sensor (MB-OX-EC) to be used with aqueous systems.

Enzyme Expression
Expression of required proteins was performed with E. coli using the plasmids pEG457 and pEG458 (dhaf4610 MTase I and dhaf4610-strep tag MTaseI) and pEG459 and pEG460 (dhaf4611 CP and dhaf4611-strep tag CP) as previously reported. [1]Over-night cultures (ONCs) were prepared in 50 mL falcon tubes by supplementing LB-medium (20 mL) with ampicillin (100 µg/mL).After inoculation with a single colony of transformed cells the cultures were incubated overnight at 37 °C and 120 rpm.Main cultures (500 mL volume in 2 L non-baffled Erlenmeyer flask) supplemented with ampicillin (100 µg/mL) were inoculated with the appropriate volume of ONC to an initial OD600 of 0.05.After incubation at 37 °C and 120 rpm (2 hours) until an OD600 of 0.6-0.8 was reached, expression of the target protein was induced by the addition of AHTC (0.2 μg/mL).Shaking was continued at 120 rpm and 25 °C overnight.After shaking for 24 h, the cells were harvested by centrifugation (4,000 rpm, 4 °C, 20 min).The cell pellets were re-suspended in 50 mM TRIS-HCl buffer pH 7 yielding a 15 wt % cell suspension.Next, the cells were disrupted by sonication (Branson Digital Sonifier®) using the following settings: duration 2 min, 2.0 s sonicate, 1.0 s pause, 40% amplitude.While sonication was performed (at least 3 times with 30 seconds breaks in between), the cells were constantly cooled with ice.Afterwards by centrifugation for 15 min at 14,500 rpm, the cell fragments were removed from the extract.The cell-free extract was finally lyophilized and stored at 4 °C, ready to be used for biotransformations or further used for the purification step.The protein content of the single fraction of MTI (Figure S1) amounted to 0.81 µg which was calculated by densitometry (ImageJ).had been equilibrated with 2 column bed volumes (CVs) of Buffer W (100 mM TRIS/HCl, pH 8, 150 mM NaCl), the cell-free extract was applied to the column and the flow-through (FT) was collected.Then unbound protein was removed by washing the column with 5 CVs of Buffer W and the wash fraction (W) was again collected.The bound protein was eluted by adding 6x 0.5 CVs of Buffer BTX (100 mM TRIS/HCl, pH 8, 150 mM NaCl, 2.5 mM biotin) to the column and collected in 2.5 mL fractions.The protein content of the fractions was determined by means of a photometric Bradford assay.The purified protein solution of dhaf4610 and/or dhaf4611 was rebuffered into MOPS/KOH buffer (50 mM, pH 7, 150 mM KCl) using a PD10 column and concentrated to about 2.5 mL using Vivaspin vials.Afterwards, the purified protein was finally lyophilized and stored at 4°C until further use.

HPLC analysis
After the biotransformation (usually in 120 µL total volume, unless stated otherwise), an aliquot of 90 µL from the sample was taken and the reaction was quenched by the addition of acetonitrile (MeCN, 540 μL).After incubation (30 min) at room temperature, HPLC grade H2O (270 μL) was added, and denatured protein was removed by centrifugation (14,000 rpm, 15 min).The supernatant was filtrated through a pipette tip filled with cotton and then the HPLC analysis was performed.A reverse phase method was used employing LUNA C18 as a stationary phase and water and MeCN both containing 0.1% trifluoroacetic acid (TFA) as mobile phase with a flow rate of 1 mL/min.In the standard method (method A) 100% H2O were held for 2 min, then a gradient from 0% MeCN to 40% MeCN over 13 min was applied, followed by a gradient to 100% MeCN within 5 min, which was held for 2 min.Finally, 100% H2O were held for 3 min.Another method used (method C) was as follows: the column was rinsed with 100% H2O for 2 min, then a gradient from 0% MeCN to 30% MeCN over 13 min was applied, followed by a gradient to 100% MeCN within 5 min, which was held for 2 min.Lastly 100% H2O were detained for 3 min.Other two methods used for the new substrates screened are method D (0-70 gradient of MeCN, 25 min in total).

Biotransformation
Preparation of holo-CP.The functional holo-CP was obtained by reconstitution of the corrinoid protein with exogenous cofactor (methyl cobalamin) under inert atmosphere.For this purpose, methyl cobalamin (2 mM) was dissolved in the presence of betaine (3 M) and DTT (2 mM) in TRIS/HCl buffer (50 mM, pH 7, 0.5 mM DTT, 0.1 mM PMSF) to form a reconstitution buffer.Then, freeze-dried CP (100 mg CFE) was loaded with the freshly prepared reconstitution buffer (1 mL) and incubated for at least 2 h at 4 °C to allow incorporation of the cofactor.Afterwards salts and unbound cobalamin were removed using a PD MidiTrap TM G-25 column (GE Healthcare) according to the manual provided by the manufacturer.Finally, reconstituted holo-CP was eluted with MOPS/KOH buffer (100 mM, pH 6.5, 150 mM KCl) yielding a red colored protein solution which was stored at 4 °C until further use.The content of holo-CP in CFE (21 mg/mL) was taken from literature. [2]otransformation transmethylation analytical scale.Biocatalytic reactions were performed in degassed buffers under inert atmosphere (N2 5.0) in a MBraun LABstar glove box equipped with a MB-OX-EC O2-sensor.Analytical biotransformations were carried out in 1 mL Eppendorf Tubes ® in 120 µL scale.The lyophilized MT I (crude extract 50 mg/mL ≡ 1.95 mg/mL dhaf-MTase I; 13 mg/mL for the experiment with purified enzyme) was rehydrated in holo-CP solution (500 µL/mL reconstituted holo-CP solution ≡ 21 mg/mL CP) and donor (10 mM) and acceptor (variable concentration, ranging from 10 to 50 mM) dissolved in MOPS/KOH buffer (50 mM, pH 6.5, containing 150 mM KCl) were finally added.The reaction mixture was then shaken in the glovebox at 30 °C and 800 rpm for 24 h.Afterwards, an aliquot of 90 µL from each sample was taken out and the reaction was quenched by adding acetonitrile (MeCN, 540 μL).After incubation (30 min) at room temperature HPLC grade H2O (270 μL) was added, and denatured protein was removed by centrifugation (14,000 rpm, 15 min).The supernatant was filtrated through a pipette tip filled with cotton and then the HPLC analysis was performed.

Figure S1 .
Figure S1.SDS-PAGE analysis of the expression of MTase I in E. coli BL21 (DE3) pLysS for its evaluation with ImageJ.After cell disruption via sonication, the soluble lysate was analyzed and it is reported as (L).Std.: PageRulerTM Prestained Protein Ladder.Protein PurificationThe strep-tag purification of dhaf4610 and dhaf4611 was performed with Strep-Tactin® XT High-Capacity Purification kit according to the manual provided by the supplier.After the strep-tag column

Figure S20 .
Figure S20.GC-MS spectra testing the hydrolysis of 4c and reversibility of the reaction employing 4c as methyl donor and catechol 2a as methyl acceptor.Only the peaks for the methylated acceptor 4c and catechol 2a were observed.

Figure S21 .
Figure S21.GC-MS spectra testing the hydrolysis of 4b promoted by the CFE.Both the peaks for the methylated acceptor 4b and the product of hydrolysis 4a were observed.