Oxygen-Free Regioselective Biocatalytic Demethylation of Methyl-phenyl Ethers via Methyltransfer Employing Veratrol-O-demethylase

The cleavage of aryl methyl ethers is a common reaction in chemistry requiring rather harsh conditions; consequently, it is prone to undesired reactions and lacks regioselectivity. Nevertheless, O-demethylation of aryl methyl ethers is a tool to valorize natural and pharmaceutical compounds by deprotecting reactive hydroxyl moieties. Various oxidative enzymes are known to catalyze this reaction at the expense of molecular oxygen, which may lead in the case of phenols/catechols to undesired side reactions (e.g., oxidation, polymerization). Here an oxygen-independent demethylation via methyl transfer is presented employing a cobalamin-dependent veratrol-O-demethylase (vdmB). The biocatalytic demethylation transforms a variety of aryl methyl ethers with two functional methoxy moieties either in 1,2-position or in 1,3-position. Biocatalytic reactions enabled, for instance, the regioselective monodemethylation of substituted 3,4-dimethoxy phenol as well as the monodemethylation of 1,3,5-trimethoxybenzene. The methyltransferase vdmB was also successfully applied for the regioselective demethylation of natural compounds such as papaverine and rac-yatein. The approach presented here represents an alternative to chemical and enzymatic demethylation concepts and allows performing regioselective demethylation in the absence of oxygen under mild conditions, representing a valuable extension of the synthetic repertoire to modify pharmaceuticals and diversify natural products.


Additional data
Optimization of the demethylation of the vdmB system The first parameter investigated was the buffer composition and the pH. Four buffers (HEPES, MOPS, MES and PIPES) were analyzed in a pH range from 6.5 to 8 ( Figure S1). The reaction worked best in HEPES buffer (50 mM, pH 6.5, 150 mM KCl, dark blue, Figure S1). Additionally, vdmB and CP also tolerated higher pH values with the CHES buffer (50 mM, pH 9.5 and 10, 150 mM KCl, Figure S2). The methyltransferase vdmB possesses zinc binding motifs according to literature. 1 A concentration of 100 µM zinc was reported as most beneficial for the demethylation reaction whereas our optimization study showed that a 20 µM zinc concentration was sufficient ( Figure S3). In order to improve the bioavailability of apolar substrates, varied concentrations of DMSO (up to 10% v/v) were tested with vdmB. A concentration of 2% v/v DMSO led to the best product formation ( Figure S5). Other co-solvents such as MeOH and EtOH (2% v/v) were screened for better acceptance, but none was as good as DMSO ( Figure S6).

S7
The last parameter to be optimized was the ratio of vdmB to CP whereby for the experiment crude protein preparations were used, of which the content of pure vdmB and CP was known (Table S1). The best results were obtained by employing at least a two-fold excess of CP over vdmB concerning the amount of crude preparations (Entries 2,3 and 5). Looking at the corresponding ratio of pure proteins, most product was formed at a 14-21 fold excess of CP in comparison to vdmB (Entries 2,3 and 5). By incorporating vdmB in a two-fold excess over CP within crude preparations, the reaction produced half as much for the two demethylated products compared to the optimized conditions (Entries 6 and 8). Concluding, the carrier protein was the limiting factor for the demethylation reaction and the use of an excess was beneficial. Therefore, it was used for all subsequent experiments. Reaction conditions: substrate 1b (10 mM, 1.5 mg/mL), methyl acceptor 1n (20 mM, 2.5 mg/mL), CP ("1" = 21 mg/mL, ≡ 1 mM pure CP with MeCob; "1.5" = 26 mg/mL, ≡ 1.2 mM pure CP with MeCob; "2" = 31 mg/mL, ≡ 1.5 mM pure CP with MeCob; "3" = 33 mg/mL, ≡ 1.6 mM pure CP with MeCob) and vdmB ("1" = 40 mg/mL CFE, ≡ 0.077 mM pure vdmB, "1.5" = 60 mg/mL CFE, ≡ 0.12 mM pure vdmB, "2" = 80 mg/mL CFE, ≡ 0.154 mM pure vdmB, "3" = 120 mg/mL CFE, ≡ 0.231 mM pure vdmB) in HEPES buffer (50 mM, pH 6.5, 150 mM KCl, 20 µM ZnCl 2 ) at 30 °C, 800 rpm in Eppendorf Ther-momixer® (1.5 mL) for 24 h. All reactions were quenched by the addition of MeCN (60 vol. %) after 24 h and the conversions were analyzed via calibration curves of the corresponding reference compounds on HPLC-UV.

Materials
All starting materials were obtained from Sigma-Aldrich, Alfa Aesor or TCI-Chemicals and used as received unless stated otherwise. Yatein 1k was chemically synthesized according to literature. 2 The following compounds were obtained by the mentioned suppliers and used as corresponding references for HPLC-

Protein expression
The plasmids containing the respective genes (Table S3) were transformed into chemically competent E. coli BL21/Lemo21 (DE3) cells. The over-night cultures were prepared by picking one colony from an agar plate and suspending it in LB-medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) supplemented with ampicillin (100 µg/mL) followed by an over-night incubation at 37 °C and 120 rpm. The LB-medium (0.5-1 L, non-baffled flasks) supplemented with the ampicillin was inoculated with the over-night culture and incubated at 37 °C and 140 rpm until an OD600 of 0.6-0.8. The protein expression was induced by the appropriate addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) or anhydrotetracycline (AHTC , Table S4). Subsequently, the cell suspension was incubated for 24 h at 25 °C prior to its harvest by centrifugation (5,000 rpm, 4 °C, 10 min). In order to prepare the cell free extract (CFE) cells were resuspended in HEPES-buffer (50 mM, pH 6.5, 150 mM KCl, 7 mL buffer to 1 g wet cells) and disrupted by ultrasonication on ice (40% amplitude, 3x 6 min, pulse 1 sec, pause 2 sec) using a Sonics & Materials Vibra Cell CV26 (13 mm tip, amplitude range 36-240) . After the crude cell extract was separated from the cell debris by centrifugation (30 min, 14,000 rpm, 23,519 g), the extract was frozen in liquid nitrogen and lyophilized over-night. The CFE was analyzed on SDS-PAGE ( Figure S9) and stored at -20 °C or instantly used for further experiments.

S15
The protein content of the single fraction of vdmB-MT ( Figure S9, lane 1, red box) amounted to 0.7 µg which was calculated by densitometry (ImageJ).

Preparation of holo-CP (loading of methylcobalamin)
The loading of the CP with its cofactor methylcobalamin hydrate was performed under inert atmosphere accordingly to the optimized protocol in literature. [4][5][6] As a first step, the reconstitution buffer was prepared consisting of methylcobalamin (6 mM; four times excess to CP) and betaine (3 M) which were dissolved in Tris/HCl buffer (50 mM, pH 7, 2.5 mM DTT, 0.1 mM PMSF). The lyophilized crude CP (133 mg/mL CFE or 31 mg/mL, ≡ 1.5 mM pure CP with MeCob; a lyophilized powder was used to simplify the transfer of the protein to an inert atmosphere) was mixed with the reconstitution buffer (1mL) and incubated for at least 2 h at 4°C. This incubation step was crucial for complete loading of the CP with the methylcobalamin. The removal of salts and unbound cofactor was performed via a desalting step using a PD MidiTrap TM G-25 column (GE Healthcare) or PD 10 TM G-25 column (GE Healthcare) according to the manufacturer's manual. During this step, the buffer was exchanged to HEPES buffer (50 mM, pH 6.5, 150 mM KCl, 20 µM ZnCl2) yielding a red colored protein solution (66.7 mg/mL CFE or 21 mg/mL pure CP). Prior to biocatalytic reactions, this holo-CP solution was stored at 4 °C.

Biocatalyst preparation for demethylation
All biotransformation reactions were performed on a 0.5 or 1 mL scale under inert atmosphere (99.8% N2 gas, 200 bar) in a glove box. The lyophilized vdmB (40 mg/mL final concentration cellfree extract) was dissolved in the holo-CP solution (400 µL/mL, 21 mg/mL pure CP). As a model methyl donor veratrol 1a (10 mM) or 3,4-dimethoxytoluene 1b (10 mM) was used with either 3,4dihydroxybenzaldehyde 1m (50 mM) or later with orcinol 1n (20 mM) in HEPES buffer (50 mM, pH 6.5, 150 mM KCl, 20 µM ZnCl2). For all screened substrates, stock solutions were prepared in HEPES buffer containing DMSO (20% for dimethoxy substrates, 50% for compounds with at least S17 three methoxy groups). All demethylation reaction samples containing the proteins with the methyl donor and acceptor in HEPES buffer were shaken in a vertical position in an Eppendorf Thermomixer® at 35 °C and 800 rpm for 24 h in the glove-box (inert atmosphere). Samples (90 µL) were quenched with acetonitrile (MeCN, 540 µL), mixed thoroughly and incubated for 20 min at room temperature. Afterwards, water (270 µL, HPLC pure) was added and the denatured protein was removed by centrifugation (14,000 rpm, 10 min). The supernatant was filtered through a pipette tip filled with cotton. The samples were analyzed on HPLC using the LUNA C18 column with a mobile phase consisting of water and MeCN with 0.1% trifluoroacetic acid (TFA). The flow rate was set to 1 mL/min. In the standard method the column was rinsed for 2 min with 100% H2O, then a gradient from 0% MeCN to 40% MeCN over 13 min was applied, followed by a gradient to 100% MeCN for within 5 min, which was held for 2 min. All retention times and the corresponding k-values of the methyl donors and acceptors are summarized in Table S5.

Semi-preparative scale of yatein for NMR analytics
Special approaches were used for the demethylation of yatein 1k (100 mg, 0.15 mmol, 60% isolated yield) on a 25 mL semi-preparative scale. After all reagents were set up in a 100 mL round bottom flask under inert atmosphere, the flask was sealed by a rubber plug, parafilm and a clamp. The biotransformation was incubated for 24 h at 25 °C and 160 rpm in the incubator shaker (Multitron Infors Ht®) outside the glove box. The reaction was quenched, extracted with EtOAC (3x 25 mL), prepared for preparative HPLC as stated above (Method C) and the product 4-O-demethylyatein 2k (9% isolated yield) analyzed in CDCl3 by 1 H NMR, 13 C{ 1 H} NMR.

Determination of the pure holo-CP content in the CFE
First, different spectra of methylcobalamin (MeCob, square, dark red) and the pure holo-CP (pure holo-CP, triangle, red, Figure S11) were measured between 350 to 650 nm on the UVspectrophotometer under inert atmosphere by sealing the UV-cuvettes with parafilm. The HEPES buffer (50 mM, pH 6.5, 150 mM KCl, 20 µM ZnCl2) was used as a blank for all measurements. Both spectra of methylcobalamin and the pure holo-CP share the same absorption maximum at 520 nm which refers to the [Cob III -Me] state. The unloaded and reduced state [Cob I ] appears at 375 nm according to literature. 7 The extinction coefficient of pure holo-CP (ε520-600nm= 0.0834 mg -1 mL -1 cm -1 ) was calculated by different concentrations of this protein ( Figure S12). With this extinction coefficient the total amount of pure holo-CP in the CFE could be calculated.        The impurity peak at 14.1 min referred to orcinol 4n in small quantity which could not be fully separated from the product. The peak at 20 min appears independently and is assigned as system peak. Minor traces of impurities were found which could not be fully separated from the product. The peak at 20 min appears independently and is assigned as system peak.

S40
Due to the limited amount of product obtained and to the consequent lack of 1 H-13 C-HSQC NMR and 1 H-13 C-HMBC NMR, the assignment of the 1 H NMR peaks was done accordingly to the 6desmethylpapaverine (2j) 1 H NMR (considering the high similarity of the two molecules). Consequently the disappearance of the NOE of the H 5'with the methoxy group 12 (demethylation in position 4') in the NOESY NMR spectrum allowed the confirmation of the 4',6-didesmethyl papaverine (4j) compound. Figure S32. 1 Figure S34. 13 Figure S37. 1 H-NMR of the 3-methoxy-5-methylphenol (2n) isolated from a biotransformation, namely the demethylation of 2,4,6-trimethoxytoluene (1i). For reaction conditions see Figure S15. The insert displays the reference material (3-methoxy-5-methylphenol, Sigma Aldrich). The NMR corresponds to data reported in literature. 9