Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Biocatalytic methylation and demethylation via a shuttle catalysis concept involving corrinoid proteins


Synthetically established methods for methylation of phenols and demethylation of methyl phenyl ethers rely in general on hazardous reagents or/and harsh reaction conditions and are irreversible. Consequently, alternative regioselective methods for the reversible formation and breakage of C-O-ether bonds to be performed under mild and sustainable conditions are highly desired. Here we present a biocatalytic shuttle concept making use of corrinoid-dependent methyl transferases from anaerobic bacteria. The two-component enzymatic system consists of a corrinoid protein carrying the cofactor and acting as methyl group shuttle, and a methyltransferase catalyzing both methylation and demethylation in a reversible fashion. Various phenyl methyl ethers are successfully demethylated and serve in addition as sustainable methylating agents for the functionalization of various substituted catechols. Therefore, this methyl transfer approach represents a promising alternative to common chemical protocols and a valuable add-on for the toolbox of available biocatalysts.


From a synthetic point of view formation as well as cleavage of alkyl aryl ethers are valuable transformations contributing to the structural diversity of pharmaceuticals and natural products1,2,3. Beyond that, O-methylated phenol derivatives are highly demanded building blocks for the manufacture of antioxidants, flavoring agents, fragrances, dyes, agrochemicals, and fine chemicals4,5. Although numerous chemical procedures6 for methylation and demethylation are available (Fig. 1) and have been broadly applied, none of these protocols allows for reversible C-O-ether bond formation and breakage on synthetic scale. Conventionally, the addition of methyl moieties onto heteroatoms is accomplished with methyl iodide or dimethyl sulfide as methylating agent together with a strong base7,8. Besides toxicity and corrosiveness of the reagents needed in excess and the sizable amounts of inorganic salts formed as by-products, these traditional procedures are in general neither chemo- nor regioselective in case substrates possess more than one hydroxyl group or other reactive functional moieties (e.g., amine). Attempts to improve or even replace the classical Williamson ether synthesis led to the development of methods making use of less hazardous alkylating agents such as dimethyl carbonate9,10 or methanol11,12 which, however, still require high temperatures for reaction. Due to the high stability of O-methyl ethers against hydrolysis also their cleavage usually involves harsh reaction conditions and reagents such as strong Bronsted13,14 or Lewis acids15,16. Alternatively, the use of metal catalysts17,18 or nucleophilic methods employing for instance sodium thiolates19,20 belong to the most commonly applied chemical techniques.

Fig. 1

Conventional procedures for O-methylation and -demethylation. a Toxic methyl iodide (MeI) or dimethylsulfide (DMS) are commonly used methylating agents under basic conditions. Alternatives are dimethylcarbonate (DCM) or methanol (MeOH), which however require an elevated temperature. b Strong Bronsted- or Lewis acids such as hydrobromic acid (HBr) or boron tribromide (BBr3), respectively, as well as sodium ethanethiolate (NaSEt) are the traditional reagents for demethylation

In the last decades, biocatalysis has gained increasing attention as promising research field at the interface of organic chemistry and biotechnology, complementing or even replacing conventional organic synthesis methods by more selective, mild and sustainable procedures21,22,23. Due to nature’s manifold source of enzymes supported by the rapid development in molecular biology and gene technology, there is no limitation in accessing novel biocatalysts and thus, the repertoire of biocatalytic reactions is constantly increasing and broadening24,25,26. Since the transfer of methyl groups is an indispensable reaction in many aspects of life, methyl transferases (MTases) are ubiquitously distributed in nature27,28 and according to the required cofactor two main enzyme classes have evolved (Fig. 2). The most prominent one relies on S-adenosyl-l-methionine29,30 and typically catalyzes the methylation of C-, O-, N-, or S-nucleophiles through a SN2 mechanism by simultaneously generating S-adenosyl-homo-cystein (SAH) as by-product. Alternatively, methylation can take place at non-nucleophilic carbons involving a radical-based mechanism31,32. Even if well-understood and providing exceptional access to a range of methylated compounds, SAM-dependent methyl transfer occurs in an irreversible fashion and thus, the cofactor is required in stoichiometric amounts hindering its feasibility in synthetic processes. In spite of the substantial progress that has been made in this research area1,33,34,35 including the use of artificial SAM-analogs36 and an in-vitro SAM-regeneration system37, there is still need for a scalable alternative. Much less investigated is the class of corrinoid-dependent MTases38,39. Corrinoids share the structural motif of a corrin skeleton, a heterocyclic system composed of four pyrrole rings. The most prominent members of this group are cobalamins40,41 (B12-derivatives, Fig. 2b), in which the unique central cobalt-carbon bond is key to enable chemically challenging biotransformations42. Methyl cobalamin (Me-Cbl)-dependent MTases have been described to mediate the transfer of a methyl cation received upon heterolytic cleavage of the Co-C bond to nucleophilic substrates. The corrinoid cofactor cycles thereby between the CoIII and the highly reactive CoI state43 and thus functions as methyl group shuttle (Fig. 3a). Nature exploits this methyl transfer mainly for O-demethylation, playing an essential role in the amino acid metabolism of eukaryotes as well as in the energy generation of anaerobic organisms39,44,45. For instance, several acetogenic bacteria have been described to utilize methyl phenyl ethers as carbon and energy source, amongst others Acetobacterium dehalogenans46, Acetobacterium woodii47, Moorella thermoacetica48, and Desulfitobacterium hafniense49,50. These organisms encode a complex O-demethylation machinery constituted of four enzymes (Fig. 3b), mediating a methyl group transfer from a substrate onto tetrahydrofolate (FH4) via two half-reactions. In the first half reaction, MTase I binds the substrate and mediates both ether cleavage and transfer of the methyl group onto CoI of the super-reduced cofactor bound to the corrinoid protein (CP). Then, a second MTase (MTase II) transfers the methyl group from CoIII-CP onto FH4, being further metabolized by the organism. The fourth protein acts as an activating enzyme (AE) to catalyze the ATP-driven reduction of the oxidized cofactor in the inactive CoII redox state to the catalytically active CoI species. While most of the MTase systems described in the literature have primarily been studied with respect to their physiological role using crude cell extracts of wild-type organisms, just a few of them51,52 have been recombinantly produced and characterized from a biochemical point of view. Even though they displayed promising demethylation properties, their feasibility as biocatalysts for synthetic purposes has not been evaluated yet.

Fig. 2

Nature’s cofactors for enzymatic (de)methylation reactions. a S-adenosyl-l-methionine (SAM) is nature’s methylating agent and donates a methyl group while irreversibly forming S-adenosyl-homo-cystein (SAH) as by-product. b General structure of cobalamins, being the most relevant representatives of corrinoid cofactors: hydroxocobalamin (OH-Cbl, R=OH), vitamin B12 (CN-Cbl, R=CN), mehylcobalamin (Me-Cbl, R=Me), coenzyme B12 (Ado-Cbl, R=5′-deoxyadenosyl)

Fig. 3

Natural cobalamin-dependent methyl transfer. a Heterolytic cleavage of the central cobalt-carbon bond in methylcobalamin. b O-demethylation machinery in anaerobic organisms consisting of two MTases I and II, a corrinoid protein (CP) with bound cobalamin and an activating enzyme (AE). The methyl group is transferred from a substrate onto tetrahydrofolate (FH4) via two half reactions with the cofactor switching between the super-reduced CoI and the CoIII state and functioning as molecular shuttle

In the present study, we report the use of a cobalamin-dependent methyltransferase system for a shuttle catalysis concept enabling both methylation and demethylation reactions in a reversible manner. To the best of our knowledge, such a system has not previously been investigated for synthetic biocatalytic applications and may represent an alternative to SAM-dependent enzymes as well as related chemical protocols.


Concept of reversible methyl transfer

For the design of an alternative biocatalytic demethylation and methylation approach inspired by the corrinoid-dependent methyltransferase system from anaerobic bacteria, we envisioned a simpler concept than shown in Fig. 3b. It relies on the use of just one methyltransferase being the only biocatalyst together with a corrinoid protein (CP) carrying the cobalamin prosthetic group and acting as methyl group shuttle. In nature MTase I is described to transform more than one substrate, whereas MTase II is restricted to the methylation of tetrahydrofolate for the purpose of energy generation. Therefore, due to the reversibility of catalysis we propose to employ MTase I both for demethylation and methylation, coupling the cleavage of a methyl phenyl ether with the methylation of unnatural acceptor molecules in shuttle catalysis like fashion53,54,55 (Fig. 4). Thus, MTase I catalyzes the demethylation of a methyl donor molecule transferring the methyl group onto the CP bound cobalamin; in the subsequent methylation step the methyl group is transferred from the cofactor to the methyl acceptor again mediated by MTase I to yield the methylated acceptor.

Fig. 4

Envisioned concept for biocatalytic methyl transfer via reversible shuttle catalysis. The cobalamin-dependent methyl transfer system consists of MTase I and CP catalyzing the methyl group shuttle between a donor and an acceptor and can be exploited for either demethylation or methylation of a substrate

Employing a single enzyme (MTase I) for both half reactions offers considerable benefits as the number of required enzymes is reduced. Furthermore, the system can be employed for the demethylation as well as the methylation of a substrate. An undesired oxidation of CoI to CoII by molecular oxygen and thus an inactivation of the catalytic shuttle system is prevented by working in an inert atmosphere (glove-box).

Enzyme selection and production

The methyltransferase MTase I and the corrinoid protein CP from Desulfitobacterium hafniense52 (D. hafniense) were selected as model proteins for investigation of the suggested concept. The proteins were heterologously expressed in E. coli BL21 (DE3) pLysS using synthetic codon-optimized genes cloned into the pASK-IBA3plus vector. The use of this vector enabled the production of C-terminal Strep-tag®-fusion-proteins, which could be purified by affinity chromatography. Similarly, MTase II from D. hafniense was recombinantly produced in E. coli in order to verify the natural reaction in vitro. For a detailed description of cloning strategy and expression results, see Supplementary Fig. 13 and Supplementary Table 1. Since E. coli is lacking the ability to synthesize any of the cobalamin derivatives56, the corrinoid cofactor was incorporated into the apo-protein after expression in order to yield active holo-CP51.

Characterization of the MTase system from Desulfitobacterium hafniense

Having the required enzymes in hand the MTase system from D. hafniense was tested for its applicability for biocatalytic methyl transfer. Based on the reported demethylation properties of MTase I52, guaiacol 1a was chosen as model methyl donor. Since it is obtainable from renewable resources57,58 it represents an attractive substrate to demonstrate the concept and serves as a cheap and sustainable methylating agent. Due to the oxygen and light-sensitive cobalt cofactor all biotransformations were performed under inert atmosphere and light exposure was kept to a minimum. Initially, titanium(III) citrate and methyl viologen were used to reduce inadvertently formed CoII to the active CoI species. However, in an oxygen-free environment the use of this O2-scavenger system became obsolete and was omitted if not indicated otherwise.

As a first step the natural reaction involving both MTases (I and II) and CP was successfully reconstituted in vitro by transferring the methyl group from 1a onto the natural substrate tetrahydrofolic acid (see Supplementary Fig. 4 and Supplementary Table 2). Then, MTase I was investigated to act as the only biocatalyst mediating both demethylation and methylation in a reversible fashion. For this purpose 3,4-dihydroxybenzoic acid 2b was chosen as non-natural model substrate for accepting the methyl group (acceptor). Detection of the demethylated product catechol 2a as well as the mono-methylated carboxylic acid 1b (regioisomeric mixture meta/para = 2/1, see Supplementary Fig. 5) proofed the feasibility of employing MTase I as the only biocatalyst for both methylation and demethylation. Initial conversions of 2–3% were improved by optimizing various parameters such as temperature, enzyme and substrate concentration (see Supplementary Fig. 69). An excess of the acceptor molecule instead of equimolar amounts pushed the demethylation of the model substrate 1a to conversions of up to 80%. Employing 3,4-dihydroxybenzaldehyde 2c as alternative model acceptor gave identical results, yielding vanillin and isovanillin 1c in a ratio of 3/1. The significant increase of conversion obtained by using an excess of one substrate indicated an equilibrium between demethylation and methylation caused by the reversible nature of the reaction (Fig. 5a). It can be assumed that the via demethylation of guaiacol 1a obtained product (catechol 2a) functions subsequently as methyl acceptor for a methylation in the reverse direction consuming the methyl group from the methylated product (vanillin/isovanillin 1c). Following mentioned transformations in both directions over time using equimolar amounts of substrates (Fig. 5b) proved this hypothesis. After having a fast methyl transfer at the beginning of the reaction, it rapidly reached an equilibrium after 4–6 h, ending up with a comparable ratio of substrates and products in any case. In contrast, employing a 5-fold molar excess of methyl acceptor 2c significantly pushed the demethylation of 1a by diminishing the reverse reaction at the same time (Fig. 5c). Hence, in order to exploit cobalamin-dependent methyl transfer either for demethylation or methylation of a substrate in an efficient way, pushing the reaction equilibrium toward either side is crucial.

Fig. 5

Reversibility of corrinoid-dependent methyl transfer catalyzed by MTase I and CP. a General reaction scheme of the reversible model reaction, in which product and demethylated acceptor obtained by demethylation (black) serve again as substrate and methyl donor for a reverse methylation (gray). b Time course of forward and reverse reaction using equimolar amounts of substrates. c Time course of demethylation using an excess amount of acceptor. Conversions at indicated time points determined by HPLC-UV from respective areas using calibration curves. Error bars represent the standard error of the mean (s.e.m., n = 3). Reaction conditions in panel (b): substrate 1a (10 mM) and methyl acceptor 2c (10 mM) in case of demethylation; substrate 2a (10 mM) and methyl donor m-1c (10 mM) in case of methylation. MTase I (freeze-dried pure enzyme, 13 mg mL−1), CP (400 µL mL−1 reconstituted solution, 22 mg mL−1 pure CP) in MOPS/KOH buffer (50 mM, pH 6.5, 150 mM KCl) at 30 °C, 800 rpm for 24 h. Reaction conditions in panel (c): substrate 1a (10 mM) and methyl acceptor 2c (50 mM), MTase I (freeze-dried cell-free extract CFE, 40 mg mL−1), CP (reconstituted solution, 400 µL mL−1) in MOPS/KOH buffer (50 mM, pH 6.5, 150 mM KCl) at 30 °C, 800 rpm for 24 h

Substrate examples for demethylation and methylation

To demonstrate the feasibility of the concept we focused at first on demethylation. Compounds turning out to be easily demethylated might then serve as suitable methyl donors for the methylation reaction in a second step. For initial experiments with MTase I from D. hafniense catechol 2a and derivatives 2b-2c were employed in excess as methyl acceptors. In addition to the model substrate 1a various other aromatic compounds (Fig. 6) fitting the catechol pattern of published natural substrates52 as well as a series of aromatic and aliphatic compounds with alternative substrate pattern such as veratrol derivatives as well as 2-(methylamino)- and 2-(methylthio)phenol were tested for demethylation (Supplementary Table 3). Aromatic substrates with a hydroxy moiety in ortho-position to the methoxy were efficiently demethylated. Furthermore, a number of anisol derivatives (Fig. 6, compounds 1a-h, 2h) bearing various substituents such as an amine-, hydroxymethyl-, aldehyde-, or acid-group in ortho-position were converted as well. Remarkably, o-anidisine 1d was the only substrate which was demethylated with higher conversions when the acceptor was present in equimolar amounts instead of excess (Supplementary Table 4). This indicated a reaction equilibrium lying on the demethylation reaction, most likely because the formed product 2-aminophenol 2d turned out to be not a suitable substrate for methylation. Generally, the choice of the proper acceptor is an important parameter to reach high conversion; interestingly, the most suitable acceptor seemed to vary depending on the substrate. Vanillin and vanillic acid as well their regioisomers (compounds 1b-c) were demethylated with moderate conversions ranging from 25–45%. Substrates such as guaiacol 1a, 3-methoxycatechol 3-2h and 2-methoxyresorcinol 2–2h were demethylated with up to 80% conversion depending on the used co-substrate. Even higher conversions than for 1a were achieved for 2,3-dimethoxyphenol 2,3-1h (up to 93%) and 2,6-1h (up to 97%). Featuring two methoxy moieties, 2,3-1h and 2,6-1h represent particularly useful methyl sources for a possible methylation reaction.

Fig. 6

Biocatalytic demethylation of various methyl phenyl ethers employing cobalamin-dependent MTase I from D. hafniense. Reaction conditions: substrate (10 mM), methyl acceptor 2a-c (50 mM, if not indicated otherwise), MTase I (freeze-dried CFE, 40 mg mL−1), CP (400 µL mL−1 reconstituted solution) in MOPS/KOH buffer (50 mM, pH 6.5, 150 mM KCl) at 30 °C, 800 rpm for 24 h. Conversions determined by HPLC-UV from areas for substrate and demethylated product, if not indicated otherwise, using calibration curves. Since the reaction has no by-products, thus the chemoselectivity was >99%, the conversion corresponds to the amount of product formed. Deviation values (s.e.m., n = 3) lie between 0.1 and 3.3 % for the substrates tested. [i] Reaction conditions as described but using methyl acceptor 2a-c at 10 mM concentration. [ii] Conversion determined by HPLC-UV from areas for the methylated acceptor using calibration curves and related to the limiting substrate concentration. Deviation values (s.e.m., n = 3) lie between 0.2 and 0.4% for the substrates tested

Transfer of one methyl moiety leads to the formation of 3-methoxycatechol 3-2h, which is further converted to pyrogallol 3h upon twofold demethylation. Therefore, they were employed together with 1a as methyl-donating co-substrates for exploration of corrinoid-dependent methylation in the next step (Fig. 7).

Fig. 7

Biocatalytic methylation of substituted catechols employing cobalamin-dependent MTase I from D. hafniense. Reaction conditions: substrate (10 mM) and methyl donor 1a/2,3-1h/2,6-1h (2× = 2 equivalents, 5× = 5 equivalents), MTase I (freeze-dried CFE, 40 mg mL−1), CP (reconstituted solution, 400 µL mL−1) in MOPS/KOH buffer (50 mM, pH 6.5, 150 mM KCl) at 30 °C, 800 rpm for 24 h. Conversions determined by HPLC-UV from areas for substrate and methylated product using calibration curves (mono-methylation; m = meta, p = para refers to the position of the introduced methyl group in relation to the substituent at the catechol chore). Since the reaction has no by-products, thus the chemoselectivity was >99%, the conversion corresponds to the amount of product formed. Deviation values (s.e.m., n = 3) lie between 0.1 and 2.7% for the substrates tested. [i] Reaction conditions as described but with MTase I (freeze-dried pure enzyme, 13 mg mL−1) and CP (400 µL mL−1 reconstituted solution, 22 mg mL−1 pure CP) in pure form. [ii] Addition of 10% (v/v) EtOH. [iii] Preparative scale: substrate (50 mM), methyl donor 2,6-1h (50 mM). [iv] Preparative scale: substrate (20 mM), methyl donor 2,6-1h (20 mM)

Besides catechol 2a, 3,4-dihydroxybenzoic acid 2b and related aldehyde 2c, also 3,4-dihydroxybenzyl alcohol 2i and caffeic acid 2j were successfully methylated. Although a number of aliphatic, cyclic and aromatic compounds including also S-,N-, and C-nucleophiles (see supplementary Table 5) turned out to be non-substrates, numerous enzyme engineering techniques59 that have emerged in recent years might tune the MTase I from D. hafniense to convert an extended range of different substrates with high activity and selectivity. When using the mono-methyl donor 1a as co-substrate, it had to be employed in excess to achieve an efficient reaction. For instance, the amount of methylated product doubled in case of 2b, 2i, and 2j by using a 5-fold excess of donor, reaching up to 84% conversion. Interestingly, when bis-methyl donors capable of donating two methyl groups such as dimethoxyphenols 2,3-1h and 2,6-1h were used as methylating reagent, conversions up to 75% were already achieved with equimolar amounts of donor, indicating their potential as efficient donors. It seemed that the availability of two transferable methyl groups in a single molecule had a similar effect on the reaction as using excess of donor. It is worth mentioning, that exclusive mono-methylation was observed for all substrates investigated. For those substrates where the formation of regioisomers was possible, product mixtures of meta- and para-methylated isomers were identified. Generally, meta-preference was observed for substrates 2b, 2c, and 2j with a regioisomeric excess of approximately = 65/35 or higher. Therefore, the corrinoid-dependent MTase I from D. hafniense displayed a similar regioselectivity than observed for various SAM-dependent catechol O-MTases (COMT)1,60,61. One exception was 3,4-dihydroxybenzyl alcohol 2i for which almost an 1/1 mixture of isomers with marginal p-preference was obtained. Since related compound 2c on the contrary was methylated with reasonable regioselectivity (m/p up to 76/24) it can be assumed that the side chain at the aromatic catechol chore has an effect on substrate binding within the enzyme and thus on discrimination of regioisomeric groups1,44. However, the regioselectivity of MTase I could be increased in favor of the m-methylated product by medium engineering. For instance, in the presence of an organic co-solvent such as ethanol (EtOH, 10% v/v) the ratio of ferulic/isoferulic acid obtained from substrate 2j was significantly increased from 71/29 to 85/15. An even more pronounced effect of EtOH addition on the regioselectivity was observed in case of substrate 2i. Here, the minimal preference for the p-methylated product (m/p = 48/52) turned into a surplus formation of the meta-isomer (m/p = 81/19) without any loss of activity. Finally, the corrinoid-dependent methylation was performed in semi-preparative reactions (24 mL). Both 3,4-dihydroxybenzyl alcohol 2i and caffeic acid 2j were methylated employing equimolar amounts of 2,6-2h giving the methylated products with 72% conversion (38% isol. yield) and 76% conv. (45% isol. yield), respectively.


This study demonstrates the feasibility of a methyl transfer concept based on corrinoid-mediated shuttle catalysis. Being sustainable, safe, scalable and not restricted by cofactor recycling issues, this protocol may lay the base for an alternative method for methylation as well as demethylation. With the substrate scope identified, the applicability and possible future impact has already been demonstrated. While the concept might enable (i) tailoring of natural products, (ii) valorization of waste materials or biomass (e.g. lignin), and (iii) deprotection by demethylation on the one hand, methylation on the other hand will allow the preparation of value-added products. The substrate scope can be expanded by common rational engineering techniques and furthermore, other MTase systems with different substrate specificities can be chosen from nature’s vast supply. Therefore, corrinoid-dependent MTases may represent a valuable and promising add-on to the enzymatic toolbox applicable for organic synthesis.


General information

For DNA- and amino acid sequences of the enzymes used see Supplementary Note 1. Biocatalysts were recombinantly expressed in E. coli and used as lyophilized cell-free extract (CFE) or purified preparation. Since E. coli is not able to synthesize cobalamins56, the corrinoid protein (CP) was reconstituted with Me-Cbl prior to use in order to yield active holo-CP. General material and detailed experimental procedures related to protein expression and cofactor reconstitution are given in Supplementary Methods. Biocatalytic reactions were performed at least in triplicates in degassed buffers under inert atmosphere, using a Labstar MB10-compact Glove-box (MBraun, Germany) equipped with a special O2-sensor (MB-OX-EC) to be used with aqueous systems.

Determination of enzyme activity

See Supplementary Methods and Supplementary Fig. 1012.

Representative biotransformation procedure

Analytical biotransformation reactions were carried out on 120 µL scale as follows: MTase I (10–20 mU, 40 mg mL−1 CFE or 13 mg mL−1 pure enzyme, respectively) was dissolved in holo-CP solution (400 µL mL−1, containing 67 mg mL−1 CFE or 22 mg mL−1 pure enzyme, respectively). Then appropriate amounts of substrate (10–50 mM final concentration) and co-substrate (10–50 mM final concentration) dissolved in MOPS/KOH buffer (50 mM, pH 6.5, 150 mM KCl) were added and the reaction samples were shaken at 30 °C and 800 rpm. See Supplementary Note 2 for details with respect to the use of an O2-scavenger system during initial experiments. After 24 h an aliquot of the reaction was withdrawn and diluted (1:10) with MeCN (6 parts) and deionized water (3 parts). Precipitated protein was removed by centrifugation (14,000 rpm, 15 min) and the clear supernatant was filtered and directly analyzed by reversed-phase HLPC.

Analytical methods

See Supplementary Methods, Supplementary Table 67, and Supplementary Fig. 13. HPLC-spectra are also available (Supplementary Fig. 1416).

Preparative methylation reactions

Semi-preparative reactions were carried out on a 24 mL scale in accordance to the procedure described above using equimolar amounts of substrate and methyl donor. The reaction mixture was shaken at 30 °C and 120 rpm for 24 h. Work-up, isolation and purification were performed as follows.

Methylation of 3,4-dihydroxybenzyl alcohol

Substrate 2i (1.2 mmol, 168 mg) was methylated according to the described procedure using 2,6-1h (1.2 mmol, 185 mg) as methyl donor. The reaction mixture was quenched by adding MeCN (24 mL) and the precipitated catalyst was removed by centrifugation (4000 rpm, 30 min). Then the supernatant was extracted with EtOAc (2× 25 mL), combined organic phases were dried over Na2SO4 and the solvent evaporated under reduced pressure. Purification by column chromatography on silica (eluent: EtOAc/Hexanes = 1/1) afforded the product mixture (vanillyl/isovanillyl alcohol = 60/40) as colorless solid in 38% yield (70 mg, 0.45 mmol). Rf (EtOAc/Hexanes = 1/1) = 0.26.

Vanillyl alcohol m-2i. 1H-NMR (300 MHz, d4-MeOH): δ 6.94 (s, 1H), 6.76 (s, 2H), 4.50 (s, 2H), 3.85 (s, 3H); 13C-NMR (75 MHz, d4-MeOH): δ 148.9. 146.9, 134.2, 121.1, 116.0, 112.1, 65.3, 56.3. NMR spectra are in agreement with literature62.

Isovanillyl alcohol p-2i. 1H-NMR (300 MHz, d4-MeOH): δ 6.88 (s, 1H), 6.86 (s, 1H), 6.83 (s, 1H), 4.46 (s, 2H), 3.83 (s, 3H); 13C-NMR (75 MHz, d4-MeOH): δ 148.4, 147.5, 135.6, 119.6, 115.4, 112.5, 65.1, 56.4. NMR spectra are in agreement with literature63.

Methylation of caffeic acid

Substrate 2j (0.48 mmol, 87 mg) was methylated according to the described procedure using 2,6-1h (0.48 mmol, 74 mg) as methyl donor. The reaction mixture was basified with aqueous NaOH (10% w/v) to pH 14 and extracted with EtOAc (2 × 25 mL). After acidifying the aqueous phase with HCl (6N) to pH 1 the mixture was extracted with EtOAc (2 × 25 mL), combined organic phases were dried over Na2SO4 and the solvent evaporated. Column chromatography on silica (eluent: EtOAc/Hexanes = 1/3, AcOH 1% v/v) afforded the product mixture (ferulic/isoferulic acid = 60/40) as brownish solid in 45% yield (42 mg, 0.23 mmol). Rf (EtOAc/Hexanes = 1/1, AcOH 1% v/v) = 0.71.

Ferulic acid m-2j. 1H-NMR (300 MHz, d4-MeOH): δH [ppm] = 7.60 (d, J = 15.9 Hz, 1H), 7.18 (d, J = 1.9 Hz, 1H), 7.06 (dd, J = 8.2, 1.9 Hz, 1H), 6.81 (d, J = 8.2 Hz, 1H), 6.31 (d, J = 15.9 Hz, 1H), 3.89 (s, 3H); 13C-NMR (75 MHz, d4-MeOH): δC [ppm] = 171.0, 150.5, 149.4, 146.9, 127.8, 124.0, 116.4, 115.9, 111.6, 56.4. NMR spectra are in agreement with literature64.

Isoferulic acid p-2j. 1H-NMR (300 MHz, d4-MeOH): δH [ppm] = 7.54 (d, J = 15.9 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 6.94 (dd, J = 8.2, 2.1 Hz, 1H), 6.81 (d, J = 8.2 Hz, 1H), 6.27 (d, J = 15.9 Hz, 1H), 3.88 (s, 3H); 13C-NMR (75 MHz, d4-MeOH): δC [ppm] = 170.8, 151.4, 148.0, 146.6, 128.9, 122.7, 116.4, 114.7, 112.5, 56.3. NMR spectra are in agreement with literature65.

Characterization of products

See Supplementary Fig. 1720 for NMR-spectra.

Data availability

The data generated and/or analyzed during this study are available within the article and the supplementary information file or obtainable from the corresponding author upon reasonable request.


  1. 1.

    Law, B. J. C. et al. Effects of active-site modification and quaternary structure on the regioselectivity of catechol-O-methyltransferase. Angew. Chem. Int. Ed. 55, 2683–2687 (2016).

    CAS  Article  Google Scholar 

  2. 2.

    Zhang, M.-X., Hu, X.-H., Xu, Y.-H. & Loh, T.-P. Selective dealkylation of alkyl aryl ethers. Asian J. Org. Chem. 4, 1047–1049 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Lee, D., Park, H. L., Lee, S. W., Bhoo, S. H. & Cho, M. H. Biotechnological production of dimethoxyflavonoids using a fusion flavonoid O-methyltransferase possessing both 3′- and 7-O-methyltransferase activities. J. Nat. Prod. 80, 1467–1474 (2017).

    CAS  Article  Google Scholar 

  4. 4.

    Bjorsvik, H. R., Liguori, L. & Minisci, F. High selectivity in the oxidation of mandelic acid derivatives and in O-methylation of protocatechualdehyde: new processes for synthesis of vanillin, iso-vanillin, and heliotropin. Org. Process Res. Dev. 4, 534–543 (2000).

    CAS  Article  Google Scholar 

  5. 5.

    Zhang, H. & Tsao, R. Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Curr. Opin. Food Sci. 8, 33–42 (2016).

    Article  Google Scholar 

  6. 6.

    Wuts, P. G. M. & Greene, T. W. in Greene’s Protective Groups in Organic Synthesis 367–430 (John Wiley & Sons, Inc., 2006).

  7. 7.

    Selva, M. & Perosa, A. Green chemistry metrics: a comparative evaluation of dimethyl carbonate, methyl iodide, dimethyl sulfate and methanol as methylating agents. Green. Chem. 10, 457–464 (2008).

    CAS  Article  Google Scholar 

  8. 8.

    Wymann, W. E., Davis, R., Patterson, J. W. & Pfister, J. R. Selective alkylations of certain phenolic and enolic functions with lithium-carbonate alkyl halide. Synth. Commun. 18, 1379–1384 (1988).

    CAS  Article  Google Scholar 

  9. 9.

    Perosa, A., Selva, M., Tundo, P. & Zordan, F. Alkyl methyl carbonates as methylating agents. The O-methylation of phenols. Synlett 2000, 272–274 (2000).

    Article  Google Scholar 

  10. 10.

    Luque, R. et al. Catechol O-methylation with dimethyl carbonate over different acid-base catalysts. New J. Chem. 30, 1228–1234 (2006).

    CAS  Article  Google Scholar 

  11. 11.

    Attolini, M., Boxus, T., Biltresse, S. & Marchand-Brynaert, J. Chemoselective O-methylation of N-acylated/sulfonylated tyrosine derivatives. Tetrahedron Lett. 43, 1187–1188 (2002).

    CAS  Article  Google Scholar 

  12. 12.

    Fuhrmann, E. & Talbiersky, J. Synthesis of alkyl aryl ethers by catalytic Williamson ether synthesis with weak alkylation agents. Org. Process Res. Dev. 9, 206–211 (2005).

    CAS  Article  Google Scholar 

  13. 13.

    Fredriksson, A. & Stone-Elander, S. Rapid microwave-assisted cleavage of methyl phenyl ethers: new method for synthesizing desmethyl precursors and for removing protecting groups. J. Label. Compd. Rad. 45, 529–538 (2002).

    CAS  Article  Google Scholar 

  14. 14.

    Hart, W. E. S., Aldous, L. & Harper, J. B. Cleavage of ethers in an ionic liquid. Enhancement, selectivity and potential application. Org. Biomol. Chem. 15, 5556–5563 (2017).

    CAS  Article  Google Scholar 

  15. 15.

    McOmie, J. F. W., Watts, M. L. & West, D. E. Demethylation of aryl methyl ethers by boron tribromide. Tetrahedron 24, 2289–2292 (1968).

    CAS  Article  Google Scholar 

  16. 16.

    Pasquini, C., Coniglio, A. & Bassetti, M. Controlled dealkylation by BBr3: efficient synthesis of para-alkoxy-phenols. Tetrahedron Lett. 53, 6191–6194 (2012).

    CAS  Article  Google Scholar 

  17. 17.

    Cornella, J., Zarate, C. & Martin, R. Metal-catalyzed activation of ethers via C-O bond cleavage: a new strategy for molecular diversity. Chem. Soc. Rev. 43, 8081–8097 (2014).

    CAS  Article  Google Scholar 

  18. 18.

    Yang, B. et al. Catalytic dealkylation of ethers to alcohols on metal surfaces. Angew. Chem. Int. Ed. 55, 9881–9885 (2016).

    CAS  Article  Google Scholar 

  19. 19.

    Ahmad, R., Saa, J. M. & Cava, M. P. Regioselective O-demethylation in aporphine alkaloid series. J. Org. Chem. 42, 1228–1230 (1977).

    CAS  Article  Google Scholar 

  20. 20.

    Dodge, J. A., Stocksdale, M. G., Fahey, K. J. & Jones, C. D. Regioselectivity in the alkaline thiolate deprotection of aryl methyl ethers. J. Org. Chem. 60, 739–741 (1995).

    CAS  Article  Google Scholar 

  21. 21.

    Patel, R. N. Biocatalysis: synthesis of key intermediates for development of pharmaceuticals. ACS Catal. 1, 1056–1074 (2011).

    CAS  Article  Google Scholar 

  22. 22.

    Honig, M., Sondermann, P., Turner, N. J. & Carreira, E. M. Enantioselective chemo- and biocatalysis: partners in retrosynthesis. Angew. Chem. Int. Ed. 56, 8942–8973 (2017).

    Article  Google Scholar 

  23. 23.

    Sheldon, R. A. & Woodley, J. M. Role of biocatalysis in sustainable chemistry. Chem. Rev. 118, 801–838 (2018).

    CAS  Article  Google Scholar 

  24. 24.

    Bornscheuer, U. T. The fourth wave of biocatalysis is approaching. Philos. Trans. A. Math. Phys. Eng. Sci. 376, pii: 20170063 (2018).

    Article  Google Scholar 

  25. 25.

    Turner, N. J. & O’Reilly, E. Biocatalytic retrosynthesis. Nat. Chem. Biol. 9, 285–288 (2013).

    CAS  Article  Google Scholar 

  26. 26.

    Chen, K., Huang, X., Kan, S. B. J., Zhang, R. K. & Arnold, F. H. Enzymatic construction of highly strained carbocycles. Science 360, 71–75 (2018).

    CAS  Article  Google Scholar 

  27. 27.

    Copeland, R. A., Solomon, M. E. & Richon, V. M. Protein methyltransferases as a target class for drug discovery. Nat. Rev. Drug Discov. 8, 724–732 (2009).

    CAS  Article  Google Scholar 

  28. 28.

    Liscombe, D. K., Louie, G. V. & Noel, J. P. Architectures, mechanisms and molecular evolution of natural product methyltransferases. Nat. Prod. Rep. 29, 1238–1250 (2012).

    CAS  Article  Google Scholar 

  29. 29.

    Struck, A. W., Thompson, M. L., Wong, L. S. & Micklefield, J. S-Adenosyl-methionine-dependent methyltransferases: highly versatile enzymes in biocatalysis, biosynthesis and other biotechnological applications. ChemBioChem 13, 2642–2655 (2012).

    CAS  Article  Google Scholar 

  30. 30.

    Bennett, M. R., Shepherd, S. A., Cronin, V. A. & Micklefield, J. Recent advances in methyltransferase biocatalysis. Curr. Opin. Chem. Biol. 37, 97–106 (2017).

    CAS  Article  Google Scholar 

  31. 31.

    Ding, W. et al. The catalytic mechanism of the class C radical s-adenosylmethionine methyltransferase NosN. Angew. Chem. Int. Ed. 56, 3857–3861 (2017).

    CAS  Article  Google Scholar 

  32. 32.

    Zhang, Q., Van der Donk, W. A. & Liu, W. Radical-mediated enzymatic methylation: a tale of two SAMS. Acc. Chem. Res. 45, 555–564 (2012).

    Article  Google Scholar 

  33. 33.

    Sommer-Kamann, C., Fries, A., Mordhorst, S., Andexer, J. N. & Muller, M. Asymmetric C-alkylation by the S-adenosylmethionine-dependent methyltransferase SgvM. Angew. Chem. Int. Ed. 56, 4033–4036 (2017).

    CAS  Article  Google Scholar 

  34. 34.

    Sadler, J. C., Humphreys, L. D., Snajdrova, R. & Burley, G. A. A tandem enzymatic sp(2)-C-methylation process: coupling in situ S-adenosyl-l-methionine formation with methyl transfer. ChemBioChem 18, 992–995 (2017).

    CAS  Article  Google Scholar 

  35. 35.

    Bai, L. et al. A water-bridged H-bonding network contributes to the catalysis of the SAM-dependent C-methyltransferase HcgC. Angew. Chem. Int. Ed. 56, 10806–10809 (2017).

    CAS  Article  Google Scholar 

  36. 36.

    Zhang, J. & Zheng, Y. G. SAM/SAH Analogs as versatile tools for SAM-dependent methyltransferases. ACS Chem. Biol. 11, 583–597 (2016).

    CAS  Article  Google Scholar 

  37. 37.

    Mordhorst, S., Siegrist, J., Muller, M., Richter, M. & Andexer, J. N. Catalytic alkylation using a cyclic S-adenosylmethionine regeneration system. Angew. Chem. Int. Ed. 56, 4037–4041 (2017).

    CAS  Article  Google Scholar 

  38. 38.

    Matthews, R. G., Koutmos, M. & Datta, S. Cobalamin-dependent and cobamide-dependent methyltransferases. Curr. Opin. Struct. Biol. 18, 658–666 (2008).

    CAS  Article  Google Scholar 

  39. 39.

    Ragsdale, S. W. Catalysis of methyl group transfers involving tetrahydrofolate and B(12). Vitam. Horm. 79, 293–324 (2008).

    CAS  Article  Google Scholar 

  40. 40.

    Gruber, K., Puffer, B. & Krautler, B. Vitamin B12-derivatives-enzyme cofactors and ligands of proteins and nucleic acids. Chem. Soc. Rev. 40, 4346–4363 (2011).

    CAS  Article  Google Scholar 

  41. 41.

    Bridwell-Rabb, J. & Drennan, C. L. Vitamin B12 in the spotlight again. Curr. Opin. Chem. Biol. 37, 63–70 (2017).

    CAS  Article  Google Scholar 

  42. 42.

    Giedyk, M., Goliszewska, K. & Gryko, D. Vitamin B12 catalysed reactions. Chem. Soc. Rev. 44, 3391–3404 (2015).

    CAS  Article  Google Scholar 

  43. 43.

    Kornobis, K., Ruud, K. & Kozlowski, P. M. Cob(I)alamin: Insight Into the Nature of Electronically Excited States Elucidated via Quantum Chemical Computations and Analysis of Absorption, CD and MCD Data. J. Phys. Chem. A 117, 863–876 (2013).

    CAS  Article  Google Scholar 

  44. 44.

    Ludwig, M. L. & Matthews, R. G. Structures and Mechanisms: From Ashes to Enzymes. ACS Symposium Series 827 (American Chemical Society, Cambridge, 2002; 186–201.

    Book  Google Scholar 

  45. 45.

    Richter, N., Zepeck, F. & Kroutil, W. Cobalamin-dependent enzymatic O-, N-, and S-demethylation. Trends Biotechnol. 33, 371–373 (2015).

    CAS  Article  Google Scholar 

  46. 46.

    Kaufmann, F., Wohlfarth, G. & Diekert, G. O-demethylase from Acetobacterium dehalogenans. Substrate specificity and function of the participating proteins. Eur. J. Biochem. 253, 706–711 (1998).

    CAS  Article  Google Scholar 

  47. 47.

    Kalil, M. S. & Stephens, G. M. Catechol production by O-demethylation of 2-methoxyphenol using the obligate anaerobe Acetobacterium woodii. Biotechnol. Lett. 19, 1165–1168 (1997).

    CAS  Article  Google Scholar 

  48. 48.

    Naidu, D. & Ragsdale, S. W. Characterization of a three-component vanillate O-demethylase from Moorella thermoacetica. J. Bacteriol. 183, 3276–3281 (2001).

    CAS  Article  Google Scholar 

  49. 49.

    Kreher, S., Schilhabel, A. & Diekert, G. Enzymes involved in the anoxic utilization of phenyl methyl ethers by Desulfitobacterium hafniense DCB2 and Desulfitobacterium hafniense PCE-S. Arch. Microbiol. 190, 489–495 (2008).

    CAS  Article  Google Scholar 

  50. 50.

    Mingo, F. S., Studenik, S. & Diekert, G. Conversion of phenyl methyl ethers by Desulfitobacterium spp. and screening for the genes involved. FEMS Microbiol Ecol. 90, 783–790 (2014).

    CAS  Article  Google Scholar 

  51. 51.

    Schilhabel, A. et al. The ether-cleaving methyltransferase system of the strict anaerobe Acetobacterium dehalogenans: Analysis and expression of the encoding genes. J. Bacteriol. 191, 588–599 (2009).

    CAS  Article  Google Scholar 

  52. 52.

    Studenik, S., Vogel, M. & Diekert, G. Characterization of an O-demethylase of desulfitobacterium hafniense DCB-2. J. Bacteriol. 194, 3317–3326 (2012).

    CAS  Article  Google Scholar 

  53. 53.

    Bhawal, B. N. & Morandi, B. Catalytic transfer functionalization through shuttle catalysis. ACS Catal. 6, 7528–7535 (2016).

    CAS  Article  Google Scholar 

  54. 54.

    Bhawal, B. N. & Morandi, B. Shuttle catalysis-new strategies in organic synthesis. Chem. Eur. J. 23, 12004–12013 (2017).

    CAS  Article  Google Scholar 

  55. 55.

    Fang, X. J., Cacherat, B. & Morandi, B. CO- and HCl-free synthesis of acid chlorides from unsaturated hydrocarbons via shuttle catalysis. Nat. Chem. 9, 1105–1109 (2017).

    CAS  Article  Google Scholar 

  56. 56.

    Roth, J. R., Lawrence, J. G. & Bobik, T. A. Cobalamin (coenzyme B-12): synthesis and biological significance. Annu. Rev. Microbiol. 50, 137–181 (1996).

    CAS  Article  Google Scholar 

  57. 57.

    Marinović, M. et al. Selective cleavage of lignin β-O-4 aryl ether bond by β-etherase of the white-rot fungus Dichomitus squalens. ACS Sustain. Chem. Eng. 6, 2878–2882 (2018).

    Article  Google Scholar 

  58. 58.

    Lan, W., Amiri, M. T., Hunston, C. M. & Luterbacher, J. S. Protection group effects during alpha,gamma-Diol lignin stabilization promote high-selectivity monomer production. Angew. Chem. Int. Ed. 57, 1356–1360 (2018).

    CAS  Article  Google Scholar 

  59. 59.

    Hammer, S. C., Knight, A. M. & Arnold, F. H. Design and evolution of enzymes for non-natural chemistry. Curr. Opin. Green. Sustain. Chem. 7, 23–30 (2017).

    Article  Google Scholar 

  60. 60.

    Creveling, C. R., Daly, J., Shimizu, H., Morris, N. & Ong, H. H. Catechol O-methyltransferase.IV. Factors Affecting meta methylation and para methylation of substituted catechols. Mol. Pharmacol. 8, 398–409 (1972).

    CAS  PubMed  Google Scholar 

  61. 61.

    Siegrist, J. et al. Functional and structural characterisation of a bacterial O-methyltransferase and factors determining regioselectivity. FEBS Lett. 591, 312–321 (2017).

    CAS  Article  Google Scholar 

  62. 62.

    Sharma, U., Kumar, N., Verma, P. K., Kumar, V. & Singh, B. Zinc phthalocyanine with PEG-400 as a recyclable catalytic system for selective reduction of aromatic nitro compounds. Green. Chem. 14, 2289–2293 (2012).

    CAS  Article  Google Scholar 

  63. 63.

    Sun, B. et al. Design, synthesis and biological evaluation of novel macrocyclic bisbibenzyl analogues as tubulin polymerization inhibitors. Eur. J. Med. Chem. 121, 484–499 (2016).

    CAS  Article  Google Scholar 

  64. 64.

    Jarrige, L., Blanchard, F. & Masson, G. Enantioselective organocatalytic intramolecular Aza-Diels-Alder reaction. Angew. Chem. Int. Ed. 56, 10573–10576 (2017).

    CAS  Article  Google Scholar 

  65. 65.

    Mo, E.J. et al. Inositol derivatives and phenolic compounds from the roots of Taraxacum coreanum. Molecules 22, pii: E1349 (2017).

    Article  Google Scholar 

Download references


This study was financed by the Austrian FFG, BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT through the Austrian FFG-COMET- Funding Program (Austrian Centre of Industrial Biotechnology). Financial support by the Austrian Science Fund (FWF) within the DK “Molecular Enzymology” program (project no. W901) is gratefully acknowledged. Financial support by the Austrian Science fund is acknowledged (P30920-B21). The drawing of the shuttle bus of the graphical abstract was taken from vecteezy ( under the common licence BY-SA and modified.

Author information




W.K. conceived the project idea; J.E.F., N.R., F.Z. and W.K. designed the study; J.E.F., N.R., K.H., S.B. and M.P. performed the experiments; N.R. and J.E.F developed methods, J.E.F. and N.R. analyzed and interpreted the data; J.E.F. and W.K. wrote the manuscript. Funding acquisition: W.K. F.Z. and W.S.; supervision: W. K. All the authors discussed the results and approved the final manuscript.

Corresponding author

Correspondence to Wolfgang Kroutil.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Farnberger, J.E., Richter, N., Hiebler, K. et al. Biocatalytic methylation and demethylation via a shuttle catalysis concept involving corrinoid proteins. Commun Chem 1, 82 (2018).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing