Biocatalytic Methyl Ether Cleavage: Characterization of the Corrinoid‐Dependent Methyl Transfer System from Desulfitobacterium hafniense

Abstract

The ether functionality represents a very common motif in organic chemistry and especially the methyl ether is commonly found in natural products. Its formation and cleavage can be achieved via countless chemical procedures. Nevertheless, since in particular the cleavage often involves harsh reaction conditions, milder alternatives are highly demanded. Very recently, we have reported on a biocatalytic shuttle catalysis concept for reversible cleavage and formation of phenolic O-methyl ethers employing a corrinoid-dependent methyl transferase system from the anaerobic organism Desulfitobacterium hafniense.Here we report the technical study of this system, focusing on the demethylation of guaiacol as model reaction. The optimal buffer-, pH-, temperature-and cofactor-preferences were determined as well as the influence of organic co-solvents. Beside methyl cobalamin also hydroxocobalamin turned out to be a suitable cofactor species, although the latter required activation. Various O-methyl phenyl ethers were successfully demethylated with conversions up to 82% at 10 mM substrate concentration.

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

“…This type of substrate was not accepted by the previously described MTase from D. hafniense . 31 , 34 − 36 However, when MT-vdmB was used in combination with the carrier protein vdmA from the same organism as described in literature, only very low conversion for demethylation of veratrol 1a (10 mM substrate concentration) was observed (3%) when using a 5-fold molar excess of 3,4-dihydroxybenzaldehyde 4c as a methyl acceptor ( Scheme 1 ). The acceptor was converted to the regioisomers vanillin 2c and isovanillin 3c in a 3:1 ratio.…”

“… 32 , 33 A specific methyltransferase dhaf4610 from Desulfitobacterium hafniense ( D. hafniense ) has been recently employed for the methylation of catechols and demethylation of guaiacol derivatives. 34 − 36 Unlike the well-investigated S -adenosyl methionine (SAM)-dependent methyltransferases, 37 − 39 which are restricted toward methylation only, this MTase 29 , 40 has been shown to enable both methylation and demethylation in a reversible manner. 34 , 35 Thereby, the MTase transfers the methyl group from the cofactor methylcobalamin (Me-Cob III ) 41 − 44 to the substrate giving cobalt in the oxidation state I (Cob I ).…”

“…Thus, in a typical reaction, the methyl transfer occurred from the substrate guaiacol 2a as methyl donor to 3,4-dihydroxybenzoic acid 4c as methyl acceptor. 31 , 34 , 35 …”

Regioselectivity of Cobalamin‐Dependent Methyltransferase Can Be Tuned by Reaction Conditions and Substrate

“…Preparation of holo ‐CP. The functional holo ‐CP was obtained by reconstitution of the corrinoid protein with exogenous cofactor (methyl cobalamin) under inert atmosphere as already described [16b] . 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.…”

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

“…[13] Previously, we showed that these cobalamin methyltransferases (cob-MT) are able to shuttle the methyl group between structurally related molecules, thus from guaiacol derivatives to catechol derivatives. However, that reaction was limited by its equilibrium (Scheme 1 A); [14] By omitting a methyl acceptor, isomerization, and thus intramolecular methyl transfer, was observed (Scheme 1 B). [15] This isomerization was also a prominent side reaction in the case of the intermolecular methyl transfer due to equilibria.…”