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.

Non-Native Site-Selective Enzyme Catalysis

“…178,179 Multi-component B12-dependent methyl transferases (MTs) catalyze this reaction with concomitant methylation of an acceptor substrate. 180 While the native methyl donors for these enzymes include substrates like CH3-H4folate, methanol, methylamine, and dimethylsulfide, [180][181][182] some can also accept various methyl phenyl ethers as substrates. Demethylation proceeds via attack of the methyl group by the nucleophilic cob(I)alamin form of a cobalamin-binding protein (e.g.…”

“…This capability enabled chemoenzymatic functionalization of different sugars via subsequent manipulation of the single deprotected hydroxyl group. 204 Screening previously engineered P450BM3 variants to identify hits for different sugars followed by further directed evolution yielded a panel of enzymes with high selectivity (~50-100% in terms of product distribution) for the demethylation of protected hexoses (182)(183)(184).…”

New Trends and Future Opportunities in the Enzymatic Formation of C−C, C−N, and C−O bonds

“…However, the need for a large excess of the methyl group acceptor to drive the equilibrium towards the formation of the desired product as well as side products observed due to isomerisation [265] represent a limitation for large scale applications. Very recently, the same authors have reported the use of thiols as methyl acceptors [266] . The use of these compounds represents a step forward towards the applicability of these systems in synthetic chemistry as they cannot be demethylated, therefore a large excess of the acceptor is no longer needed to drive the equilibrium.…”

“…Very recently, the same authors have reported the use of thiols as methyl acceptors. [266] The use of these compounds represents a step forward towards the applicability of these systems in synthetic chemistry as they cannot be demethylated, therefore a large excess of the acceptor is no longer needed to drive the equilibrium. Using the enzymatic demethylation of guaiacol as the model system, different thiols were screened, finding mercaptopropionic acid ethyl ester as the best methyl acceptor.…”

Anaerobic demethylation of guaiacyl-derived monolignols enabled by a designed artificial cobalamin methyltransferase fusion enzyme

“…[60][61][62][63][64] Depending on the methyl acceptor used, irreversible demethylation has been shown. 65 It is worth to note, that in contrast to oxidative demethylating enzymes, [42][43][44][45][46][47][48][49][50] cobalamin-dependent methyltransferases do not require molecular oxygen for demethylation. This is important, as molecular oxygen may initiate undesired side reactions of the phenolic substrates/products such as polymerisation especially for catechol derivatives.…”