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

Abstract

Regioselective reactions represent a significant challenge for organic chemistry. Here the regioselective methylation of a single hydroxy group of 4-substituted catechols was investigated employing the cobalamin-dependent methyltransferase from Desulfitobacterium hafniense. Catechols substituted in position four were methylated either in meta-or para-position to the substituent depending whether the substituent was polar or apolar. While the biocatalytic cobalamin dependent methylation was meta-selective with 4-substituted catechols bearing hydrophilic groups, it was para-selective for hydrophobic substituents. Furthermore, the presence of water miscible co-solvents had a clear improving influence, whereby THF turned out to enable the formation of a single regioisomer in selected cases. Finally, it was found that also the pH led to an enhancement of regioselectivity for the cases investigated.

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

“…For this purpose the cobalamin-dependent methyltransferase (dhaf-MT, 37.5 kDa, 327 amino acids, expression level 3.5%) and the corrinoidbinding protein (dhaf-CP, 21.4 kDa, 212 amino acids, expression level 3%) originating from the strictly anaerobic bacterium Desultobacterium hafniense were investigated. [60][61][62][63] To decide whether the MT is at the N-terminus or at the C-terminus, dhaf-MT and dhaf-CP were aligned to the natural methyltransfer protein cmuA (chloromethane utilisation) 75,76 from Hyphomicrobium chloromethanicum, that contains a methyltransfer domain and a cobalamin domain. 77 It is worth to note that this enzyme cmuA has never been expressed in E. coli before and also all our trials to express cmuA failed.…”

“…57–59 catalyze both reactions, the methylation of phenols and the demethylation of methyl phenyl ethers in a reversible manner (Scheme 1). 60–64 Depending on the methyl acceptor used, irreversible demethylation has been shown. 65…”

“…[57][58][59] catalyze both reactions, the methylation of phenols and the demethylation of methyl phenyl ethers in a reversible manner (Scheme 1). [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.…”

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

“…Their scope to date is very similar to that of SAM-dependent MTs although, unlike SAMdependent enzymes which can only catalyse the methylation/ alkylation reactions, cobalamin-dependent MTs can also be employed to perform the cleavage of CÀ O bonds and have been recently used as a complementary tool to P450s for enzymatic demethylation. [259,260] Methylation of phenols is a common strategy in organic synthesis to protect the phenol moiety and perform further chemistry. Subsequent deprotection often involves the use of strong acids such as HBr or hazardous reagents such as BBr 3 so alternative strategies to perform this process under mild conditions are advantageous.…”

“…Alternatively, cobalamin‐dependent MTs have also emerged as a valuable tool for O ‐methylation. Their scope to date is very similar to that of SAM‐dependent MTs although, unlike SAM‐dependent enzymes which can only catalyse the methylation/alkylation reactions, cobalamin‐dependent MTs can also be employed to perform the cleavage of C−O bonds and have been recently used as a complementary tool to P450s for enzymatic demethylation [259,260] . Methylation of phenols is a common strategy in organic synthesis to protect the phenol moiety and perform further chemistry.…”