Regiocomplementary O‐Methylation of Catechols by Using Three‐Enzyme Cascades

Abstract

S-Adenosylmethionine (SAM)-dependent enzymes have great potential for selective alkylation processes. In this study we investigated the regiocomplementary O-methylation of catechols. Enzymatic methylation is often hampered by the need for a stoichiometric supply of SAM and the inhibitory effect of the SAM-derived byproduct on most methyltransferases. To counteract these issues we set up an enzyme cascade. Firstly, SAM was generated from l-methionine and ATP by use of an archaeal methionine adenosyltransferase. Secondly, 4-O-methylation of the substrates dopamine and dihydrocaffeic acid was achieved by use of SafC from the saframycin biosynthesis pathway in 40-70 % yield and high selectivity. The regiocomplementary 3-O-methylation was catalysed by catechol O-methyltransferase from rat. Thirdly, the beneficial influence of a nucleosidase on the overall conversion was demonstrated. The results of this study are important milestones on the pathway to catalytic SAM-dependent alkylation processes.

Functional and structural characterisation of a bacterial O‐methyltransferase and factors determining regioselectivity

“…1). The beneficial influence of EcMTAN, especially for MxSafC-catalysed reactions, has been shown previously [26]. In comparison to dihydrocaffeic acid 1d (three-carbon acidic side chain), the MxSafC-catalysed methylation of the 3,4-dihydroxybenzoic acid 1b (onecarbon acidic side chain) resulted in an unexpected meta preference.…”

“…Recent results from our group confirmed the general para selectivity of MxSafC for the substrates dopamine 1a and dihydrocaffeic acid 1d [7,26]. However, for the case of 3,4-dihydroxybenzoic acid 1b, MxSafC showed a strong preference for the meta position of the substrate, which was even more pronounced than in the case of RnCOMT [26]. …”

“…The closely related (49% identity on amino acid level) MxSafC from the saframycin biosynthetic pathway in Myxococcus xanthus is strictly para selective for its physiological substrate L-dopa and structurally similar substances such as dopamine 1a, whereas for caffeic acid 1e a lower re but still a preference for para methylation was described [7]. Recent results from our group confirmed the general para selectivity of MxSafC for the substrates dopamine 1a and dihydrocaffeic acid 1d [7,26]. However, for the case of 3,4-dihydroxybenzoic acid 1b, MxSafC showed a strong preference for the meta position of the substrate, which was even more pronounced than in the case of RnCOMT [26].…”

“…MxSafC, RnCOMT and EcMTAN were produced and purified as described previously [26]. Inserts for the variants MxSafC-K145A and RnCOMT-K144A were generated by overlap extension PCR using the following primers:…”

Asymmetric C‐Alkylation by the S‐Adenosylmethionine‐Dependent Methyltransferase SgvM

“…[17] Thec osubstrate SAM was produced in situ from l-methionine and ATP by the methionine adenosyltransferase from Thermococcus kodakarensis (TkMAT), and the SAM-derived byproduct S-adenosylhomocysteine (SAH) was degraded using methylthioadenosine/SAH nucleosidase from E. coli (EcMTAN). [18] In time-dependent LC-MS analysis,a ni ncreasing product peak with the mass of the expected 3,4-dimethyl-2-oxovalerate (2; m/z 142.7) was observed while the substrate peak decreased accordingly.After 18 hours,the total conversion of 1 was determined (Scheme 1). [19] This protocol was also used to investigate other substrates.…”