Synthetic Reagents for Enzyme‐Catalyzed Methylation

Abstract

Late-stage methylation is a key technology in the development of pharmaceutical compounds. Methyltransferase biocatalysis may provide powerful options to insert methyl groups into complex molecules with high regio-and chemoselectivity. The challenge of a large-scale application of methyltransferases is their dependence on S-adenosylmethionine (SAM) as a stoichiometric, and thus exceedingly expensive co-substrate. As a solution to this problem, we and others have explored the use of methyl halides as reagents for the in situ regeneration of SAM. However, the need to handle volatile electrophiles, such as methyl iodide (MeI), may also hamper applications at scale. As a more practical solution, we have now developed an enzyme-catalyzed process for the regeneration of SAM with methyl toluene sulfonate. Herein, we describe enzymes from the thiopurine methyltransferase family that accept sulfate-and sulfonate-based methyl donors to convert Sadenosylhomocysteine into SAM with efficiencies that rival MeI-based reactions.

Selective Biocatalytic N‐Methylation of Unsaturated Heterocycles

“…[37] These conclusions on the role of the disordered N-terminus in more efficient binding of bulkier substrates is in agreement with results from the Seebeck group obtained through X-ray crystallography. [14] Having selective enzymes and simple reagents in hand, we finally aimed to understand the scope and limitation of this approach in preparative scale reactions. We synthesized the methylated benzimidazoles 2 f and 2 g using either MeI…”

Selective Biocatalytic N‐Methylation of Unsaturated Heterocycles

“…[12] The cascade is driven by a promiscuous anion MT that uses iodo-and bromoalkanes as well as methylsulfates and -sulfonates as simple precursors to generate SAM or analogs thereof through enzymatic alkylation of S-adenosyl-Lhomocysteine. [9,11,13,14] A second MT utilizes SAM (or its analogue) as electrophile in highly selective methylation/ alkylation reactions. In a first study, we reported that this biocatalytic system can be engineered to methylate pyrazoles with high regioselectivity using iodomethane as reagent.…”

“…[9,11] Very recently the Seebeck group has also reported that methylsulfates and -sulfonates can be used as methyl source. [14] Since anion MTs and related enzymes do not only bind halides, [37] but also hydrogen sulfide anion, [38,39] thiocyanate [37,40] and phosphonates, [41] we postulated that such enzymes could also activate a broader range of leaving groups for SAH methylation (and SAM regeneration). We studied the promiscuous activity of our in-house anion MT library to activate phosphates, mesylates and tosylates as leaving groups (Figure S14).…”

“…We found that methyl tosylate (MeOTs) is well accepted to synthesize SAM from SAH (Figure 3A), which further confirms the original work from the Seebeck group. [14] To us, this is of interest as MeOTs is a "hard" reagent that may show different non-enzymatic background methylation behavior in biotransformations than the "soft" MeI, therefore expanding this methodology towards a broader range of potential nucleophiles. In addition, tosylates are not only commercially available but also directly accessible from alcohols, thus, generating a link between abundant and cheap alcohols and selective biocatalytic alkylation chemistry using MTs.…”

“…[37] These conclusions on the role of the disordered N-terminus in more efficient binding of bulkier substrates is in agreement with results from the Seebeck group obtained through X-ray crystallography. [14] Having selective enzymes and simple reagents in hand, we finally aimed to understand the scope and limitation of this approach in preparative scale reactions. We synthesized the methylated benzimidazoles 2 f and 2 g using either MeI or MeOTs with excellent isolated yield (96 % and 89 %, respectively) and high regioselectivity (Figure 4).…”