Selective Biocatalytic N-Methylation of Unsaturated Heterocycles

Abstract

Methods for regioselective N-methylation and -alkylation of unsaturated heterocycles with "off the shelf" reagents are highly sought-after. This reaction could drastically simplify synthesis of privileged bioactive molecules. Here we report engineered and natural methyltransferases for challenging N-(m)ethylation of heterocycles, including benzimidazoles, benzotriazoles, imidazoles and indazoles. The reactions are performed through a cyclic enzyme cascade that consists of two methyltransferases using only iodoalkanes or methyl tosylate as simple reagents. This method enables the selective synthesis of important molecules that are otherwise difficult to access, proceeds with high regioselectivity (r.r. up to >99 %), yield (up to 99 %), on a preparative scale, and with nearly equimolar concentrations of simple starting materials.

Keywords: Alkylation; Biocatalysis; Heterocycles; Methyltransferase; SAM Recycling.

Figure 1

N‐(m)ethylated heterocyclic compounds. A) Examples of pharmaceuticals containing N‐(m)ethylated heterocycles. B) Synthetic strategies to access N‐substituted heterocycles typically involve de novo ring construction, here shown in the selective synthesis of N‐methylated benzimidazoles and indazoles as an example. Such syntheses proceed via multistep reaction sequences, with the N‐methylation/‐alkylation pattern being set right in the beginning. This can be laborious, especially in the direct conversion of lead structures into several derivatives. C) Catalytic regioselective alkylation: As countless N‐heterocycles are commercially available, a direct regioselective methylation/alkylation with synthetic reagents would significantly shorten synthesis.

Figure 2

Regioselective biocatalytic methylation. A) Cyclic enzyme cascade applied in this study. SAM is used as cosubstrate by methyltransferases in selective alkylation reactions. The produced SAH byproduct can be methylated by an anion MT using simple methyl iodide. B) We identified a panel of MTs (engineered variants and wildtype enzymes) that show broad activity‐selectivity profiles for different N‐heterocycles. The figure shows such a profile for 5‐bromobenzimidazole as substrate. While the wildtype NNMT (grey) shows low activity and selectivity in the methylation reaction using SAM as cosubstrate, various variants of NMMT (v20, v31, v53) and the wildtype HNMT revealed high activity and selectivity in the promiscuous C−N bond formation. C) Regioselective (m)ethylation of various N‐heterocycles using the cascade of Figure 2A. Each product is shown with the corresponding enzyme. The yield is given as mean value from triplicate experiments. The regioselectivity is reported as regioisomeric ratios (r.r., in blue). Standard reaction conditions: 2 mM substrate, 1 mol % of both enzymes and SAH, 5 equiv haloalkane, 2 % DMSO, 20 h, RT. ^a) 2 mol % enzyme variant, 48 h. ^b) 1 mM substrate. ^c) 5 mol % enzyme variant, 48 h. ^d) 3.5 % DMSO. acp=(S)‐3‐amino‐3‐carboxypropyl. ado=adenosyl.

Figure 3

Enzymatic SAH alkylation using methyl tosylate as reagent. A) Reaction scheme of the enzymatic SAM formation by SAH methylation using MeOTs. B) Time course for the acl‐catalyzed SAM formation using MeI (red) and MeOTs (blue). The reaction leads to 100 % conversion within minutes. Conversion is shown as mean value (n=3) with standard deviation as error bars. Control experiments without enzyme (acl‐MT) are shown in green (MeI) and orange (MeOTs). Reaction conditions: 1 mM SAH, 1 mol % acl‐MT, 1.5 equiv MeI or MeOTs, RT. C) Molecular Dynamics (MD) simulations, a total of 2.5 μs of MD simulation time from 5 independent replicas, were used to characterize the conformational dynamics of acl‐MT with SAH bound. The flexible N‐terminus loop 1 (residues 1–8) and loop 2 (residues 35–47) are highlighted in yellow and green, respectively. The accessible empty space in the acl‐MT with SAH bound active site is shown as a blue surface in a representative snapshot obtained from MD simulations. The catalytically relevant binding modes of MeOTs, bound in a near attack conformation (NAC) for an efficient methyl transfer to SAH, were characterized using restrained‐MD simulations. MeOTs can occupy the space generated in the active site by the displacement of the flexible loops. See Supporting Information (Figures S15–S23) for more details.

Figure 4

Enzymatic preparative scale methylation. 5‐bromobenzimidazole was selectively methylated using either MeI or MeOTs as methyl source. The GC chromatograms on the right highlight the selectivities achieved. Reaction conditions with MeI: 1 mM substrate, 2 % i‐PrOH, 0.5 mol % acl‐MT, 2 mol % v31, 1 mol % SAH. Reaction conditions with MeOTs: 2 mM substrate, 2 % i‐PrOH, 0.5 mol % acl‐MT, 1 mol % HNMT and SAH. All enzymes have been used in purified form.