Engineered Enzymes Enable Selective N‐Alkylation of Pyrazoles With Simple Haloalkanes

Selective alkylation of pyrazoles could solve a challenge in chemistry and streamline synthesis of important molecules. Here we report catalyst‐controlled pyrazole alkylation by a cyclic two‐enzyme cascade. In this enzymatic system, a promiscuous enzyme uses haloalkanes as precursors to generate non‐natural analogs of the common cosubstrate S‐adenosyl‐l‐methionine. A second engineered enzyme transfers the alkyl group in highly selective C−N bond formations to the pyrazole substrate. The cosubstrate is recycled and only used in catalytic amounts. Key is a computational enzyme‐library design tool that converted a promiscuous methyltransferase into a small enzyme family of pyrazole‐alkylating enzymes in one round of mutagenesis and screening. With this enzymatic system, pyrazole alkylation (methylation, ethylation, propylation) was achieved with unprecedented regioselectivity (>99 %), regiodivergence, and in a first example on preparative scale.