biostudies-literatureBengel LLBundesministerium für Bildung und ForschungDeutsche Forschungsgemeinschaft5554-5560https://www.ebi.ac.uk/biostudies/studies/S-EPMC7986378biostudies-literatureUnknown60(10)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 (mAngewandte Chemie (International ed. in English)Engineered Enzymes Enable Selective N-Alkylation of Pyrazoles With Simple Haloalkanes.PMC7986378420112577031B0359ABengel LLKienzle SHammer SCAberle BHauer BEgler-Kemmerer ANfalseEngineered 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 (m2021-01-01T00:00:00Z2021 Mar2025-04-05T15:25:50.317Z2025-04-05T15:25:50.317ZS-EPMC79863783330064610.1002/anie.202014239