RESUMO
Reactions that were once the exclusive province of synthetic catalysts can increasingly be addressed using biocatalysis. Through discovery of unnatural enzyme reactions, biochemists have significantly expanded the reach of enzymatic catalysis to include carbene transfer chemistries including olefin cyclopropanation. Here we describe hemoprotein cyclopropanation catalysts derived from thermophilic bacterial globins that react with diazoacetone and an unactivated olefin substrate to furnish a cyclopropyl ketone, a previously unreported reaction for enzyme catalysts. We further demonstrate that the resulting cyclopropyl ketone can be converted to a key cyclopropanol intermediate that occurs en route to the anti-hepatitis C drug grazoprevir.
Assuntos
Proteínas de Bactérias/química , Ciclopropanos/síntese química , Hemeproteínas/química , Propanóis/síntese química , Alcenos/química , Amidas , Compostos Azo/química , Proteínas de Bactérias/genética , Biocatálise , Carbamatos , Ciclização , Evolução Molecular Direcionada , Hemeproteínas/genética , Estrutura Molecular , Mutagênese Sítio-Dirigida , Estudo de Prova de Conceito , Quinoxalinas/química , Sulfonamidas , Verrucomicrobia/químicaRESUMO
Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4''-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4''-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.
