A Chemoselective and Stereodivergent Platform of Heme-Nitrene Transferases to Access Chiral Aryl-B-Amino Esters and Investigation of the Sequence-Activity Landscape

Engineered biocatalysts can utilize nitrene precursors to access enantioenriched amination products, yet they have not been applied to produce valuable, enantiomerically enriched non-canonical b-amino esters. Current approaches to synthesizing β-amino acids rely on pre-oxidized precursors and multistep synthetic approaches involving various protecting groups. We engineered a platform of heme enzymes for stereoselective C–H bond amination of readily available carboxylic ester derivatives to install primary amines. A directed evolution campaign coupled with sequencing of over 1000 variants enabled us to develop three engineered variants using either O-pivaloylhydroxylamine triflic acid (PONT) or hydroxylamine hydrochloride (H2NOH∙HCl) as aminating reagents. A simple analysis of the resulting sequence–activity dataset revealed additional improvements to the final variant, highlighting the utility of sequencing data to guide future steps in directed evolution campaigns. The evolved nitrene transferases expand the scope of accessible chiral β-amino acid building blocks for peptidomimetic applications and provide new starting points for the design and synthesis of enantioenriched b-amino acid motifs.