Biocatalytic Synthesis\nof α‑Amino Esters\nvia Nitrene C–H Insertion

α-Amino esters are precursors\nto noncanonical amino\nacids\nused in developing small-molecule therapeutics, biologics, and tools\nin chemical biology. α-C–H amination of abundant and\ninexpensive carboxylic acid esters through nitrene transfer presents\na direct approach to α-amino esters. Methods for nitrene-mediated\namination of the protic α-C–H bonds in carboxylic acid\nesters, however, are underdeveloped. This gap arises because hydrogen\natom abstraction (HAA) of protic C–H bonds by electrophilic\nmetal-nitrenoids is slow: metal-nitrenoids preferentially react with\npolarity-matched, hydridic C–H bonds, even when weaker protic\nC–H bonds are present. This study describes the discovery and\nevolution of highly stable protoglobin nitrene transferases that catalyze\nthe enantioselective intermolecular amination of the α-C–H\nbonds in carboxylic acid esters. We developed a high-throughput assay\nto evaluate the activity and enantioselectivity of mutant enzymes\ntogether with their sequences using the Every Variant Sequencing (evSeq)\nmethod. The assay enabled the identification of enantiodivergent enzymes\nthat function at ambient conditions in <i>Escherichia coli</i> whole cells and whose activities can be enhanced by directed evolution\nfor the amination of a range of substrates.