摘要：Edwin Alfonzo, Deirdre Hanley, Zi-Qi Li,Kathleen M. Sicinski, Shilong Gao,and Frances H. Arnold*

转自：康龙化成

Biocatalytic Synthesis of α‑AminoEsters via Nitrene C−H Insertion

Edwin Alfonzo, Deirdre Hanley, Zi-Qi Li,Kathleen M. Sicinski, Shilong Gao,and Frances H. Arnold*

Division of Chemistry and ChemicalEngineering, California Institute of Technology, Pasadena, California 91125,United States;

—J.Am. Chem. Soc. 2024146, 27267−27273

Recommended by Yuquan Liu_PT

KEYWORDS:Enzyme chemistry (反应类型),Biocatalysis(反应类型),C(sp3)-N (成键类型), carboxylic acid esters (底物), α-amino esters (产物)

ABSTRACT: α-Aminoesters are precursors to noncanonical amino acids used in developingsmall-molecule therapeutics, biologics, and tools in chemical biology. α-C−Hamination of abundant and inexpensive carboxylic acid esters through nitrenetransfer presents a direct approach to α-amino esters. Methods fornitrene-mediated amination of the protic α-C−H bonds in carboxylic acid esters,however, are underdeveloped. This gap arises because hydrogen atom abstraction(HAA) of protic C−H bonds by electrophilic metal-nitrenoids is slow: metal-nitrenoids preferentially react with polarity-matched, hydridic C−H bonds, even whenweaker protic C−H bonds are present. This study describes the discovery andevolution of highly stable protoglobin nitrene transferasesthat catalyze the enantioselective intermolecular amination of the α-C−H bondsin carboxylic acid esters. We developed a high-throughput assay to evaluate theactivity and enantioselectivity of mutant enzymes together with their sequencesusing theEvery Variant Sequencing (evSeq)method. The assay enabled the identification of enantiodivergent enzymes thatfunction at ambient conditions in Escherichia coli whole cells and whoseactivities can be enhanced by directed evolution for the amination of a rangeof substrates.

Privious work

Thiswork

Selected substrate scope

(A)Enzyme variant survey for amination of electron-rich aryl carboxylic acidesters and (B) Identified improved variants

Prof.Frances H. Arnold etal developed protoglobin nitrene transferases thatcatalyze the enantioselective intermolecular α-C−H primary amination of carboxylicacid esters, enabling the synthesis of unprotected chiral α-aminoesters. Ahigh-throughput assay, critical in evolving a highly enantioselective enzymefor L-α-amino ester synthesis and discovering one favoring D-α-aminoesters, assessed the activity and enantioselectivity of mutant enzymes. The engineeredenzymes offer a valuable substrate scope, including access to an intermediateused for synthesizing an active pharmaceutical ingredient. Notably, theyoperate aerobically and at room temperature, unlike previous nitrenetransferases that require anaerobic environments or subambient temperatures to perform optimally. This work expands the substrates known toundergo enzymatic amination andshowcases the extent to which an enzyme’s active site can be reshaped throughDE. They anticipate that these enzymes will facilitate the functionalization ofother protic C−H bonds for synthesizing important chiral amines.

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