Biocatalytic Synthesis of α-Amino Esters via Nitrene C–H Insertion

Creators: Alfonzo, Edwin¹; Hanley, Deirdre¹; Li, Zi-Qi¹; Sicinski, Kathleen M.¹; Gao, Shilong¹; Arnold, Frances H.¹

1. California Institute of Technology

Abstract

α-Amino esters are precursors to noncanonical amino acids used in developing small-molecule therapeutics, biologics, and tools in chemical biology. α-C–H amination of abundant and inexpensive carboxylic acid esters through nitrene transfer presents a direct approach to α-amino esters. Methods for nitrene-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 when weaker protic C–H bonds are present. This study describes the discovery and evolution of highly stable protoglobin nitrene transferases that catalyze the enantioselective intermolecular amination of the α-C–H bonds in carboxylic acid esters. We developed a high-throughput assay to evaluate the activity and enantioselectivity of mutant enzymes together with their sequences using the Every Variant Sequencing (evSeq) method. The assay enabled the identification of enantiodivergent enzymes that function at ambient conditions in Escherichia coli whole cells and whose activities can be enhanced by directed evolution for the amination of a range of substrates.

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Acknowledgement

This work is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under award number DE-SC0021141 to F.H.A. E.A. acknowledges support from the National Institute for General Medical Sciences (K99GM152783). D.H. was supported by a Presidential Graduate Fellowship from the California Institute of Technology. K.M.S. acknowledges support from NIH Ruth L. Kirschstein National Research Service Award (1F32GM145123-01A1). We thank Dr. K. E. Johnston for providing vector graphics for Figure 2 and Dr. S. Brinkmann-Chen, Dr. A. Das, Dr. J. L. Kennemur, and Mr. R. Lal for critically reading the manuscript. This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.

Supplemental Material

Materials and methods; cloning and mutagenesis; protocol for high-throughput screening using E. coli whole cells in a 96-well deep-well plates; protocol for protein expression and analytical-scale reactions; hemochromagen assay; analytical-scale whole cell reactions and their calibration curves; scale-up enzymatic reaction; and ¹H and ¹³C NMR spectra

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