Enzymatic Nitrogen Incorporation Using Hydroxylamine
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1.
California Institute of Technology
Abstract
Hydroxylamine-derived reagents have enabled versatile nitrene transfer reactions for introducing nitrogen-containing functionalities in small-molecule catalysis, as well as biocatalysis. These reagents, however, result in a poor atom economy and stoichiometric organic waste. Activating hydroxylamine (NH₂OH) for nitrene transfer offers a low-cost and sustainable route to amine synthesis, since water is the sole byproduct. Despite its presence in nature, hydroxylamine is not known to be used for enzymatic nitrogen incorporation in biosynthesis. Here, we report an engineered heme enzyme that can utilize hydroxylammonium chloride, an inexpensive commodity chemical, for nitrene transfer. Directed evolution of Pyrobaculum arsenaticum protoglobin generated efficient enzymes for benzylic C–H primary amination and styrene aminohydroxylation. Mechanistic studies supported a stepwise radical pathway involving rate-limiting hydrogen atom transfer. This unprecedented activity is a useful addition to the "nitrene transferase" repertoire and hints at possible future discovery of natural enzymes that use hydroxylamine for amination chemistry.
Copyright and License
© 2023 American Chemical Society.
Acknowledgement
This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Basic Sciences (DE-SC0021141 to F.H.A.). This research was also supported by the G. Harold & Leila Y. Mathers Foundation (MF-2111-02065 to F.H.A.). E.A. acknowledges support from an NIH Ruth L. Kirschstein NRSA Postdoctoral Fellowship (F32GM143799 to E.A.). K.M.S. acknowledges support from an NIH Ruth L. Kirschstein NRSA Postdoctoral Fellowship (F32GM145123 to K.M.S.). The authors also thank Alex Johnson for assistance with recombination experiment and Nicholas J. Porter, Patrick J. Almhjell, Kadina E. Johnston, and Sabine Brinkmann-Chen for helpful discussions and comments on the manuscript.
Conflict of Interest
The authors declare no competing financial interest.
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.
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Additional details
- ISSN
- 1520-5126
- PMCID
- PMC10560455
- United States Department of Energy
- DE-SC0021141
- G. Harold and Leila Y. Mathers Foundation
- MF-2111-02065
- National Institutes of Health
- F32GM143799
- National Institutes of Health
- F32GM145123
- Caltech groups
- Division of Biology and Biological Engineering