Reaction Discovery Using Spectroscopic Insights from an Enzymatic C–H Amination Intermediate

Das, Anuvab; Gao, Shilong; Lal, Ravi G.; Hicks, Madeline H.; Oyala, Paul H.; Arnold, Frances H.

doi:10.1021/jacs.4c05761

Reaction Discovery Using Spectroscopic Insights from an Enzymatic C–H Amination Intermediate

Journal Article · Mon Jul 22 00:00:00 EDT 2024 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/jacs.4c05761· OSTI ID:2406462

^[1]; ^[2]; Lal, Ravi G. ^[3]; Hicks, Madeline H. ^[3]; ^[3]; ^[3]

California Institute of Technology (CalTech), Pasadena, CA (United States); California Institute of Technology
California Institute of Technology (CalTech), Pasadena, CA (United States); Moderna, Boston, MA (United States)
California Institute of Technology (CalTech), Pasadena, CA (United States)

Engineered hemoproteins can selectively incorporate nitrogen from nitrene precursors like hydroxylamine, O-substituted hydroxylamines, and organic azides into organic molecules. Although iron-nitrenoids are often invoked as the reactive intermediates in these reactions, their innate reactivity and transient nature have made their characterization challenging. Here we characterize an iron-nitrosyl intermediate generated from NH₂OH within a protoglobin active site that can undergo nitrogen-group transfer catalysis, using UV–vis, electron paramagnetic resonance (EPR) spectroscopy, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) techniques. The mechanistic insights gained led to the discovery of aminating reagents—nitrite (NO₂^–), nitric oxide (NO), and nitroxyl (HNO)—that are new to both nature and synthetic chemistry. Based on the findings, we propose a catalytic cycle for C–H amination inspired by the nitrite reductase pathway. Furthermore, this study highlights the potential of engineered hemoproteins to access natural nitrogen sources for sustainable chemical synthesis and offers a new perspective on the use of biological nitrogen cycle intermediates in biocatalysis.

View Accepted Manuscript (DOE)

Research Organization:: California Institute of Technology (CalTech), Pasadena, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: SC0021141

OSTI ID:: 2406462

Journal Information:: Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Journal Issue: 30 Vol. 146; ISSN 0002-7863

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (70)

Enantioselective Intramolecular CH Amination Catalyzed by Engineered Cytochrome P450 Enzymes In Vitro and In Vivo McIntosh, John A.; Coelho, Pedro S.; Farwell, Christopher C. Angewandte Chemie International Edition, Vol. 52, Issue 35 https://doi.org/10.1002/anie.201304401	journal	July 2013
Enantioselective Aminohydroxylation of Styrenyl Olefins Catalyzed by an Engineered Hemoprotein Cho, Inha; Prier, Christopher K.; Jia, Zhi‐Jun Angewandte Chemie International Edition, Vol. 58, Issue 10 https://doi.org/10.1002/anie.201812968	journal	March 2019
Nitrene Photochemistry of Manganese N‐Haloamides** Van Trieste, Gerard P.; Reid, Kaleb A.; Hicks, Madeline H. Angewandte Chemie International Edition, Vol. 60, Issue 51 https://doi.org/10.1002/anie.202108304	journal	November 2021
Biocatalytic, Intermolecular C−H Bond Functionalization for the Synthesis of Enantioenriched Amides Athavale, Soumitra V.; Gao, Shilong; Liu, Zhen Angewandte Chemie International Edition, Vol. 60, Issue 47 https://doi.org/10.1002/anie.202110873	journal	October 2021
NO and HNO donors, nitrones, and nitroxides: Past, present, and future Oliveira, Catarina; Benfeito, Sofia; Fernandes, Carlos Medicinal Research Reviews, Vol. 38, Issue 4 https://doi.org/10.1002/med.21461	journal	November 2017
Disproportionation of O-Benzylhydroxylamine Catalyzed by a Ferric Bis-Picket Fence Porphyrin Complex McQuarters, Ashley B.; Goodrich, Lauren E.; Goodrich, Claire M. Zeitschrift für anorganische und allgemeine Chemie, Vol. 639, Issue 8-9 https://doi.org/10.1002/zaac.201300125	journal	May 2013
Nature's nitrite-to-ammonia expressway, with no stop at dinitrogen Kroneck, Peter M. H. JBIC Journal of Biological Inorganic Chemistry, Vol. 27, Issue 1 https://doi.org/10.1007/s00775-021-01921-4	journal	December 2021
Electron spin resonance of iron-nitric oxide complexes with amino acids, peptides and proteins Woolum, John C.; Tiezzi, Enzo; Commoner, Barry Biochimica et Biophysica Acta (BBA) - Protein Structure, Vol. 160, Issue 3 https://doi.org/10.1016/0005-2795(68)90204-3	journal	August 1968
Structure of nitric oxide hemoglobin Deatherage, James F.; Moffat, Keith Journal of Molecular Biology, Vol. 134, Issue 3 https://doi.org/10.1016/0022-2836(79)90360-7	journal	November 1979
Single crystal EPR of myoglobin nitroxide. Freezing-induced reversible changes in the molecular orientation of the ligand. Hori, H.; Ikeda-Saito, M.; Yonetani, T. Journal of Biological Chemistry, Vol. 256, Issue 15 https://doi.org/10.1016/S0021-9258(18)43356-X	journal	August 1981
Paramagnetic Resonance Study of Nitric Oxide Hemoglobin Kon, H. Journal of Biological Chemistry, Vol. 243, Issue 16 https://doi.org/10.1016/S0021-9258(18)93262-X	journal	August 1968
Electromagnetic Properties of Hemoproteins Yonetani, T.; Yamamoto, H.; Erman, J. E. Journal of Biological Chemistry, Vol. 247, Issue 8 https://doi.org/10.1016/S0021-9258(19)45449-5	journal	April 1972
Comparison of the reactivity of nitric oxide and nitroxyl with heme proteins Miranda, Katrina M.; Nims, Raymond W.; Thomas, Douglas D. Journal of Inorganic Biochemistry, Vol. 93, Issue 1-2 https://doi.org/10.1016/S0162-0134(02)00498-1	journal	January 2003
The emergence of nitric oxide in the biosynthesis of bacterial natural products Caranto, Jonathan D. Current Opinion in Chemical Biology, Vol. 49 https://doi.org/10.1016/j.cbpa.2018.11.007	journal	April 2019
Nitroxyl (azanone) trapping by metalloporphyrins Doctorovich, Fabio; Bikiel, Damian; Pellegrino, Juan Coordination Chemistry Reviews, Vol. 255, Issue 23-24 https://doi.org/10.1016/j.ccr.2011.04.012	journal	December 2011
New additions to the arsenal of biocatalysts for noncanonical amino acid synthesis Alfonzo, Edwin; Das, Anuvab; Arnold, Frances H. Current Opinion in Green and Sustainable Chemistry, Vol. 38 https://doi.org/10.1016/j.cogsc.2022.100701	journal	December 2022
Study on C H amination reactions catalyzed by iron porphyrin nitrene complexs with different nitrogen sources Zhang, Jiahua; Hou, Cheng; Li, Weikang Inorganic Chemistry Communications, Vol. 114 https://doi.org/10.1016/j.inoche.2020.107787	journal	April 2020
Disproportionation of hydroxylamine by water-soluble iron(III) porphyrinate compounds Bari, Sara E.; Amorebieta, Valentín T.; Gutiérrez, María M. Journal of Inorganic Biochemistry, Vol. 104, Issue 1 https://doi.org/10.1016/j.jinorgbio.2009.09.024	journal	January 2010
Hydroxylamine and the nitrogen cycle: A review Soler-Jofra, Aina; Pérez, Julio; van Loosdrecht, Mark C. M. Water Research, Vol. 190 https://doi.org/10.1016/j.watres.2020.116723	journal	February 2021
Heme P460: A (Cross) Link to Nitric Oxide Coleman, Rachael E.; Lancaster, Kyle M. Accounts of Chemical Research, Vol. 53, Issue 12 https://doi.org/10.1021/acs.accounts.0c00573	journal	November 2020
The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity Lehnert, Nicolai; Kim, Eunsuk; Dong, Hai T. Chemical Reviews, Vol. 121, Issue 24 https://doi.org/10.1021/acs.chemrev.1c00253	journal	December 2021
Biological and Bioinspired Inorganic N–N Bond-Forming Reactions Ferousi, Christina; Majer, Sean H.; DiMucci, Ida M. Chemical Reviews, Vol. 120, Issue 12 https://doi.org/10.1021/acs.chemrev.9b00629	journal	February 2020
Synthesis of Higher-Order Nitrogen-Containing Organic Compounds from Butylamine Using a Microsecond Pulse Dielectric Barrier Discharge Banerjee, Avishek; Armstrong, Owen; Girard-Lauriault, Pierre-Luc Industrial & Engineering Chemistry Research, Vol. 62, Issue 36 https://doi.org/10.1021/acs.iecr.3c01564	journal	September 2023
Six-Electron Reduction of Nitrite to Ammonia by Cytochrome c Nitrite Reductase: Insights from Density Functional Theory Studies Bykov, Dmytro; Neese, Frank Inorganic Chemistry, Vol. 54, Issue 19 https://doi.org/10.1021/acs.inorgchem.5b01506	journal	August 2015
Nature’s Machinery, Repurposed: Expanding the Repertoire of Iron-Dependent Oxygenases Dunham, Noah P.; Arnold, Frances H. ACS Catalysis, Vol. 10, Issue 20 https://doi.org/10.1021/acscatal.0c03606	journal	September 2020
Expanding Biocatalysis for Organosilane Functionalization: Enantioselective Nitrene Transfer to Benzylic Si–C–H Bonds Das, Anuvab; Long, Yueming; Maar, Ryan R. ACS Catalysis, Vol. 14, Issue 1 https://doi.org/10.1021/acscatal.3c05370	journal	December 2023
Enantioselective Enzyme-Catalyzed Aziridination Enabled by Active-Site Evolution of a Cytochrome P450 Farwell, Christopher C.; Zhang, Ruijie K.; McIntosh, John A. ACS Central Science, Vol. 1, Issue 2 https://doi.org/10.1021/acscentsci.5b00056	journal	April 2015
Reactions of Spinach Nitrite Reductase with Its Substrate, Nitrite, and a Putative Intermediate, Hydroxylamine Kuznetsova, Sofya; Knaff, David B.; Hirasawa, Masakazu Biochemistry, Vol. 43, Issue 33 https://doi.org/10.1021/bi048826r	journal	July 2004
Involvement of a Novel Hydroxylamine Oxidoreductase in Anaerobic Ammonium Oxidation Schalk, Jos; de Vries, Simon; Kuenen, J. Gijs Biochemistry, Vol. 39, Issue 18 https://doi.org/10.1021/bi992721k	journal	April 2000
P450-Catalyzed Intramolecular sp ³ C–H Amination with Arylsulfonyl Azide Substrates Singh, Ritesh; Bordeaux, Melanie; Fasan, Rudi ACS Catalysis, Vol. 4, Issue 2 https://doi.org/10.1021/cs400893n	journal	December 2013
Enzymatic C(sp ³ )-H Amination: P450-Catalyzed Conversion of Carbonazidates into Oxazolidinones Singh, Ritesh; Kolev, Joshua N.; Sutera, Philip A. ACS Catalysis, Vol. 5, Issue 3 https://doi.org/10.1021/cs5018612	journal	February 2015
Functionalized nitrogen atom transfer catalyzed by cytochrome P-450 Svastits, Edmund W.; Dawson, John H.; Breslow, Ronald Journal of the American Chemical Society, Vol. 107, Issue 22 https://doi.org/10.1021/ja00308a064	journal	October 1985
An Electron Spin Resonance Study of Some Complexes of Iron, Nitric Oxide, and Anionic Ligands McDonald, C. C.; Phillips, W. D.; Mower, H. F. Journal of the American Chemical Society, Vol. 87, Issue 15 https://doi.org/10.1021/ja01093a007	journal	August 1965
Mechanism of Reaction of Hydrogen Peroxide with Horseradish Peroxidase: Identification of Intermediates in the Catalytic Cycle Rodríguez-López, Jose Neptuno; Lowe, David J.; Hernández-Ruiz, Josefa Journal of the American Chemical Society, Vol. 123, Issue 48 https://doi.org/10.1021/ja011853+	journal	November 2001
Mechanism of the Six-Electron Reduction of Nitrite to Ammonia by Cytochrome c Nitrite Reductase Einsle, Oliver; Messerschmidt, Albrecht; Huber, Robert Journal of the American Chemical Society, Vol. 124, Issue 39 https://doi.org/10.1021/ja0206487	journal	October 2002
Enzyme-Controlled Nitrogen-Atom Transfer Enables Regiodivergent C–H Amination Hyster, Todd K.; Farwell, Christopher C.; Buller, Andrew R. Journal of the American Chemical Society, Vol. 136, Issue 44 https://doi.org/10.1021/ja509308v	journal	October 2014
Enzymatic Primary Amination of Benzylic and Allylic C(sp ³ )–H Bonds Jia, Zhi-Jun; Gao, Shilong; Arnold, Frances H. Journal of the American Chemical Society, Vol. 142, Issue 23 https://doi.org/10.1021/jacs.0c03428	journal	May 2020
A Combined Spectroscopic and Computational Study on the Mechanism of Iron-Catalyzed Aminofunctionalization of Olefins Using Hydroxylamine Derived N–O Reagent as the “Amino” Source and “Oxidant” Chatterjee, Sayanti; Harden, Ingolf; Bistoni, Giovanni Journal of the American Chemical Society, Vol. 144, Issue 6 https://doi.org/10.1021/jacs.1c11083	journal	February 2022
An Enzymatic Platform for Primary Amination of 1-Aryl-2-alkyl Alkynes Liu, Zhen; Qin, Zi-Yang; Zhu, Ledong Journal of the American Chemical Society, Vol. 144, Issue 1 https://doi.org/10.1021/jacs.1c11340	journal	December 2021
C–N Bond Forming Radical Rebound Is the Enantioselectivity-Determining Step in P411-Catalyzed Enantioselective C(sp³)–H Amination: A Combined Computational and Experimental Investigation Mai, Binh Khanh; Neris, Natalia M.; Yang, Yang Journal of the American Chemical Society, Vol. 144, Issue 25 https://doi.org/10.1021/jacs.2c02283	journal	May 2022
Enzymatic Nitrogen Insertion into Unactivated C–H Bonds Athavale, Soumitra V.; Gao, Shilong; Das, Anuvab Journal of the American Chemical Society, Vol. 144, Issue 41 https://doi.org/10.1021/jacs.2c08285	journal	October 2022
Asymmetric Radical Bicyclization for Stereoselective Construction of Tricyclic Chromanones and Chromanes with Fused Cyclopropanes Lee, Wan-Chen Cindy; Wang, Jingyi; Zhu, Yiling Journal of the American Chemical Society, Vol. 145, Issue 21 https://doi.org/10.1021/jacs.3c01618	journal	May 2023
Enzymatic Nitrogen Incorporation Using Hydroxylamine Gao, Shilong; Das, Anuvab; Alfonzo, Edwin Journal of the American Chemical Society, Vol. 145, Issue 37 https://doi.org/10.1021/jacs.3c08053	journal	September 2023
Myoglobin-Catalyzed Azide Reduction Proceeds via an Anionic Metal Amide Intermediate Tinzl, Matthias; Diedrich, Johannes V.; Mittl, Peer R. E. Journal of the American Chemical Society, Vol. 146, Issue 3 https://doi.org/10.1021/jacs.3c09279	journal	January 2024
Characterization of Porphyrin-Co(III)-‘Nitrene Radical’ Species Relevant in Catalytic Nitrene Transfer Reactions Goswami, Monalisa; Lyaskovskyy, Volodymyr; Domingos, Sérgio R. Journal of the American Chemical Society, Vol. 137, Issue 16 https://doi.org/10.1021/jacs.5b01197	journal	April 2015
Trapping a Highly Reactive Nonheme Iron Intermediate That Oxygenates Strong C—H Bonds with Stereoretention Serrano-Plana, Joan; Oloo, Williamson N.; Acosta-Rueda, Laura Journal of the American Chemical Society, Vol. 137, Issue 50 https://doi.org/10.1021/jacs.5b09904	journal	December 2015
Unprecedented Cyclization Catalyzed by a Cytochrome P450 in Benzastatin Biosynthesis Tsutsumi, Hayama; Katsuyama, Yohei; Izumikawa, Miho Journal of the American Chemical Society, Vol. 140, Issue 21 https://doi.org/10.1021/jacs.8b02769	journal	May 2018
Observation of a Photogenerated Rh ₂ Nitrenoid Intermediate in C–H Amination Das, Anuvab; Maher, Andrew G.; Telser, Joshua Journal of the American Chemical Society, Vol. 140, Issue 33 https://doi.org/10.1021/jacs.8b05599	journal	July 2018
Mechanism-Guided Design and Discovery of Efficient Cytochrome P450-Derived C–H Amination Biocatalysts Steck, Viktoria; Kolev, Joshua N.; Ren, Xinkun Journal of the American Chemical Society, Vol. 142, Issue 23 https://doi.org/10.1021/jacs.9b12859	journal	May 2020
Analysis of the Structural Diversity, Substitution Patterns, and Frequency of Nitrogen Heterocycles among U.S. FDA Approved Pharmaceuticals: Miniperspective Vitaku, Edon; Smith, David T.; Njardarson, Jon T. Journal of Medicinal Chemistry, Vol. 57, Issue 24 https://doi.org/10.1021/jm501100b	journal	October 2014
Reactive Nitrogen Species Reactivities with Nitrones: Theoretical and Experimental Studies Nash, Kevin M.; Rockenbauer, Antal; Villamena, Frederick A. Chemical Research in Toxicology, Vol. 25, Issue 8 https://doi.org/10.1021/tx200526y	journal	July 2012
Structure of cytochrome c nitrite reductase Einsle, Oliver; Messerschmidt, Albrecht; Stach, Petra Nature, Vol. 400, Issue 6743 https://doi.org/10.1038/22802	journal	July 1999
Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivity Onderko, Elizabeth L.; Silakov, Alexey; Yosca, Timothy H. Nature Chemistry, Vol. 9, Issue 7 https://doi.org/10.1038/nchem.2781	journal	May 2017
Enantioselective, intermolecular benzylic C–H amination catalysed by an engineered iron-haem enzyme Prier, Christopher K.; Zhang, Ruijie K.; Buller, Andrew R. Nature Chemistry, Vol. 9, Issue 7 https://doi.org/10.1038/nchem.2783	journal	May 2017
Reversing nitrogen fixation Lehnert, Nicolai; Dong, Hai T.; Harland, Jill B. Nature Reviews Chemistry, Vol. 2, Issue 10 https://doi.org/10.1038/s41570-018-0041-7	journal	September 2018
Addressing nitrogenous gases from croplands toward low-emission agriculture Pan, Shu-Yuan; He, Kung-Hui; Lin, Kuan-Ting npj Climate and Atmospheric Science, Vol. 5, Issue 1 https://doi.org/10.1038/s41612-022-00265-3	journal	June 2022
Capture and characterization of a reactive haem–carbenoid complex in an artificial metalloenzyme Hayashi, Takahiro; Tinzl, Matthias; Mori, Takahiro Nature Catalysis, Vol. 1, Issue 8 https://doi.org/10.1038/s41929-018-0105-6	journal	July 2018
Stereoselective construction of β-, γ- and δ-lactam rings via enzymatic C–H amidation Roy, Satyajit; Vargas, David A.; Ma, Pengchen Nature Catalysis, Vol. 7, Issue 1 https://doi.org/10.1038/s41929-023-01068-2	journal	December 2023
Metal-Diazo Radicals of α-Carbonyl Diazomethanes Li, Feifei; Xiao, Longqiang; Liu, Lijian Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep22876	journal	March 2016
Controlling a burn: outer-sphere gating of hydroxylamine oxidation by a distal base in cytochrome P460 Smith, Meghan A.; Majer, Sean H.; Vilbert, Avery C. Chemical Science, Vol. 10, Issue 13 https://doi.org/10.1039/C9SC00195F	journal	January 2019
Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460 Bollmeyer, Melissa M.; Majer, Sean H.; Coleman, Rachael E. Chemical Science, Vol. 14, Issue 31 https://doi.org/10.1039/D3SC02288A	journal	January 2023
Oxidative heme protein-mediated nitroxyl (HNO) generation Reisz, Julie A.; Bechtold, Erika; King, S. Bruce Dalton Transactions, Vol. 39, Issue 22 https://doi.org/10.1039/c000980f	journal	January 2010
Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase Lewis, Russell D.; Garcia-Borràs, Marc; Chalkley, Matthew J. Proceedings of the National Academy of Sciences, Vol. 115, Issue 28 https://doi.org/10.1073/pnas.1807027115	journal	June 2018
Kinetic Properties and Electron Paramagnetic Resonance Spectra of the Nitric Oxide Derivative of Hemoglobin Components of Trout (Salmo irideus) Brunori, M.; Falcioni, G.; Rotilio, G. Proceedings of the National Academy of Sciences, Vol. 71, Issue 6 https://doi.org/10.1073/pnas.71.6.2470	journal	June 1974
Mechanistic Enzymology of Oxygen Activation by the Cytochromes p450 Makris, Thomas M.; Davydov, Roman; Denisov, Ilia G. Drug Metabolism Reviews, Vol. 34, Issue 4 https://doi.org/10.1081/DMR-120015691	journal	January 2002
Transformation of the Nitrogen Cycle: Recent Trends, Questions, and Potential Solutions Galloway, J. N.; Townsend, A. R.; Erisman, J. W. Science, Vol. 320, Issue 5878 https://doi.org/10.1126/science.1136674	journal	May 2008
The Evolution and Future of Earth’s Nitrogen Cycle Canfield, Donald E.; Glazer, Alexander N.; Falkowski, Paul G. Science, Vol. 330, Issue 6001 https://doi.org/10.1126/science.1186120	journal	October 2010
Electron Spin Resonance of Nitric Oxide-Hemoglobin Complexes in Solution Sancier, K. M.; Freeman, G.; Mills, J. S. Science, Vol. 137, Issue 3532 https://doi.org/10.1126/science.137.3532.752	journal	September 1962
Hydroxylamine-induced oxidation of ferrous CO-bound carboxymethylated-cytochrome c Ascenzi, Paolo; De Simone, Giovanna; Ciaccio, Chiara Journal of Porphyrins and Phthalocyanines, Vol. 22, Issue 12 https://doi.org/10.1142/S1088424618501055	journal	December 2018
Fate of nitrogen in agriculture and environment: agronomic, eco-physiological and molecular approaches to improve nitrogen use efficiency Anas, Muhammad; Liao, Fen; Verma, Krishan K. Biological Research, Vol. 53, Issue 1 https://doi.org/10.1186/s40659-020-00312-4	journal	October 2020

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
biocatalysis
directed evolution
electron paramagnetic resonance
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Reaction Discovery Using Spectroscopic Insights from an Enzymatic C–H Amination Intermediate

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