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Biocatalytic, enantioenriched primary amination of tertiary C–H bonds

A preprint version of the article is available at ChemRxiv.


Intermolecular functionalization of tertiary C–H bonds to construct fully substituted stereogenic carbon centres represents a formidable challenge: without the assistance of directing groups, state-of-the-art catalysts struggle to introduce chirality to racemic tertiary sp3-carbon centres. Direct asymmetric functionalization of such centres is a worthy reactivity and selectivity goal for modern biocatalysis. Here we present an engineered nitrene transferase (P411-TEA-5274), derived from a bacterial cytochrome P450, that is capable of aminating tertiary C–H bonds to provide chiral α-tertiary primary amines with high efficiency (up to 2,300 total turnovers) and selectivity (up to >99% enantiomeric excess). The construction of fully substituted stereocentres with methyl and ethyl groups underscores the enzyme’s remarkable selectivity. A comprehensive substrate scope study demonstrates the biocatalyst’s compatibility with diverse functional groups and tertiary C–H bonds. Mechanistic studies explain how active-site residues distinguish between the enantiomers and enable the enzyme to perform this transformation with excellent enantioselectivity.

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Fig. 1: Direct enantioselective functionalization of C(sp3)–H bonds.
Fig. 2: Directed evolution for enzymatic primary amination of tertiary C–H bonds.
Fig. 3: Substrate scope study.
Fig. 4: Mechanistic studies of enantioenriched enzymatic primary amination of tertiary C–H bonds.
Fig. 5: Molecular dynamics simulations of iron-haem-catalysed tertiary C–H amination.

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Data availability

Crystallographic data are available free of charge from the Cambridge Crystallographic Data Centre under no. CCDC 2287786 (3f derivative (S)-N-(2-(4-methoxyphenyl)butan-2-yl)benzamide). The original materials and data that support the findings of this study are available within the paper and its Supplementary Information or can be obtained from the corresponding author upon reasonable request.


  1. Chu, J. C. K. & Rovis, T. Complementary strategies for directed C(sp3)–H functionalization: a comparison of transition-metal-catalyzed activation, hydrogen atom transfer, and carbene/nitrene transfer. Angew. Chem. Int. Ed. 57, 62–101 (2018).

    Article  CAS  Google Scholar 

  2. Dalton, T., Faber, T. & Glorius, F. C–H activation: toward sustainability and applications. ACS Cent. Sci. 7, 245–261 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Newton, C. G., Wang, S. G., Oliveira, C. C. & Cramer, N. Catalytic enantioselective transformations involving C–H bond cleavage by transition-metal complexes. Chem. Rev. 117, 8908–8976 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Saint-Denis, T. G., Zhu, R. Y., Chen, G., Wu, Q. F. & Yu, J. Q. Enantioselective C(sp3)–H bond activation by chiral transition metal catalysts. Science 359, eaao4798 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Rogge, T. et al. C–H activation. Nat. Rev. Methods Primers 1, 43 (2021).

    Article  CAS  Google Scholar 

  6. Davies, H. M. L. & Beckwith, R. E. J. Catalytic enantioselective C–H activation by means of metal-carbenoid-induced C–H insertion. Chem. Rev. 103, 2861–2904 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Zhang, C., Li, Z. L., Gu, Q. S. & Liu, X. Y. Catalytic enantioselective C(sp3)–H functionalization involving radical intermediates. Nat. Commun. 12, 475 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Yang, Y. & Arnold, F. H. Navigating the unnatural reaction space: directed evolution of heme proteins for selective carbene and nitrene transfer. Acc. Chem. Res. 54, 1209–1225 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang, R. K., Huang, X. & Arnold, F. H. Selective C–H bond functionalization with engineered heme proteins: new tools to generate complexity. Curr. Opin. Chem. Biol. 49, 67–75 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Lewis, J. C., Coelho, P. S. & Arnold, F. H. Enzymatic functionalization of carbon–hydrogen bonds. Chem. Soc. Rev. 40, 2003–2021 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Yang, Y., Cho, I., Qi, X., Liu, P. & Arnold, F. H. An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp3)–H bonds. Nat. Chem. 11, 987–993 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yang, C.-J. et al. Cu-catalysed intramolecular radical enantioconvergent tertiary β-C(sp3)–H amination of racemic ketones. Nat. Catal. 3, 539–546 (2020).

    Article  CAS  Google Scholar 

  13. Lang, K., Li, C., Kim, I. & Zhang, X. P. Enantioconvergent amination of racemic tertiary C–H bonds. J. Am. Chem. Soc. 142, 20902–20911 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ye, C. X., Shen, X., Chen, S. & Meggers, E. Stereocontrolled 1,3-nitrogen migration to access chiral α-amino acids. Nat. Chem. 14, 566–573 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ye, C.-X., Dansby, D. R., Chen, S. & Meggers, E. Expedited synthesis of α-amino acids by single-step enantioselective α-amination of carboxylic acids. Nat. Synth. 2, 645–652 (2023).

    Article  Google Scholar 

  16. Hahn, C. J. et al. Crystal structure of a key enzyme for anaerobic ethane activation. Science 373, 118–121 (2021).

    Article  CAS  PubMed  Google Scholar 

  17. Zanger, U. M. & Schwab, M. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol. Ther. 138, 103–141 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Huang, X. & Groves, J. T. Oxygen activation and radical transformations in heme proteins and metalloporphyrins. Chem. Rev. 118, 2491–2553 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Poulos, T. L. Cytochrome P450 flexibility. Proc. Natl Acad. Sci. USA 100, 13121–13122 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Brandenberg, O. F., Fasan, R. & Arnold, F. H. Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions. Curr. Opin. Biotechnol. 47, 102–111 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chen, K. & Arnold, F. H. Engineering new catalytic activities in enzymes. Nat. Catal. 3, 203–213 (2020).

    Article  CAS  Google Scholar 

  22. Hager, A., Vrielink, N., Hager, D., Lefranc, J. & Trauner, D. Synthetic approaches towards alkaloids bearing α-tertiary amines. Nat. Prod. Rep. 33, 491–522 (2016).

    Article  CAS  PubMed  Google Scholar 

  23. Liu, G., Cogan, D. A. & Ellman, J. A. Catalytic asymmetric synthesis oftert-butanesulfinamide. Application to the asymmetric synthesis of amines. J. Am. Chem. Soc. 119, 9913–9914 (1997).

    Article  CAS  Google Scholar 

  24. Ellman, J. A., Owens, T. D. & Tang, T. P. N-tert-butanesulfinyl imines: versatile intermediates for the asymmetric synthesis of amines. Acc. Chem. Res. 35, 984–995 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Gao, X., Turek-Herman, J. R., Choi, Y. J., Cohen, R. D. & Hyster, T. K. Photoenzymatic synthesis of α-tertiary amines by engineered flavin-dependent ‘ene’-reductases. J. Am. Chem. Soc. 143, 19643–19647 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Barrios-Rivera, J., Xu, Y., Wills, M. & Vyas, V. K. A diversity of recently reported methodology for asymmetric imine reduction. Org. Chem. Front. 7, 3312–3342 (2020).

    Article  CAS  Google Scholar 

  27. Slabu, I., Galman, J. L., Lloyd, R. C. & Turner, N. J. Discovery, engineering, and synthetic application of transaminase biocatalysts. ACS Catal. 7, 8263–8284 (2017).

    Article  CAS  Google Scholar 

  28. Afanasyev, O. I., Kuchuk, E., Usanov, D. L. & Chusov, D. Reductive amination in the synthesis of pharmaceuticals. Chem. Rev. 119, 11857–11911 (2019).

    Article  CAS  PubMed  Google Scholar 

  29. Jia, Z. J., Gao, S. & Arnold, F. H. Enzymatic primary amination of benzylic and allylic C(sp3)–H bonds. J. Am. Chem. Soc. 142, 10279–10283 (2020).

    Article  CAS  PubMed  Google Scholar 

  30. Liu, Z. et al. An enzymatic platform for primary amination of 1-aryl-2-alkyl alkynes. J. Am. Chem. Soc. 144, 80–85 (2022).

    Article  CAS  PubMed  Google Scholar 

  31. Yang, Y., Shi, S.-L., Niu, D., Liu, P. & Buchwald, S. L. Catalytic asymmetric hydroamination of unactivated internal olefins to aliphatic amines. Science 349, 62–66 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Fandrick, K. R. et al. A general copper-BINAP-catalyzed asymmetric propargylation of ketones with propargyl boronates. J. Am. Chem. Soc. 133, 10332–10335 (2011).

    Article  CAS  PubMed  Google Scholar 

  33. Kille, S. et al. Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis. ACS Synth. Biol. 2, 83–92 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Reetz, M. T. & Carballeira, J. D. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protoc. 2, 891–903 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Prier, C. K., Zhang, R. K., Buller, A. R., Brinkmann-Chen, S. & Arnold, F. H. Enantioselective, intermolecular benzylic C–H amination catalysed by an engineered iron-haem enzyme. Nat. Chem. 9, 629–634 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. D’Amato, E. M., Borgel, J. & Ritter, T. Aromatic C–H amination in hexafluoroisopropanol. Chem. Sci. 10, 2424–2428 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Legnani, L., Prina Cerai, G. & Morandi, B. Direct and practical synthesis of primary anilines through iron-catalyzed C–H bond amination. ACS Catal. 6, 8162–8165 (2016).

    Article  CAS  Google Scholar 

  38. Liu, J. et al. Fe-catalyzed amination of (hetero)arenes with a redox-active aminating reagent under mild conditions. Eur. J. Chem. 23, 563–567 (2017).

    Article  CAS  Google Scholar 

  39. Luo, Y.-R. Comprehensive Handbook of Chemical Bond Energies (Taylor & Francis, 2007).

  40. Pan, Y. et al. Kinetic resolution of α-tertiary propargylic amines through asymmetric remote aminations of anilines. ACS Catal. 11, 8443–8448 (2021).

    Article  CAS  Google Scholar 

  41. Hennessy, E. T., Liu, R. Y., Iovan, D. A., Duncan, R. A. & Betley, T. A. Iron-mediated intermolecular N-group transfer chemistry with olefinic substrates. Chem. Sci. 5, 1526–1532 (2014).

    Article  CAS  Google Scholar 

  42. Jacobs, B. P., Wolczanski, P. T., Jiang, Q., Cundari, T. R. & MacMillan, S. N. Rare examples of Fe(IV) alkyl-imide migratory insertions: impact of Fe–C covalency in (Me2IPr)Fe(=NAd)R2 (R=neoPe, 1-nor). J. Am. Chem. Soc. 139, 12145–12148 (2017).

    Article  CAS  PubMed  Google Scholar 

  43. Singh, R., Kolev, J. N., Sutera, P. A. & Fasan, R. Enzymatic C(sp3)–H amination: P450-catalyzed conversion of carbonazidates into oxazolidinones. ACS Catal. 5, 1685–1691 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Athavale, S. V. et al. Enzymatic nitrogen insertion into unactivated C–H bonds. J. Am. Chem. Soc. 144, 19097–19105 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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This work is supported by the National Institute of General Medical Science of the National Institutes of Health (grant no. R01GM138740). Support by National Science Foundation Division of Chemistry (grant no. CHE-2153972 to K.N.H.) and the Alexander von Humboldt-Foundation (Feodor Lynen Fellowship, T.R.) is gratefully acknowledged. We thank S. C. Virgil for the maintenance of the Caltech Center for Catalysis and Chemical Synthesis (3CS). We thank M. Shahgoli for mass spectrometry assistance. We thank D. Vander Velde for the maintenance of the Caltech nuclear magnetic resonance facility. We thank M. K. Takase and L. M. Henling for assistance with X-ray crystallographic data collection. We also thank S. Brinkmann-Chen for the helpful discussions and comments on the manuscript.

Author information

Authors and Affiliations



R.M. conceptualized and designed the project under the guidance of F.H.A. R.M. and S.G. carried out the initial screening of haem proteins. R.M., S.G. and S.J.W. performed the directed evolution experiments, with support from Z.-Y.Q. for the validation. R.M., S.G. and Z.-Y.Q. investigated the substrate scope and reaction mechanism, with support from Z.-Q.L. T.R. carried out the computational studies with K.N.H. providing guidance. A.D. conducted crystallization of a 3f derivative. R.M. and F.H.A. wrote the manuscript with input from all authors.

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Correspondence to Frances H. Arnold.

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Nature Catalysis thanks Bernhard Hauer, Nicholas Turner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Methods 1–23, Tables 1–8, Figs. 1–26 and References.

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Supplementary Data 1

Compound 3f derivative.cif Crystallographic data for compound 3f derivative. CCDC reference 2287786.

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Mao, R., Gao, S., Qin, ZY. et al. Biocatalytic, enantioenriched primary amination of tertiary C–H bonds. Nat Catal 7, 585–592 (2024).

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