Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Enantioselective, intermolecular benzylic C–H amination catalysed by an engineered iron-haem enzyme

Nature Chemistry volume 9, pages 629–634 (2017)Cite this article

Subjects

Abstract

C–H bonds are ubiquitous structural units of organic molecules. Although these bonds are generally considered to be chemically inert, the recent emergence of methods for C–H functionalization promises to transform the way synthetic chemistry is performed. The intermolecular amination of C–H bonds represents a particularly desirable and challenging transformation for which no efficient, highly selective, and renewable catalysts exist. Here we report the directed evolution of an iron-containing enzymatic catalyst—based on a cytochrome P450 monooxygenase—for the highly enantioselective intermolecular amination of benzylic C–H bonds. The biocatalyst is capable of up to 1,300 turnovers, exhibits excellent enantioselectivities, and provides access to valuable benzylic amines. Iron complexes are generally poor catalysts for C–H amination: in this catalyst, the enzyme's protein framework confers activity on an otherwise unreactive iron-haem cofactor.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Intermolecular C–H amination, a simplifying transformation for chiral amine synthesis.
Figure 2: Proposed mechanism of cytochrome P411-catalysed intermolecular C–H amination.
Figure 3: Evolution of a cytochrome P411 catalyst for enantioselective C–H amination on increasingly challenging substrates.
Figure 4: Kinetic isotope effect and enzyme structural studies.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Hartwig, J. F. Evolution of C–H bond functionalization from methane to methodology. J. Am. Chem. Soc. 138, 2–24 (2016).

    Article  CAS  Google Scholar 

  2. Godula, K. & Sames, D. C–H bond functionalization in complex organic synthesis. Science 312, 67–72 (2006).

    Article  CAS  Google Scholar 

  3. Yamaguchi, J., Yamaguchi, A. D. & Itami, K. C–H bond functionalization: emerging synthetic tools for natural products and pharmaceuticals. Angew. Chem. Int. Ed. 51, 8960–9009 (2012).

    Article  CAS  Google Scholar 

  4. Bertini, I., Gray, H. B., Lippard, S. J. & Valentine, J. S. (eds) Bioinorganic Chemistry (University Science Books, 1994).

    Google Scholar 

  5. Zalatan, D. N. & Du Bois, J. Metal-catalyzed oxidations of C–H to C–N bonds. Top. Curr. Chem. 292, 347–378 (2010).

    Article  CAS  Google Scholar 

  6. Davies, H. M. L. & Manning, J. R. Catalytic C–H functionalization by metal carbenoid and nitrenoid insertion. Nature 451, 417–424 (2008).

    Article  CAS  Google Scholar 

  7. Collet, F., Lescot, C. & Dauban, P. Catalytic C–H amination: the stereoselectivity issue. Chem. Soc. Rev. 40, 1926–1936 (2011).

    Article  CAS  Google Scholar 

  8. Wu, W.-T., Yang, Z.-P. & You, S.-L. in Asymmetric Functionalization of C–H Bonds (ed. You, S.-L.) 1–66 (RSC Catalysis Series No. 25, 2015).

  9. Jeffrey, J. L. & Sarpong, R. Intramolecular C(sp3)–H amination. Chem. Sci. 4, 4092–4106 (2013).

    Article  CAS  Google Scholar 

  10. Ochiai, M., Miyamoto, K., Kaneaki, T., Hayashi, S. & Nakanishi, W. Highly regioselective amination of unactivated alkanes by hypervalent sulfonylimino-λ3-bromane. Science 332, 448–451 (2011).

    Article  CAS  Google Scholar 

  11. 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 

  12. Sharma, A. & Hartwig, J. F. Metal-catalysed azidation of tertiary C–H bonds suitable for late-stage functionalization. Nature 517, 600–604 (2015).

    Article  CAS  Google Scholar 

  13. Huang, X., Bergsten, T. M. & Groves, J. T. Manganese-catalyzed late-stage aliphatic C–H azidation. J. Am. Chem. Soc. 137, 5300–5303 (2015).

    Article  CAS  Google Scholar 

  14. Michaudel, Q., Thevenet, D. & Baran, P. S. Intermolecular Ritter-type C–H amination of unactivated sp3 carbons. J. Am. Chem. Soc. 134, 2547–2550 (2012).

    Article  CAS  Google Scholar 

  15. Nägeli, I. et al. Rhodium(II)-catalyzed CH Insertions with {[(4-Nitrophenyl)sulfonyl]imino}phenyl-λ3-iodane. Helv. Chim. Acta 80, 1087–1105 (1997).

    Article  Google Scholar 

  16. Yamawaki, M., Tsutsui, H., Kitagaki, S., Anada, M. & Hashimoto, S. Dirhodium(II) tetrakis[N-tetrachlorophthaloyl-(S)-tert-leucinate]: a new chiral Rh(II) catalyst for enantioselective amidation of C–H bonds. Tetrahedron Lett. 43, 9561–9564 (2002).

    Article  CAS  Google Scholar 

  17. Reddy, R. P. & Davies, H. M. L. Dirhodium tetracarboxylates derived from adamantylglycine as chiral catalysts for enantioselective C–H aminations. Org. Lett. 8, 5013–5016 (2006).

    Article  CAS  Google Scholar 

  18. Nishioka, Y., Uchida, T. & Katsuki, T. Enantio- and regioselective intermolecular benzylic and allylic C–H bond amination. Angew. Chem. Int. Ed. 52, 1739–1742 (2013).

    Article  CAS  Google Scholar 

  19. Zhou, X.-G., Yu, X.-Q., Huang, J.-S. & Che, C.-M. Asymmetric amidation of saturated C–H bonds catalysed by chiral ruthenium and manganese porphyrins. Chem. Commun. 2377–2378 (1999).

  20. Kohmura, Y. & Katsuki, T. Mn(salen)-catalyzed enantioselective C–H amination. Tetrahedron Lett. 42, 3339–3342 (2001).

    Article  CAS  Google Scholar 

  21. Liang, C. et al. Efficient diastereoselective intermolecular rhodium-catalyzed C–H amination. Angew. Chem. Int. Ed. 45, 4641–4644 (2006).

    Article  CAS  Google Scholar 

  22. Urlacher, V. B. & Girhard, M. Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol. 30, 26–36 (2012).

    Article  CAS  Google Scholar 

  23. Podust, L. M. & Sherman, D. H. Diversity of P450 enzymes in the biosynthesis of natural products. Nat. Prod. Rep. 29, 1251–1266 (2012).

    Article  CAS  Google Scholar 

  24. Both, P. et al. Whole-cell biocatalysts for stereoselective C–H amination reactions. Angew. Chem. Int. Ed. 55, 1511–1513 (2016).

    Article  CAS  Google Scholar 

  25. Schrewe, M., Ladkau, N., Bühler, B. & Schmid, A. Direct terminal alkylamino-functionalization via multistep biocatalysis in one recombinant whole-cell catalyst. Adv. Synth. Catal. 355, 1693–1697 (2013).

    Article  CAS  Google Scholar 

  26. McIntosh, J. A. et al. Enantioselective intramolecular C–H amination catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo. Angew. Chem. Int. Ed. 52, 9309–9312 (2013).

    Article  CAS  Google Scholar 

  27. Hyster, T. K., Farwell, C. C., Buller, A. R., McIntosh, J. A. & Arnold, F. H. Enzyme-controlled nitrogen-atom transfer enables regiodivergent C–H amination. J. Am. Chem. Soc. 136, 15505–15508 (2014).

    Article  CAS  Google Scholar 

  28. Farwell, C. C., Zhang, R. K., McIntosh, J. A., Hyster, T. K. & Arnold, F. H. Enantioselective enzyme-catalyzed aziridination enabled by active-site evolution of a cytochrome P450. ACS Cent. Sci. 1, 89–93 (2015).

    Article  CAS  Google Scholar 

  29. Prier, C. K., Hyster, T. K., Farwell, C. C., Huang, A. & Arnold, F. H. Asymmetric enzymatic synthesis of allylic amines: a sigmatropic rearrangement strategy. Angew. Chem. Int. Ed. 55, 4711–4715 (2016).

    Article  CAS  Google Scholar 

  30. Singh, R., Bordeaux, M. & Fasan, R. P450-catalyzed intramolecular sp3 C–H amination with arylsulfonyl azide substrates. ACS Catal. 4, 546–552 (2014).

    Article  CAS  Google Scholar 

  31. Bordeaux, M., Singh, R. & Fasan, R. Intramolecular C(sp3)–H amination of arylsulfonyl azides with engineered and artificial myoglobin-based catalysts. Bioorg. Med. Chem. 22, 5697–5704 (2014).

    Article  CAS  Google Scholar 

  32. Svastits, E. W., Dawson, J. H., Breslow, R. & Gellman, S. H. Functionalized nitrogen atom transfer catalyzed by cytochrome P-450. J. Am. Chem. Soc. 107, 6427–6428 (1985).

    Article  CAS  Google Scholar 

  33. Coelho, P. S. et al. A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo. Nat. Chem. Biol. 9, 485–487 (2013).

    Article  CAS  Google Scholar 

  34. Whitehouse, C. J. C., Bell, S. G. & Wong, L.-L. P450BM3 (CYP102A1): connecting the dots. Chem. Soc. Rev. 41, 1218–1260 (2012).

    Article  CAS  Google Scholar 

  35. Barniol-Xicota, M., Leiva, R., Escolano, C. & Vázquez, S. Synthesis of cinacalcet: an enantiopure active pharmaceutical ingredient (API). Synthesis 48, 783–803 (2016).

    Article  CAS  Google Scholar 

  36. Bloom, J. D., Labthavikul, S. T., Otey, C. R. & Arnold, F. H. Protein stability promotes evolvability. Proc. Natl Acad. Sci. USA 103, 5869–5874 (2006).

    Article  CAS  Google Scholar 

  37. Ankner, T. & Hilmersson, G. Instantaneous deprotection of tosylamides and esters with SmI2/Amine/Water. Org. Lett. 11, 503–506 (2009).

    Article  CAS  Google Scholar 

  38. Poulos, T. L. Cytochrome P450. Curr. Opin. Struct. Biol. 5, 767–774 (1995).

    Article  CAS  Google Scholar 

  39. Li, H. & Poulos, T. L. The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid. Nat. Struct. Biol. 4, 140–146 (1997).

    Article  CAS  Google Scholar 

  40. Roiban, G.-D. & Reetz, M. T. Expanding the toolbox of organic chemists: directed evolution of P450 monooxygenases as catalysts in regio- and stereoselective oxidative hydroxylation. Chem. Commun. 51, 2208–2224 (2015).

    Article  CAS  Google Scholar 

  41. Key, H. M., Dydio, P., Clark, D. S. & Hartwig, J. F. Abiological catalysis by artificial haem proteins containing noble metals in place of iron. Nature 534, 534–537 (2016).

    Article  CAS  Google Scholar 

  42. Srivastava, P., Yang, H., Ellis-Guardiola, K. & Lewis, J. C. Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation. Nat. Commun. 6, 7789 (2015).

    Article  CAS  Google Scholar 

  43. Hyster, T. K., Knörr, L., Ward, T. R. & Rovis, T. Biotinylated Rh(III) complexes in engineered streptavidin for accelerated asymmetric C–H activation. Science 338, 500–503 (2012).

    Article  CAS  Google Scholar 

  44. Dydio, P., Key, H. M., Hayashi, H., Clark, D. S. & Hartwig, J. F. Chemoselective, enzymatic C–H bond amination catalyzed by a cytochrome P450 containing an Ir(Me)-PIX cofactor. J. Am. Chem. Soc. 139, 1750–1753 (2017).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Our research is supported by the National Science Foundation, Division of Molecular and Cellular Biosciences (grant MCB-1513007) and by funds from the American Recovery and Reinvestment Act (ARRA) through the National Institutes of Health Shared Instrumentation Grant Program (S10RR027203). C.K.P. thanks the Resnick Sustainability Institute for a postdoctoral fellowship. R.K.Z. was supported by a National Science Foundation Graduate Research Fellowship (NSF GRFP; DGE-1144469), is a trainee in the Caltech Biotechnology Leadership Program, and has received financial support from the Donna and Benjamin M. Rosen Bioengineering Center. A.R.B. is funded by a Ruth Kirschstein NIH Postdoctoral Fellowship F32G110851. We thank S. Virgil, J. Kaiser, and R. D. Lewis for experimental assistance, and O. F. Brandenberg, S. C. Hammer, and S. B. J. Kan for helpful discussion and comments on the manuscript.

Author information

Author notes

  1. Christopher K. Prier and Ruijie K. Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, MC 210-41, Pasadena, 91125, California, USA

    Christopher K. Prier, Ruijie K. Zhang, Andrew R. Buller, Sabine Brinkmann-Chen & Frances H. Arnold

Authors
  1. Christopher K. Prier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ruijie K. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew R. Buller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sabine Brinkmann-Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Frances H. Arnold
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.K.P. and R.K.Z. designed, carried out, and analysed all amination experiments, with F.H.A. providing guidance. C.K.P., R.K.Z. and S.B.-C. obtained protein crystals. R.K.Z. and A.R.B. solved the crystal structure. C.K.P. and F.H.A. wrote the manuscript with input from all of the authors.

Corresponding author

Correspondence to Frances H. Arnold.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 5564 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Prier, C., Zhang, R., Buller, A. et al. Enantioselective, intermolecular benzylic C–H amination catalysed by an engineered iron-haem enzyme. Nature Chem 9, 629–634 (2017). https://doi.org/10.1038/nchem.2783

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.2783

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing