The ability to selectively functionalize ubiquitous C–H bonds streamlines the construction of complex molecular architectures from easily available precursors. Here we report enzyme catalysts derived from a cytochrome P450 that use a nitrene transfer mechanism for the enantioselective amination of primary, secondary and tertiary C(sp3)–H bonds. These fully genetically encoded enzymes are produced and function in bacteria, where they can be optimized by directed evolution for a broad spectrum of enantioselective C(sp3)–H amination reactions. These catalysts can aminate a variety of benzylic, allylic and aliphatic C–H bonds in excellent enantioselectivity with access to either antipode of product. Enantioselective amination of primary C(sp3)–H bonds in substrates that bear geminal dimethyl substituents furnished chiral amines that feature a quaternary stereocentre. Moreover, these enzymes enabled the enantioconvergent transformation of racemic substrates that possess a tertiary C(sp3)–H bond to afford products that bear a tetrasubstituted stereocentre, a process that has eluded small-molecule catalysts. Further engineering allowed for the enantioselective construction of methyl–ethyl stereocentres, which is notoriously challenging in asymmetric catalysis.
Subscribe to Journal
Get full journal access for 1 year
only $13.33 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
All data necessary to support the paper’s conclusions are available in the main text and the Supplementary Information. Solid-state structures of 2a, 4a, 5a and 5f are available free of charge from the Cambridge Crystallographic Data Centre under reference nos CCDC 1905551, 1905553, 1905552 and 1905554. Plasmids encoding the enzymes reported in this study are available for research purposes from F.H.A. under a material transfer agreement with the California Institute of Technology.
Saint-Denis, T. G., Zhu, R.-Y., Chen, G., Wu, Q.-F. & Yu, J.-Q. Enantioselective C(sp 3)‒H bond activation by chiral transition metal catalysts. Science 359, eaao4798 (2018).
Gutekunst, W. R. & Baran, P. S. C–H functionalization logic in total synthesis. Chem. Soc. Rev. 40, 1976–1991 (2011).
Hartwig, J. F. & Larsen, M. A. Undirected, homogeneous C–H bond functionalization: challenges and opportunities. ACS Cent. Sci. 2, 281–292 (2016).
Lu, H. & Zhang, X. P. Catalytic C–H functionalization by metalloporphyrins: recent developments and future directions. Chem. Soc. Rev. 40, 1899–1909 (2011).
Davies, H. M. L. & Manning, J. R. Catalytic C–H functionalization by metal carbenoid and nitrenoid insertion. Nature 451, 417–424 (2008).
Smalley, A. P., Cuthbertson, J. D. & Gaunt, M. J. Palladium-catalyzed enantioselective C–H activation of aliphatic amines using chiral anionic binol-phosphoric acid ligands. J. Am. Chem. Soc. 139, 1412–1415 (2017).
Quasdorf, K. W. & Overman, L. E. Catalytic enantioselective synthesis of quaternary carbon stereocentres. Nature 516, 181–191 (2014).
Bhat, V., Welin, E. R., Guo, X. & Stoltz, B. M. Advances in stereoconvergent catalysis from 2005 to 2015: transition-metal-mediated stereoablative reactions, dynamic kinetic resolutions, and dynamic kinetic asymmetric transformations. Chem. Rev. 117, 4528–4561 (2017).
Lewis, J. C., Coelho, P. S. & Arnold, F. H. Enzymatic functionalization of carbon–hydrogen bonds. Chem. Soc. Rev. 40, 2003–2021 (2011).
Ortiz de Montellano, P. R. Hydrocarbon hydroxylation by cytochrome P450 enzymes. Chem. Rev. 110, 932–948 (2010).
Fasan, R. Tuning P450 enzymes as oxidation catalysts. ACS Catal. 2, 647–666 (2012).
Coelho, P. S., Brustad, E. M., Kannan, A. & Arnold, F. H. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339, 307–310 (2013).
Kan, S. B. J., Lewis, R. D., Chen, K. & Arnold, F. H. Directed evolution of cytochrome c for carbon–silicon bond formation: bringing silicon to life. Science 354, 1048–1051 (2016).
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).
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).
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).
Singh, R., Bordeaux, M. & Fasan, R. P450-catalyzed intramolecular sp 3 C–H amination with arylsulfonyl azide substrates. ACS Catal. 4, 546–552 (2014).
Zhang, R. K. et al. Enzymatic assembly of carbon–carbon bonds via iron-catalysed sp 3 C–H functionalization. Nature 565, 67–72 (2019).
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).
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).
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).
Kurokawa, T., Kim, M. & Du Bois, J. Synthesis of 1,3-diamines through rhodium-catalyzed C–H insertion. Angew. Chem. Int. Ed. 48, 2777–2779 (2009).
Lu, H., Jiang, H., Wojtas, L. & Zhang, X. P. Selective intramolecular C–H amination through the metalloradical activation of azides: synthesis of 1,3-diamines under neutral and nonoxidative conditions. Angew. Chem. Int. Ed. 49, 10192–10196 (2010).
Zalatan, D. N. & Du Bois, J. A chiral rhodium carboxamidate catalyst for enantioselective C–H amination. J. Am. Chem. Soc. 130, 9220–9221 (2008).
Ichinose, M. et al. Enantioselective intramolecular benzylic C–H bond amination: efficient synthesis of optically active benzosultams. Angew. Chem. Int. Ed. 50, 9884–9887 (2011).
Roizen, J. L., Harvey, M. E. & Du Bois, J. Metal-catalyzed nitrogen-atom transfer methods for the oxidation of aliphatic C–H bonds. Acc. Chem. Res. 45, 911–922 (2012).
Paradine, S. M. & White, M. C. Iron-catalyzed intramolecular allylic C–H amination. J. Am. Chem. Soc. 134, 2036–2039 (2012).
Paradine, S. M. et al. A manganese catalyst for highly reactive yet chemoselective intramolecular C(sp 3)–H amination. Nat. Chem. 7, 987–994 (2015).
Clark, J. R., Feng, K., Sookezian, A. & White, M. C. Manganese-catalysed benzylic C(sp 3)–H amination for late-stage functionalization. Nat. Chem. 10, 583–591 (2018).
Li, C. et al. Catalytic radical process for enantioselective amination of C(sp 3)−H bonds. Angew. Chem. Int. Ed. 57, 16837–16841 (2018).
Lu, H., Lang, K., Jiang, H., Wojtas, L. & Zhang, X. P. Intramolecular 1,5-C(sp 3)–H radical amination via Co(ii)-based metalloradical catalysis for five-membered cyclic sulfamides. Chem. Sci. 7, 6934–6939 (2016).
Capdevila, J. H. et al. The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3. J. Biol. Chem. 271, 22663–22671 (1996).
Chen, K., Huang, X., Kan, S. B. J., Zhang, R. K. & Arnold, F. H. Enzymatic construction of highly strained carbocycles. Science 360, 71–75 (2018).
Nagib, D. A. Catalytic desymmetrization by C–H functionalization as a solution to the chiral methyl problem. Angew. Chem. Int. Ed. 56, 7354–7356 (2017).
Kainz, Q. M. et al. Asymmetric copper-catalyzed C–N cross-couplings induced by visible light. Science 351, 681–684 (2016).
Wendlandt, A. E., Vangal, P. & Jacobsen, E. N. Quaternary stereocentres via an enantioconvergent catalytic SN1 reaction. Nature 556, 447–451 (2018).
Zhang, X. et al. An enantioconvergent halogenophilic nucleophilic substitution (SN2X) reaction. Science 363, 400–404 (2019).
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).
Singh, R., Kolev, J. N., Sutera, P. A. & Fasan, R. Enzymatic C(sp 3)–H amination: P450-catalyzed conversion of carbonazidates into oxazolidinones. ACS Catal. 5, 1685–1691 (2015).
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).
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).
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).
This work was supported by the NSF (grant nos MCB-1513007 for F.H.A. and CHE-1654122 for P.L.). Y.Y. thanks the National Institutes of Health for a postdoctoral fellowship (grant no. 1F32GM133126-01). Calculations were performed at the Center for Research Computing at the University of Pittsburgh. We thank R. K. Zhang, K. Chen, D. C. Miller, D. K. Romney (Caltech) and Y. Wang (University of Pittsburgh) for helpful discussions and comments on the manuscript, L. Henling for X-ray diffraction analysis and S. Virgil for assistance with chiral supercritical fluid chromatography analysis.
A provisional patent application (inventors Y.Y. and I.C.) has been filed through the California Institute of Technology. The provisional patent covers the development and application of engineered cytochromes P450 for the synthesis of chiral diamine derivatives by C–H amination.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
1H, 13C and 19F NMR spectra.
Crystallographic data for compound 2a.
Crystallographic data for compound 4a.
Crystallographic data for compound 5a.
Crystallographic data for compound 5f.
About this article
Cite this article
Yang, Y., Cho, I., Qi, X. et al. An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp3)–H bonds. Nat. Chem. 11, 987–993 (2019). https://doi.org/10.1038/s41557-019-0343-5
AIChE Journal (2020)
Nature Catalysis (2020)
European Journal of Organic Chemistry (2020)