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 [2+2]-cycloadditions with triplet photoenzymes

Abstract

Naturally evolved enzymes, despite their astonishingly large variety and functional diversity, operate predominantly through thermochemical activation. Integrating prominent photocatalysis modes into proteins, such as triplet energy transfer, could create artificial photoenzymes that expand the scope of natural biocatalysis1,2,3. Here, we exploit genetically reprogrammed, chemically evolved photoenzymes embedded with a synthetic triplet photosensitizer that are capable of excited-state enantio-induction4,5,6. Structural optimization through four rounds of directed evolution afforded proficient variants for the enantioselective intramolecular [2+2]-photocycloaddition of indole derivatives with good substrate generality and excellent enantioselectivities (up to 99% enantiomeric excess). A crystal structure of the photoenzyme–substrate complex elucidated the non-covalent interactions that mediate the reaction stereochemistry. This study expands the energy transfer reactivity7,8,9,10 of artificial triplet photoenzymes in a supramolecular protein cavity and unlocks an integrated approach to valuable enantioselective photochemical synthesis that is not accessible with either the synthetic or the biological world alone.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Fig. 1: Design of triplet photoenzyme (TPe) for enantioselective [2+2]-cycloaddition.
Fig. 2: Directed evolution of TPe.
Fig. 3: TPe optimization guided by the crystal structure.
Fig. 4: The substrate scope of TPe.

Similar content being viewed by others

Data availability

All data are available in the main text or the Supplementary Information. The crystal structure data of TPe3.0 and TPe3.0 in complex with substrate 1b have been deposited in the Protein Data Bank under accession numbers 7XUP and 7XUQSource data are provided with this paper.

References

  1. Arnold, F. H. Innovation by evolution: bringing new chemistry to life (Nobel Lecture). Angew. Chem. Int. Ed. 58, 14420–14426 (2019).

    CAS  Google Scholar 

  2. Lovelock, S. L. et al. The road to fully programmable protein catalysis. Nature 606, 49–58 (2022).

    ADS  CAS  PubMed  Google Scholar 

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

    CAS  Google Scholar 

  4. Silvi, M. & Melchiorre, P. Enhancing the potential of enantioselective organocatalysis with light. Nature 554, 41–49 (2018).

    ADS  CAS  PubMed  Google Scholar 

  5. Brimioulle, R., Lenhart, D., Maturi, M. M. & Bach, T. Enantioselective catalysis of photochemical reactions. Angew. Chem. Int. Ed. 54, 3872–3890 (2015).

    CAS  Google Scholar 

  6. Genzink, M. J., Kidd, J. B., Swords, W. B. & Yoon, T. P. Chiral photocatalyst structures in asymmetric photochemical synthesis. Chem. Rev. 122, 1654–1716 (2022).

    CAS  PubMed  Google Scholar 

  7. Strieth-Kalthoff, F., James, M. J., Teders, M., Pitzer, L. & Glorius, F. Energy transfer catalysis mediated by visible light: principles, applications, directions. Chem. Soc. Rev. 47, 7190–7202 (2018).

    CAS  PubMed  Google Scholar 

  8. Großkopf, J., Kratz, T., Rigotti, T. & Bach, T. Enantioselective photochemical reactions enabled by triplet energy transfer. Chem. Rev. 122, 1626–1653 (2022).

    PubMed  Google Scholar 

  9. Zhou, Q.-Q., Zou, Y.-Q., Lu, L.-Q. & Xiao, W.-J. Visible-light-induced organic photochemical reactions through energy-transfer pathways. Angew. Chem. Int. Ed. 58, 1586–1604 (2019).

    CAS  Google Scholar 

  10. Strieth-Kalthoff, F. & Glorius, F. Triplet energy transfer photocatalysis: unlocking the next level. Chem 6, 1888–1903 (2020).

    CAS  Google Scholar 

  11. Poplata, S., Tröster, A., Zou, Y.-Q. & Bach, T. Recent advances in the synthesis of cyclobutanes by olefin [2+2] photocycloaddition reactions. Chem. Rev. 116, 9748–9815 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Münster, N., Parker, N. A., van Dijk, L., Paton, R. S. & Smith, M. D. Visible light photocatalysis of 6π heterocyclization. Angew. Chem. Int. Ed. 56, 9468–9472 (2017).

    Google Scholar 

  13. Becker, M. R., Wearing, E. R. & Schindler, C. S. Synthesis of azetidines via visible-light-mediated intermolecular [2+2] photocycloadditions. Nat. Chem. 12, 898–905 (2020).

    CAS  PubMed  Google Scholar 

  14. Candish, L. et al. Photocatalysis in the life science industry. Chem. Rev. 122, 2907–2980 (2022).

    CAS  PubMed  Google Scholar 

  15. Bach, T. & Hehn, J. Photochemical reactions as key steps in natural product synthesis. Angew. Chem. Int. Ed. 50, 1000–1045 (2011).

    CAS  Google Scholar 

  16. Kleinmans, R. et al. Intermolecular [2π+2σ]-photocycloaddition enabled by triplet energy transfer. Nature 606, 477–482 (2022).

    ADS  Google Scholar 

  17. Huang, M., Zhang, L., Pan, T. & Luo, S. Deracemization through photochemical E/Z isomerization of enamines. Science 375, 869–874 (2022).

    ADS  CAS  PubMed  Google Scholar 

  18. Müller, C., Bauer, A. & Bach, T. Light-driven enantioselective organocatalysis. Angew. Chem. Int. Ed. 48, 6640–6642 (2009).

    Google Scholar 

  19. Li, X., Großkopf, J., Jandl, C. & Bach, T. Enantioselective, visible light mediated aza Paternò–Büchi reactions of quinoxalinones. Angew. Chem. Int. Ed. 60, 2684–2688 (2021).

    CAS  Google Scholar 

  20. Alonso, R. & Bach, T. A chiral thioxanthone as organocatalyst for enantioselective [2+2] photocycloaddition reactions induced by visible light. Angew. Chem. Int. Ed. 53, 4368–4371 (2014).

    CAS  Google Scholar 

  21. Hölzl-Hobmeier, A. et al. Catalytic deracemization of chiral allenes by sensitized excitation with visible light. Nature 564, 240–243 (2018).

    ADS  PubMed  Google Scholar 

  22. Plaza, M., Großkopf, J., Breitenlechner, S., Bannwarth, C. & Bach, T. Photochemical deracemization of primary allene amides by triplet energy transfer: a combined synthetic and theoretical study. J. Am. Chem. Soc. 143, 11209–11217 (2021).

    CAS  PubMed  Google Scholar 

  23. Skubi, K. L. et al. Enantioselective excited-state photoreactions controlled by a chiral hydrogen-bonding iridium sensitizer. J. Am. Chem. Soc. 139, 17186–17192 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Brenninger, C., Jolliffe, J. D. & Bach, T. Chromophore activation of α,β-unsaturated carbonyl compounds and its application to enantioselective photochemical reactions. Angew. Chem. Int. Ed. 57, 14338–14349 (2018).

    CAS  Google Scholar 

  25. Skubi, K. L., Blum, T. R. & Yoon, T. P. Dual catalysis strategies in photochemical synthesis. Chem. Rev. 116, 10035–10074 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Pecho, F. et al. Enantioselective [2+2] photocycloaddition via iminium ions: catalysis by a sensitizing chiral Brønsted acid. J. Am. Chem. Soc. 143, 9350–9354 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Blum, T. R., Miller, Z. D., Bates, D. M., Guzei, I. A. & Yoon, T. P. Enantioselective photochemistry through Lewis acid catalyzed triplet energy transfer. Science 354, 1391–1395 (2016).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chapman, S. J. et al. Cooperative stereoinduction in asymmetric photocatalysis. J. Am. Chem. Soc. 144, 4206–4213 (2022).

    CAS  PubMed  Google Scholar 

  29. Black, M. J. et al. Asymmetric redox-neutral radical cyclization catalysed by flavin-dependent ‘ene’-reductases. Nat. Chem. 12, 71–75 (2020).

    ADS  PubMed  Google Scholar 

  30. 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).

    CAS  PubMed  Google Scholar 

  31. Biegasiewicz, K. F. et al. Photoexcitation of flavoenzymes enables a stereoselective radical cyclization. Science 364, 1166–1169 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Emmanuel, M. A., Greenberg, N. R., Oblinsky, D. G. & Hyster, T. K. Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light. Nature 540, 414–417 (2016).

    ADS  CAS  PubMed  Google Scholar 

  33. Huang, X. et al. Photoenzymatic enantioselective intermolecular radical hydroalkylation. Nature 584, 69–74 (2020).

    ADS  CAS  PubMed  Google Scholar 

  34. Harrison, W., Huang, X. & Zhao, H. Photobiocatalysis for abiological transformations. Acc. Chem. Res. 55, 1087–1096 (2022).

    CAS  PubMed  Google Scholar 

  35. Burke, A. J. et al. Design and evolution of an enzyme with a non-canonical organocatalytic mechanism. Nature 570, 219–223 (2019).

    ADS  CAS  PubMed  Google Scholar 

  36. Liu, X. et al. Genetically encoded photosensitizer protein facilitates the rational design of a miniature photocatalytic CO2-reducing enzyme. Nat. Chem. 10, 1201–1206 (2018).

    CAS  PubMed  Google Scholar 

  37. Kang, F. et al. Rational design of a miniature photocatalytic CO2-reducing enzyme. ACS Catal. 11, 5628–5635 (2021).

    CAS  Google Scholar 

  38. Fu, Y. et al. Biocatalytic cross-coupling of aryl halides with a genetically engineered photosensitizer artificial dehalogenase. J. Am. Chem. Soc. 143, 617–622 (2021).

    CAS  PubMed  Google Scholar 

  39. Madoori, P. K., Agustiandari, H., Driessen, A. J. M. & Thunnissen, A. W. H. Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition. EMBO J. 28, 156–166 (2009).

    CAS  PubMed  Google Scholar 

  40. Roelfes, G. LmrR: a privileged scaffold for artificial metalloenzymes. Acc. Chem. Res. 52, 545–556 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhu, M., Zheng, C., Zhang, X. & You, S.-L. Synthesis of cyclobutane-fused angular tetracyclic spiroindolines via visible-light-promoted intramolecular dearomatization of indole derivatives. J. Am. Chem. Soc. 141, 2636–2644 (2019).

    CAS  PubMed  Google Scholar 

  42. Xu, J. et al. Stereodivergent protein engineering of a lipase to access all possible stereoisomers of chiral esters with two stereocenters. J. Am. Chem. Soc. 141, 7934–7945 (2019).

    CAS  PubMed  Google Scholar 

  43. Cozzi, F., Ponzini, F., Annuziata, R., Cinquini, M. & Siegel, J. S. Polar interactions between stacked π systems in fluorinated 1,8-diarylnaphthalenes: importance of quadrupole moments in molecular recognition. Angew. Chem. Int. Ed. 34, 1019 (1995).

    CAS  Google Scholar 

  44. Rui, J. et al. Directed evolution of nonheme iron enzymes to access abiological radical-relay C(sp3)−H azidation. Science 376, 869–874 (2022).

    ADS  CAS  PubMed  Google Scholar 

  45. Drienovská, I. et al. A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residue. Nat. Chem. 10, 946–952 (2018).

    PubMed  Google Scholar 

Download references

Acknowledgements

We thank the National Key R&D Program of China (no. 2018YFA0903500), the National Natural Science Foundation of China (no. 22077042, 22107075) and the Natural Science Foundation of Top Talent of SZTU (20211061010013) for financial support. We thank the Analytical and Testing Centre of HUST, Analytical and Testing Centre of School of Chemistry and Chemical Engineering (HUST) and Research Core Facilities for Life Science (HUST) for instrument support. We thank X. Wan at the Shanghai Institute of Organic Chemistry for providing vibrational circular dichroism analysis, and the staff at beamlines BL02U1 and BL18U1 of the Shanghai Synchrotron Radiation Facility (SSRF) for assistance during X-ray crystal data collection. We thank T. Bach at Technical University of Munich and S. Xie at HUST for valuable discussions.

Author information

Authors and Affiliations

Authors

Contributions

Y.W. and F.Z. conceived the project and designed the experiments. N.S. and J.H. performed the experiments and interpreted the data. J.Q. and X.C. performed the crystallography study and interpreted the data. T.Z. and R.L. carried out the computational studies. J.G., L.T., W. Zhang and Y.D. assisted with the molecular biology experiments. G.W. assisted with the substrate synthesis. W. Zhao performed protein mass analysis. Y.W. and F.Z. wrote the manuscript with input from all of the authors.

Corresponding authors

Correspondence to Xi Chen, Fangrui Zhong or Yuzhou Wu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review information

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Docking of substrate 1a interacting with the LmrR dimer using AutoDock4.

The blue sticks show substrate 1a. The yellow balls and sticks show the W96 residue. The yellow dashes show the π-π interaction between W96 and the indole moiety of 1a. The red balls show the residues pointed to the inner pocket and surrounding W96, which are reserved in the first round of evolution. The green balls show the residues pointed to the inner pocket and with close spatial distances (<10 Å) to the C(2)-C(3) double bond of indole 1a, which were screened for BpA insertion.

Extended Data Fig. 2 The crystal structures of TPe3.0 and TPe3.0 in complex with substrate 1b.

a. The crystal structure of TPe3.0 cocrystalized with 1b (PDB code: 7XUQ). Two molecules forming a dimer are presented. The backbone is shown as grey cartoon. The BpA is shown as sticks with carbon atoms coloured in light blue. 1b is shown as sticks with carbon atoms coloured in orange. Oxygen and nitrogen atoms are shown in red and blue, respectively. The yellow dashes show the π-π interactions between BpA and the substrates with the distances (Å) labelled. b. The crystal structure of TPe3.0 (PDB code: 7XUP) in a monomeric form. c. Superimposition of the structure of TPe3.0 and the structure of TPe3.0 in complex with substrate 1b. TPe3.0 is shown as pink cartoon while TPe3.0 in complex with 1b is shown as grey cartoon. Interacting residues V15, L18, M89, A92, BpA and L96 are shown as sticks with carbon atoms coloured in light pink and grey respectively. Oxygen, nitrogen and sulfur atoms are shown in red, blue and yellow, respectively.

Extended Data Fig. 3 The reaction time course of photocycloaddition of 1b catalysed by TPe4.0_FBpA under different light intensity irradiation.

Light intensity is (A) 162 mW/cm2 and (B) 3.8 mW/cm2. Error bars denote the standard deviation from triplicate measurements, and they are not shown when smaller than the data point marker.

Supplementary information

Supplementary Information

Supplementary Sections 1–19, Figs. 1–19, Tables 1–10 and NMR spectra data.

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, N., Huang, J., Qian, J. et al. Enantioselective [2+2]-cycloadditions with triplet photoenzymes. Nature 611, 715–720 (2022). https://doi.org/10.1038/s41586-022-05342-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-022-05342-4

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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