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Enantioselective [2+2]-cycloadditions with triplet photoenzymes


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.

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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.

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.


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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



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.

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The authors declare no competing interests.

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

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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.

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Sun, N., Huang, J., Qian, J. et al. Enantioselective [2+2]-cycloadditions with triplet photoenzymes. Nature 611, 715–720 (2022).

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