Abstract
While natural terpenoid cyclases generate complex terpenoid structures via cationic mechanisms, alternative radical cyclization pathways are underexplored. The metal-catalysed H-atom transfer reaction (M-HAT) offers an attractive means for hydrofunctionalizing olefins, providing access to terpenoid-like structures. Artificial metalloenzymes offer a promising strategy for introducing M-HAT reactivity into a protein scaffold. Here we report our efforts towards engineering an artificial radical cyclase (ARCase), resulting from anchoring a biotinylated [Co(Schiff-base)] cofactor within an engineered chimeric streptavidin. After two rounds of directed evolution, a double mutant catalyses a radical cyclization to afford bicyclic products with a cis-5-6-fused ring structure and up to 97% enantiomeric excess. The involvement of a histidine ligation to the Co cofactor is confirmed by crystallography. A time course experiment reveals a cascade reaction catalysed by the ARCase, combining a radical cyclization with a conjugate reduction. The ARCase exhibits tolerance towards variations in the dienone substrate, highlighting its potential to access terpenoid scaffolds.
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Data availability
The data that support the findings in this study are available within this Article and its Supplementary Information and Supplementary Data. Crystallographic data for the ARCase structure of [Co(Biot-en-tBu2-Salphen)] 4 ‧ chSav*_α16 S122V-K131H reported in this article have been deposited at Protein Data Bank under the code 8QEX. Source data are provided with this paper.
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Acknowledgements
T.R.W. thanks the NCCR Catalysis (grant number 180544), a National Centre of Competence in Research funded by the Swiss National Science Foundation. Additional funding was provided by the NCCR Molecular Systems Engineering (grant number 200021_178760). X.Z. thanks the SIOC postdoc fellowship for the financial support. R.T. thanks the Naito Foundation for financial support. We acknowledge the Paul Scherrer Institut, Villigen, Switzerland, for provision of synchrotron radiation beamtime at beamline X06SA of the Swiss Light Source.
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Contributions
T.R.W., X.Z. and D.C. conceived and designed the study. X.Z. contributed to the synthesis of the substrates, products and complexes. D.C. contributed to the mutagenesis and protein expression and protein purification and protein mass. D.C. and X.Z. performed the catalytic and time course experiment, designed the screening workflow and recorded the data. T.R.W., X.Z. and D.C. analysed the data. R.P.J., T.M. and D.C. contributed to the crystallization and crystallographic structural analysis. A.A.V., B.E.C., A.S. and T.R.W. contributed to the collaboration on the computational design of chimeric streptavidin. R.T. contributed to the molecular modelling and QM/MM calculation. Z.Z. offered the molecular biology instructions. D.A.G. offered synthetic suggestions. A.L. provided critical insight regarding the M-HAT reaction. A.S. established the Chim_Sav library workflow and associated molecular biology methods. T.R.W., X.Z. and D.C. wrote the paper, which was further supplemented through contributions from R.P.J., R.T. and A.A.V. All authors have given approval to the final version of the paper.
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Extended data
Extended Data Fig. 1 Asymmetric radical cyclization of dienone 1 and kinetic resolution of the cyclized enone 2 to the corresponding ketone 8 at 10 °C.
a) [Co(Biot-en-tBu2-Salphen)] 4 · chSav*_α16 S122V-K131H catalyzed cascade reaction consisting of a radical cyclization followed by a conjugate reduction. b) Time-course monitoring at 10 °C of ARCase progress, product-distribution (top) and enantioselectivity (bottom).
Supplementary information
Supplementary Information
Supplementary Tables 1–20 and Figs. 1–86.
Supplementary Data 1
Coordinates for the ARCase [Co(Biot-en-tBu2-Salphen)] 4 ‧ Sav*_α16 S122V-K131H.
Source data
Source Data Table 1
Source data for Table 1.
Source Data Fig. 2
Source data for Fig. 2.
Source Data Fig. 3
Source data for Fig. 3.
Source Data Fig. 5
Source data for Fig. 5.
Source Data Extended Data Fig./Table 1
Source data for Extended Data Fig. 1 in the main text.
Source Data Extended Data Fig./Table 2
Source data for Extended Data Table 1 in the main text.
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Chen, D., Zhang, X., Vorobieva, A.A. et al. An evolved artificial radical cyclase enables the construction of bicyclic terpenoid scaffolds via an H-atom transfer pathway. Nat. Chem. 16, 1656–1664 (2024). https://doi.org/10.1038/s41557-024-01562-5
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DOI: https://doi.org/10.1038/s41557-024-01562-5
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