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Light-activated tetrazines enable precision live-cell bioorthogonal chemistry

Nature Chemistry volume 14, pages 1078–1085 (2022)Cite this article

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Abstract

Bioorthogonal cycloaddition reactions between tetrazines and strained dienophiles are widely used for protein, lipid and glycan labelling because of their extremely rapid kinetics. However, controlling this chemistry in the presence of living mammalian cells with a high degree of spatial and temporal precision remains a challenge. Here we demonstrate a versatile approach to light-activated formation of tetrazines from photocaged dihydrotetrazines. Photouncaging, followed by spontaneous transformation to reactive tetrazine, enables live-cell spatiotemporal control of rapid bioorthogonal cycloaddition with dienophiles such as trans-cyclooctenes. Photocaged dihydrotetrazines are stable in conditions that normally degrade tetrazines, enabling efficient early-stage incorporation of bioorthogonal handles into biomolecules such as peptides. Photocaged dihydrotetrazines allow the use of non-toxic light to trigger tetrazine ligations on living mammalian cells. By tagging reactive phospholipids with fluorophores, we demonstrate modification of HeLa cell membranes with single-cell spatial resolution. Finally, we show that photo-triggered therapy is possible by coupling tetrazine photoactivation with strategies that release prodrugs in response to tetrazine ligation.

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Fig. 1: Light-controlled bioorthogonal tetrazine ligation in living cells.
Fig. 2: Early-stage functionalization of a peptide with a photocaged dihydrotetrazine group.
Fig. 3: Single-cell remodelling of HeLa S3 cell membranes by photoactivation of tetrazine ligation.
Fig. 4: Light-activated tetrazine prodrug therapy in Hep 3B cancer cells.

Data availability

The data that support the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

Financial support for this work was provided by the National Institutes of Health (DP2DK111801, R01GM123285, R35GM141939 and T32CA009523). We thank W. Xiong and C. Wang for their assistance in measuring the emission spectra of the LED lights. We thank A. Winter and E. Gehrmann for their assistance with the synthesis of BODIPY photocaged dihydrotetrazine. We thank I. Budin and G. Riddihough for providing helpful comments.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, San Diego, CA, USA

    Luping Liu, Dongyang Zhang, Mai Johnson & Neal K. Devaraj

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  1. Luping Liu
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  2. Dongyang Zhang
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  3. Mai Johnson
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  4. Neal K. Devaraj
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Contributions

L.L. and N.K.D. conceived the project. L.L. designed and performed the synthetic experiments. L.L. and D.Z. performed microscopy experiments. L.L. and M.J. performed cell experiments. L.L., D.Z., M.J. and N.K.D. analysed the data. L.L. and N.K.D. wrote the manuscript.

Corresponding author

Correspondence to Neal K. Devaraj.

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Nature Chemistry thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Materials and methods, Supplementary text, Figs. 1–35, copies of mass and NMR spectra and references 46–51.

Reporting Summary

Supplementary Data 1

Cell membrane labelling.

Supplementary Data 2

Spatiotemporal cell membrane labelling.

Supplementary Data 3

Light-activated drug delivery by 1a.

Source data

Source Data Fig. 4

Light-activated drug delivery by 1c.

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Liu, L., Zhang, D., Johnson, M. et al. Light-activated tetrazines enable precision live-cell bioorthogonal chemistry. Nat. Chem. 14, 1078–1085 (2022). https://doi.org/10.1038/s41557-022-00963-8

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