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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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.
The authors declare no competing interests.
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Materials and methods, Supplementary text, Figs. 1–35, copies of mass and NMR spectra and references 46–51.
Supplementary Data 1
Cell membrane labelling.
Supplementary Data 2
Spatiotemporal cell membrane labelling.
Supplementary Data 3
Light-activated drug delivery by 1a.
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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