The tetrazine ligation is an inverse electron-demand Diels-Alder reaction widely used for bioorthogonal modifications due to its versatility, site specificity and fast reaction kinetics. A major limitation has been the incorporation of dienophiles in biomolecules and organisms, which relies on externally added reagents. Available methods require the incorporation of tetrazine-reactive groups by enzyme-mediated ligations or unnatural amino acid incorporation. Here we report a tetrazine ligation strategy, termed TyrEx (tyramine excision) cycloaddition, permitting autonomous dienophile generation in bacteria. It utilizes a unique aminopyruvate unit introduced by post-translational protein splicing at a short tag. Tetrazine conjugation occurs rapidly with a rate constant of 0.625 (15) M s and was applied to produce a radiolabel chelator-modified Her2-binding Affibody and intracellular, fluorescently labelled cell division protein FtsZ. We anticipate the labelling strategy to be useful for intracellular studies of proteins, as a stable conjugation method for protein therapeutics, as well as other applications.
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| http://dx.doi.org/10.1038/s41557-023-01252-8 | DOI Listing |
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