: rapid exchange between unsymmetric tetrazines and thiols for reversible, chemoselective functionalisation of biomolecules with on-demand bioorthogonal locking.

The late-stage functionalisation and diversification of complex structures including biomolecules is often achieved with the help of click chemistry. Besides employing irreversible click-like reactions, many synthetic applications benefit from reversible click reaction strategies, so called de-/trans-click approaches. Yet, the combination of both, reversible and irreversible click chemistry - while still respecting the stringent criteria of click transformations - remains so far elusive for modifications of biomolecular structures. Here, we report as a concept that enables reversible click reactions and on-demand locking of chemical entities, thus switching from reversible to irreversible modifications of complex biomolecules. For this purpose, we employ the tetrazine-thiol exchange (TeTEx) reaction as a fully traceless click reaction with second order rate constants higher than 2 M s within aqueous environments. Employing TeTEx as a reversible click reaction for the chemoselective modification of biomolecules is made possible by the use of 3,6-disubstituted 1,2,4,5-tetrazines bearing a single sulfide residue. The inherent reactivity of tetrazines towards inverse electron demand Diels-Alder (IEDDA) reactions allows to stabilize the clicked structure, switching from reversible to irreversible systems ().