Fluorogenic Bifunctional Trans‐cyclooctenes as Efficient Tools for Investigating Click‐to‐Release Kinetics

The inverse electron demand Diels-Alder pyridazine elimination reaction between tetrazines and allylic substituted trans -cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage reactions. Determining the rate of elimination on alkylamine substrates has so far proven difficult. Here, we report a fluorogenic tool consisting of a TCO-linked EDANS fluorophore and a DABCYL quencher for accurate detection of both the click and release rates for any tetrazine at physiologically relevant concentrations.