The tetrazine ligation is the reaction of a trans-cyclooctene and an s-tetrazine in an inverse electron demand Diels Alder (IEDDA) reaction followed by a retro-Diels Alder reaction to eliminate nitrogen gas. This is a fast reaction with a second order rate constant of 2000 M−1–s−1 (in 9:1 methanol/water) allowing modification of biomolecules at very low concentrations. This ligation can be performed in aqueous media with very fast speed. Reactions have also been performed using TCO or norbornenes as dienophiles at second order rates on the order of 1 M−1•s−1 in aqueous media. The reaction has been applied in labeling live cells and other bio-entities.