Many efforts have been undertaken to develop possibilities for copper-free Diels-Alder reaction based conjugation. Very promising options are Tetrazines and appropriate Dienophiles, as they are patent free and have potential for affordable bulk application. The Diels-Alder reaction between tetrazines and appropriate dienophiles happens spontaneously without addition of any catalyst. This is a big difference and advantage to the conventional Click reaction, which requires stoichiometric amounts of heavy metals (copper or ruthenium) and limits the click reaction towards use with biomolecules or bulk application. Different cyclooctyne derivatives can overcome some of these limitations, however, tetrazines bear a much broader potential, in particular for bulk applications.

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• Biomedical applications of tetrazine cycloadditions; N. K. Devaraj and R. Weissleder; Acc Chem Res 2011; 44: 816-27. doi:10.1021/ar200037t