Development of the First Tritiated Tetrazine: Facilitating Tritiation of Proteins.

Tetrazine (Tz)–trans‐cyclooctene (TCO) ligation is an ultra‐fast and highly selective reaction and it is particularly suited to label biomolecules under physiological conditions. As such, a 3H‐Tz based synthon would have wide applications for in vitro/ex vivo assays. In this study, we developed a 3H‐labeled Tz and characterized its potential for application to pretargeted autoradiography. Several strategies were explored to synthesize such a Tz. However, classical approaches such as reductive halogenation failed. For this reason, we designed a Tz containing an aldehyde and explored the possibility of reducing this group with NaBT4. This approach was successful and resulted in [3H]‐(4‐(6‐(pyridin‐2‐yl)‐1,2,4,5‐tetrazin‐3‐yl)phenyl)methan‐t‐ol with a radiochemical yield of 22 %, a radiochemical purity of 96 % and a molar activity of 0.437 GBq/μmol (11.8 Ci/mmol). The compound was successfully applied to pretargeted autoradiography. Thus, we report the synthesis of the first 3H‐labeled Tz and its successful application as a labeling building block.