Labelling cell organelles with dual orthogonal click reaction pairs

While single click reactions for cell imaging are widely applied, employing multiple orthogonal click reactions tracking multiple complex intracellular systems within one cell remains challenging. Our research combined two click reactions with different reaction mechanisms: tetrazine- alkene [4+2] inverse electron demand Diels-Alder cycloaddition, and CBT-Cys condensation reaction. Orthogonality between these two reactions was studied and the two reactions were applied for simultaneous labeling mitochondria and outer cell membrane of HepG2 cells. The results indicate that it is possible to use two (or n) orthogonal click reactions to track multiple organelles within one cell.