Click Chemistry for Cell Labeling: A Review of Pro-fluorescent Tetrazines for Live Cell Imaging

Click chemistry has advanced over the past two decades to enable the imaging of diverse biomolecules in cells, including non-genetically encoded molecules like lipids and glycans. In the toolbox of bioorthogonal click reactions for cell labeling, pro-fluorescent tetrazines stand out owing to their fast kinetics and low background fluorescence when reacting with dienophiles like trans-cyclooctene, bicyclononynes, or cyclopropenes. In this chapter, we present a brief overview of click chemistry approaches to cell labeling followed by an in-depth review of pro-fluorescent tetrazines. We review key properties for cell imaging, including ligation rates, fluorescence turn-on ratios and excitation/emission spectra. Finally, we discuss recent applications in biology for click chemistry-based cell imaging based on fluorogenic tetrazines and on activatable tetrazine ligations for cell-selective labeling.