Werther, PhilippYserentant, KlausBraun, FelixGrussmayer, KristinNavikas, VytautasYu, MiaoZhang, ZhibinZiegler, Michael J.Mayer, ChristophGralak, Antoni J.Busch, MarvinChi, WeijieRominger, FrankRadenovic, AleksandraLiu, XiaogangLemke, Edward A.Buckup, TiagoHerten, Dirk-PeterWombacher, Richard2021-10-092021-10-092021-10-092021-09-2210.1021/acscentsci.1c00703https://infoscience.epfl.ch/handle/20.500.14299/182080WOS:000700884500016Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.Chemistry, MultidisciplinaryChemistrytetrazine probesmammalian-cellsfluorophorestrategytext::journal::journal article::research article