Evaluation of bioorthogonally applicable tetrazine-Cy3 probes for fluorogenic labeling schemes

The fluorogenic features of three sets of tetrazine-Cy3 probes were evaluated in bioorthogonal tetrazine-cyclooctyne ligation schemes. These studies revealed that the more efficient, internal conversion-based quenching of fluorescence by the tetrazine modul is translated to improved fluorogenicity compared to the more conventional, energy transfer-enabled design. Furthermore, a comparison of directly conjugated probes and vinylene-linked tetrazine-Cy3 probes revealed that more intimate conjugation of the tetrazine and the chromophore results in more efficient IC-based quenching even in spectral ranges where tetrazine exhibits diminished modulation efficiency. The applicability of these tetrazine-quenched fluorogenic Cy3 probes was demonstrated in the fluorogenic labeling schemes of the extra- and intracellular proteins of live cells.