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

Abstract

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.