Xanthene fluorophores, including fluorescein, rhodol, and rhodamines, are representative classes of fluorescent probes that have been applied in the detection and visualization of biomolecules. "Turn on" activatable fluorescent probes, that can be turned on in response to enzymatic reactions, have been developed and prepared to reduce the high background signal of "always-on" fluorescent probes. However, the development of activity-based fluorescent probes for biological applications, using simple xanthene dyes, is hampered by their inefficient synthetic methods and the difficulty of chemical modifications. We have, thus, developed a highly efficient, versatile synthetic route to developing chemically more stable reduced xanthene fluorophores, based on fluorescein, rhodol, and rhodamine via continuous Pd-catalyzed cross-coupling. Their fluorescent nature was evaluated by monitoring fluorescence with variation in the concentration, pH, and solvent. As an application to activatable fluorescent probe, nitroreductase (NTR)-responsive fluorescent probes were also developed using the reduced xanthene fluorophores, and their fluorogenic properties were evaluated.
Keywords: activatable fluorescent probe; fluorescence; nitroreductase; reduced rhodafluor; xanthene fluorophore.