Fluorescence of the 5- and 6-Carboxyfluorescein Bifluorophores

Bifluorophores (5-FAM) 2 and (6-FAM) 2 were synthesized on a 3,5-diaminobenzoic acid framework, which allows the insertion of two fluorophore molecules into a single site. Despite the similarity of the structures of the 5- and 6-carboxyfluorescein isomers (5-FAM and 6-FAM), the photophysical behavior of the corresponding bifluorophores differs. The luminescence quantum yield of (6-FAM) 2 in a buffer solution with pH 8.5 is 2.4 times lower than for (5-FAM) 2 . The structural relaxation of molecules in the excited state was shown to be slower with increasing solution viscosity when using a water–glycerol mixture and with greater microviscosity in the case of a micellar solution. The slower relaxation leads to an increase in the fluorescence intensity, lifetime of the excited states, and extent of polarization, i.e., the photophysical properties of the synthesized compounds are largely by the viscosity of the medium.