Construction, photophysical properties, structure-activity relationship, and applications of fluorescein analogs

Abstract

The investigation on the construction strategy, relationship between the structure and photophysical properties of fluorescein analogs plays an important role in the rational design of specific fluorescent dyes. In this work, a fluorescein analog Fc-1, containing a reactive aldehyde group was prepared using 2′, 7′-dichlorofluorescein (DCF) as the raw material and Vilsmeier–Haack reaction mechanism. Meanwhile, two more fluorescein analogs Fc-2 and Fc-3 were synthesized based on Fc-1 and the Knoevenagel reaction mechanism. The results of Fc-1 indicated that the yield of Fc-1 was higher than 95% after a step of the low-temperature hydrolysis (40 °C) from an intermediate imine salt DCF-3. The fluorescence quantum yield (Φ) of Fc-1 was relatively high in polar protic solvents, even in H2O (Φ = 0.25). Fc-1 had large Stokes shifts in different solvents (≧ 123 nm), which was indentified by the Gaussian calculation of Fc-1. Meanwhile, Fc-1 was also used as a ratio and colorimetric fluorescent probe for H2S in vitro, and the dianion ([Fc-1-2H]2-) mechanism for detecting H2S was proposed by nuclear magnetic resonance (NMR), pH titration, and Gaussian calculation. Additionally, Fc-1 can be used in leukocyte classification because of its relatively high Φ in water, cell permeability, and cytotoxicity. All the data of dyes Fc-2 and Fc-3 indicated that the derivatization of the carboxyl group for Fc-2 did not affect the photophysical properties of Fc-2. Both of them had relatively large Stokes shifts and long emission wavelengths. The large Stokes shift of Fc-2 and Fc-3 was due to the distribution of electron clouds in the excited states of Fc-2 and Fc-3. The results of pH titration proved that dyes Fc-2 and Fc-3 were sensitive to the changes in pH and exhibited their potentials as pH probes. Thus, the strategy of constructing fluorescent dyes based on Vilsmeier–Haack and Knoevenagel reactions and the relationship between structure and properties will be beneficial to developing more dyes with large Stokes shift.