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

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 H 2 O (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 H 2 S in vitro , and the dianion ([Fc-1-2H] 2- ) mechanism for detecting H 2 S 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. In this work, three fluorescein analogs (Fc-1 , Fc-2 , and Fc-3) using DCF as raw material were designed and successfully synthesized by the Vilsmeier–Haack and Knoevenagel reaction mechanisms. Dye Fc-1 with reactive aldehyde group was obtained in high yield from the low-temperature hydrolysis. The photophysical results showed that Fc-1 had relatively high fluorescence quantum yields in polar proton solvents and large Stokes shifts in different solvents. The Gaussian calculation proved and explained the reason for the large Stokes shift of Fc-1. Meanwhile, it was found that Fc-1 could detect H 2 S in ratio and colorimetric assay. The mechanism of detecting H 2 S with Fc-1 was rationally proposed by combining the results of NMR, pH titrations, and Gaussian calculation. Additionally, Fc-1 realized the five-classification detection of leukocytes because of its good water solubility, cell permeability, and cytotoxicity. Further, the photophysical properties of dyes Fc-2 and Fc-3 were studied, which showed that both dyes exhibited large Stokes shifts and long emission wavelengths. The derivatization of the carboxyl group on the benzene ring did not affect the photophysical properties of Fc-2. Similarly, the Gaussian calculation method was used to explain the reason for the large Stokes shift. The spectral changes of two dyes at different pH values proved that dyes Fc-2 and Fc-3 had potential applications in pH fluorescent probes. The strategy of constructing fluorescent dyes with excellent photophysical properties was recommended on the basis of Vilsmeier–Haack and Knoevenagel reactions and it will be beneficial to developing more dyes. [Display omitted] • Three fluorescein analogs were constructed based on Vilsmeier-Haack and Knoevenagel condensation reactions. • The fluorescent dye Fc-1 can realize the detection of H 2 S and pH simultaneously. • The constructing strategy of fluorescent dyes based on Vilsmeier-Haack and Knoevenagel reactions will be beneficial to developing dyes with large Stokes shifts. Synopsis: Three fluorescein analogs with relatively good photophysical properties were synthesized and their relationships between structure and properties were investigated. [ABSTRACT FROM AUTHOR]