Improvement and Biological Applications of Fluorescent Probes for Zinc, ZnAFs

Abstract

The development and cellular applications of novel fluorescent probes for Zn2+, ZnAF-1F, and ZnAF-2F are described. Fluorescein is used as a fluorophore of ZnAFs, because its excitation and emission wavelengths are in the visible range, which minimizes cell damage and autofluorescence by excitation light. N,N-Bis(2-pyridylmethyl)ethylenediamine, used as an acceptor for Zn2+, is attached directly to the benzoic acid moiety of fluorescein, resulting in very low quantum yields of 0.004 for ZnAF-1F and 0.006 for ZnAF-2F under physiological conditions (pH 7.4) due to the photoinduced electron-transfer mechanism. Upon the addition of Zn2+, the fluorescence intensity is quickly increased up to 69-fold for ZnAF-1F and 60-fold for ZnAF-2F. Apparent dissociation constants (K(d)) are in the nanomolar range, which affords sufficient sensitivity for biological applications. ZnAFs do not fluoresce in the presence of other biologically important cations such as Ca2+ and Mg2+, and are insensitive to change of pH. The complexes with Zn2+ of previously developed ZnAFs, ZnAF-1, and ZnAF-2 decrease in fluorescence intensity below pH 7.0 owing to protonation of the phenolic hydroxyl group of fluorescein, whose pKa value is 6.2. On the other hand, the Zn2+ complexes of ZnAF-1F and ZnAF-2F emit stable fluorescence around neutral and slightly acidic conditions because the pKa values are shifted to 4.9 by substitution of electron-withdrawing fluorine at the ortho position of the phenolic hydroxyl group. For application to living cells, the diacetyl derivative of ZnAF-2F, ZnAF-2F DA, was synthesized. ZnAF-2F DA can permeate through the cell membrane, and is hydrolyzed by esterase in the cytosol to yield ZnAF-2F, which is retained in the cells. Using ZnAF-2F DA, we could measure the changes of intracellular Zn2+ in cultured cells and hippocampal slices.

Improvement of Fluorescence Characteristics of Coumarins: Syntheses and Fluorescence Properties of 6-Methoxycoumarin and Benzocoumarin Derivatives as Novel Fluorophores Emitting in the Longer Wavelength Region and Their Application to Analytical Reagents

“…Fluorescein is one of the most widely used fluorophore for biological experiments, since it has a high fluorescence quantum yield of 0.90 or grater in aqueous solution and its excitation and emission wavelengths are in the visible region. Recently, Nagano and coworkers have succeeded in developing fluorescent reagents for the specific detection of nitric oxide (NO), [5][6][7][8] singlet oxygen ( 1 O 2 ), 9,10) and others 11,12) using fluorescein as the fluorophore. 4,4-Difluoro-4-bora-3a,4a-diazas-indacenes (BODIPYs) are also well-known fluorophores that have very sharp and narrow fluorescence bandwidths in addition to their high fluorescence quantum yield in aqueous solution.…”

A novel small molecule fluorescent sensor for Zn2+ based on pyridine–pyridone scaffold

“…We realized that Zn 2+ selective fluorescent probes have mainly used three core structures, namely quinoline, BF 2 chelated dipyrromethene and fluorescein [21][22][23][24][25][26][27][28][29][30]. After modification of the core structure in getting selectivity toward Zn 2+ , the molecule obviously became bigger and more hydrophobic.…”

Photochemical and complexation studies for new fluorescent and colored chelator

“…Fluorescent probes for measuring biologically important ions such as Ca 2C , Mg 2C , and Zn 2C have been developed in many research groups [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. The fluorescence intensity of these metal probes changes mainly based on the differences of fluorescent properties between free form of the probe molecule and its metal complex.…”