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.

中文翻译：

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锌、ZnAFs 荧光探针的改进和生物学应用

描述了用于 Zn2+、ZnAF-1F 和 ZnAF-2F 的新型荧光探针的开发和细胞应用。荧光素用作 ZnAFs 的荧光团，因为它的激发和发射波长在可见光范围内，可最大限度地减少激发光对细胞的损伤和自发荧光。N,N-双（2-吡啶基甲基）乙二胺用作 Zn2+ 的受体，直接连接到荧光素的苯甲酸部分，导致生理条件下 ZnAF-1F 和 ZnAF-2F 的量子产率非常低，分别为 0.004 和 0.006条件 (pH 7.4) 由于光诱导电子转移机制。添加 Zn2+ 后，ZnAF-1F 的荧光强度迅速增加至 69 倍，ZnAF-2F 的荧光强度增加至 60 倍。表观解离常数 (K(d)) 在纳摩尔范围内，这为生物应用提供了足够的灵敏度。ZnAFs 在其他重要的生物阳离子如 Ca2+ 和 Mg2+ 存在下不发荧光，并且对 pH 值的变化不敏感。由于荧光素酚羟基的质子化，先前开发的 ZnAF、ZnAF-1 和 ZnAF-2 与 Zn2+ 的复合物在 pH 7.0 以下荧光强度降低，荧光素的 pKa 值为 6.2。另一方面，ZnAF-1F 和 ZnAF-2F 的 Zn2+ 复合物在中性和微酸性条件下发出稳定的荧光，因为 pKa 值通过在酚羟基的邻位取代吸电子氟而移动到 4.9。为了应用于活细胞，合成了 ZnAF-2F 的二乙酰衍生物 ZnAF-2F DA。ZnAF-2F DA 可以透过细胞膜，并被胞质溶胶中的酯酶水解产生 ZnAF-2F，其保留在细胞中。使用ZnAF-2F DA，我们可以测量培养细胞和海马切片中细胞内Zn2+的变化。