Improvement and biological applications of fluorescent probes for zinc, ZnAFs
Improvement and biological applications of fluorescent probes for zinc, ZnAFs
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DOI：
10.1021/ja025567p
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发表时间：
2002-06-12
影响因子：
15
通讯作者：
Nagano, T
中科院分区：
文献类型：
--
作者：
Hirano, T;Kikuchi, K;Nagano, T
本文介绍了新型锌离子荧光探针的发展和细胞应用。由于荧光素的激发波长和发射波长都在可见光范围内，最大限度地减少了激发光对细胞的损伤和自发荧光，因此被用作锌原子荧光材料的荧光体。N，N-二(2-吡啶甲基)乙二胺作为锌离子的受体，直接连接到荧光素的苯甲酸部分，在生理条件下(pH 7.4)，由于光诱导电子转移机制，导致锌离子的量子产率很低，分别为0.004和0.006。加入锌离子后，其荧光强度迅速增加，最高可达690倍，2F可达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。在其他生物上重要的阳离子如钙离子和镁离子的存在下，锌AFs不发光，并且对pH的变化不敏感。由于荧光素中的酚羟基发生质子化作用，与锌离子形成的络合物，其荧光强度降低到pH 7.0以下，其pK(A)值为6.2。而在中性和弱酸性条件下，由于酚羟基邻位上的吸电子氟被取代，使锌配合物的pKa值移动到4.9，因此它们在中性和弱酸性条件下都能发出稳定的荧光。为了应用于活细胞，合成了二乙酰基衍生物ZnAF-2F DA。ZnAF-2F DA能透过细胞膜，被胞浆中的酯酶降解，生成保留在细胞内的ZnAF-2F。用ZnAF-2F-DA可以检测培养细胞和海马片内锌离子的变化。
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 pK(a) value is 6.2. On the other hand, the Zn2+ complexes of ZnAF-1 F 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.