Design and synthesis of highly sensitive and selective fluorescein-derived magnesium fluorescent probes and application to intracellular 3D Mg2+ imaging
Design and synthesis of highly sensitive and selective fluorescein-derived magnesium fluorescent probes and application to intracellular 3D Mg2+ imaging
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DOI：
10.1021/ja049624l
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发表时间：
2004-12-22
影响因子：
15
通讯作者：
Suzuki, K
中科院分区：
文献类型：
--
作者：
Komatsu, H;Iwasawa, N;Suzuki, K
细胞内镁离子的作用是药理学和细胞生物学领域的高度关注。为了实现细胞内Mg2+的动态和三维成像，人们迫切需要开发优化的Mg2+荧光探针。本文介绍了三种新型Mg2+荧光探针KMG-101、-103和-104的设计、合成和细胞应用。该系列化合物具有带电荷的β -二酮作为Mg2+特异性结合位点和荧光素残基作为荧光团，可以用Ar+激光激发，如广泛用于共聚焦扫描显微镜。这种分子设计导致对Mg2+离子的强烈off-on型荧光响应。两种荧光探针KMG-103和-104具有合适的解离常数（K-d,K-Mg2+ = 2 mM），并且在细胞内镁离子浓度范围（0.1-6 mM）内荧光增强近10倍，从而实现高对比度，敏感和选择性的Mg2+测量。在细胞内应用方面，合成了膜透性探针KMG-104AM，并成功导入PC12细胞。将线粒体解耦剂FCCP应用于探针结合的细胞后，随着时间的推移，游离镁离子浓度可以随之增加。用共聚焦显微镜观察了细胞内三维镁离子浓度分布。
The role of intracellular magnesium ions is of high interest in the fields of pharmacology and cellular biology. To accomplish the dynamic and three-dimensional imaging of intracellular Mg2+, there is a strong desire for the development of optimized Mg2+ fluorescent probes. In this paper we describe the design, synthesis, and cellular application of the three novel Mg2+ fluorescent probes KMG-101, -103, and -104. The compounds of this series feature a charged beta-diketone as a binding site specific for Mg2+ and a fluorescein residue as the fluorophore that can be excited with an Ar+ laser such as is widely used in confocal scanning microscopy. This molecular design leads to an intensive off-on-type fluorescent response toward Mg2+ ions. The two fluorescent probes KMG-103 and -104 showed suitable dissociation constants (K-d,K-Mg2+ = 2 mM) and nearly a 10-fold fluorescence enhancement over the intracellular magnesium ion concentration range (0.1-6 mM), allowing high-contrast, sensitive, and selective Mg2+ measurements. For intracellular applications, the membrane-permeable probe KMG-104AM was synthesized and successfully incorporated into PC12 cells. Upon application of the mitochondria uncoupler FCCP to the probe-incorporated cells, the resulting increase in the free magnesium ion concentration could be followed over time. By using a confocal microscope, the intracellular 3D magnesium ion concentration distributions were satisfactorily observed.