Design and synthesis of highly sensitive and selective fluorescein-derived magnesium fluorescent probes and application to intracellular 3D Mg²⁺ imaging

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 β-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,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.