Near-infrared fluorescent probes for imaging of intracellular mg²⁺ and application to multi-color imaging of mg²⁺, atp, and mitochondrial membrane potential

The magnesium ion (Mg²⁺) is an essential cation to maintain proper cellular activities. To visualize the dynamics and functions of Mg²⁺, there is a great need for the development of Mg²⁺-selective fluorescent probes. However, conventional Mg²⁺ fluorescent probes are falling behind in low selectivity and poor fluorescence color variation. In this report, to make available a distinct color window for multi-color imaging, we designed and synthesized highly Mg²⁺-selective and near-infrared (NIR) fluorescent probes, the KMG-500 series consisting of a charged β-diketone as a selective binding site for Mg²⁺ and a Si-rhodamine residue as the NIR fluorophore, which showed photoinduced electron transfer (PeT)-type OFF-ON response to the concentration of Mg²⁺. Two types of KMG-500 series probes, tetramethyl substituted Si-rhodamine KMG-501 and tetraethyl substituted Si-rhodamine KMG-502, were synthesized for the evaluation of cell permeability. For intracellular application, the membrane-permeable acetoxymethyl derivative KMG-501 (KMG-501AM) was synthesized and allowed to stably stain cultured rat hippocampal neurons during imaging of intracellular Mg²⁺. On the other hand, KMG-502 was cell membrane permeable without AM modification, preventing the probe from staying inside cells during imaging. KMG-501 distributed mainly in the cytoplasm and partially localized in lysosomes and mitochondria in cultured rat hippocampal neurons. Mg²⁺ increase in response to the FCCP uncoupler inducing depolarization of the mitochondrial inner membrane potential was detected in the KMG-501 stained neurons. For the first time, KMG-501 succeeded in imaging intracellular Mg²⁺ dynamics with NIR fluorescence. Moreover, it allows one to simultaneously visualize changes in Mg²⁺ and ATP concentration and also mitochondrial inner membrane potential and their interactions.