Design and synthesis of a FlAsH-type Mg²⁺ fluorescent probe for specific protein labeling

Although the magnesium ion (Mg²⁺) is one of the most abundant divalent cations in cells and is known to play critical roles in many physiological processes, its mobilization and underlying mechanisms are still unknown. Here, we describe a novel fluorescent Mg²⁺ probe, "KMG-104-AsH", composed of a highly selective fluorescent Mg ²⁺ probe, "KMG-104", and a fluorescence-recoverable probe, "FlAsH", bound specifically to a tetracysteine peptide tag (TCtag), which can be genetically incorporated into any protein. This probe was developed for molecular imaging of local changes in intracellular Mg²⁺ concentration. KMG-104-AsH was synthesized, and its optical properties were investigated in solution. The fluorescence intensity of KMG-104-AsH (at λ_em/max = 540 nm) increases by more than 10-fold by binding to both the TCtag peptide and Mg²⁺, and the probe is highly selective for Mg²⁺ (K_d/Mg = 1.7 mM, K_d/Ca 100 mM). Application of the probe for imaging of Mg²⁺ in HeLa cells showed that this FlAsH-type Mg²⁺ sensing probe is membrane-permeable and binds specifically to tagged proteins, such as TCtag-actin and mKeima-TCtag targeted to the cytoplasm and the mitochondrial intermembrane space. KMG-104-AsH bound to TCtag responded to an increase in intracellular Mg²⁺ concentration caused by the release of Mg²⁺ from mitochondria induced by FCCP, a protonophore that eliminates the inner membrane potential of mitochondria. This probe is expected to be a strong tool for elucidating the dynamics and mechanisms of intracellular localization of Mg²⁺.