Development of Phosphinate Ligand-Based Low-Affinity Ca 2+ Fluorescent Probes and Application to Intracellular Ca 2+ Imaging.

Divalent metal cations such as calcium ion (Ca) and magnesium ion (Mg) are indispensable to the regulation of various cellular activities. In this research, we developed the KLCA series utilizing -aminophenol--diacetate--methylene-methylphosphinate (APDAP) as a target binding site, which was reported recently as a highly free Mg-selective ligand. with orange fluorescence based on a rhodamine fluorophore and with near-infrared (NIR) fluorescence based on a Si-rhodamine fluorophore were synthesized, intended for application to multicolor imaging. The evaluation of the fluorescence response to Ca and Mg of the KLCA series indicated the applicability as low-affinity Ca probes. While mainly localized in the cytosol in cultured rat hippocampal neurons, localized to the cytosol and granular organelles in neurons. Comparison of the fluorescence response of and the high-affinity Ca probe Fluo-4 upon stimulation by glutamate in stained neurons revealed that could reflect the secondary large rise of intracellular Ca, which Fluo-4 could not detect. In addition, showed a fluorescence response similar to the low-affinity Ca probe Fluo-5N upon stimulation by glutamate in stained neurons, concluding that and could be used as low-affinity Ca probes. The KLCA series offers new options for low-affinity Ca probes. Moreover, achieved simultaneous visualization of the change in Ca and ATP concentrations and also in mitochondrial inner membrane potential in neurons. is expected to be a strong tool that enables simultaneous multicolor imaging of multiple targets and elucidation of their relationship in cells.