Development of selective, visible light-excitable, fluorescent magnesium ion probes with a novel fluorescence switching mechanism

Novel fluorescent Mg²⁺ probes, 2′-carboxyfluorescein (2′-CF) and its derivatives, that are excitable by visible light, were designed, synthesized and characterized. After complexation with Mg²⁺, the absorption and emission maxima of these fluorescent probes were red-shifted and the fluorescence intensity was increased 11-fold at neutral pH. The apparent dissociation constant (K_d) of the Mg²⁺-2′-CF complex was 15.8 mM at neutral pH. The K_d of the Ca²⁺-2′-CF complex was about 10 times larger than that of the Mg²⁺ complex and the other derivatives showed similar characteristics. In contrast, all the commercially available fluorescent Mg²⁺ probes have a higher affinity for Ca²⁺ than Mg²⁺. 2′-CF fluoresced in alkaline solution (pH > 8) and the pK_a value was 8.8. The pK_a value of the Mg²⁺-2′-CF complex was 6.8, and the fluorescence intensity was increased in the neutral conditions. Thus, the addition of Mg²⁺ resulted in a lowering of the pK_a and also an increase of the fluorescence intensity.