Design and Synthesis of Highly Sensitive and Selective Fluorescein-Derived Magnesium Fluorescent Probes and Application to Intracellular 3D Mg²⁺ Imaging

Abstract

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 beta-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 (Kd,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.

Solvent Effect on the Fluorescence Response of Hydroxycoumarin Bearing a Dipicolylamine Binding Site to Metal Ions

“…5,6 Fluorescent receptors for Mg 2+ , which have diaza-18-crown-6, β-diketone, and calixarene moieties for ion recognition, heve been reported. [7][8][9] However, those receptors could not discriminate Mg 2+ from Ca 2+ due to the similar chemical properties of those two cations. 10 Thus, the development of fluorescent probes with higher specificity and selectivity is being pursued in earnest.…”

Dictyostelium Myosin-5b Is a Conditional Processive Motor

“…tion, we followed the spatial and temporal changes in free Mg 2ϩ -ion distribution in Dictyostelium cells by TIRF microscopy using the Mg 2ϩ -sensitive dye KMG-104AM that was previously described as a potent marker for measuring Mg 2ϩ -ion concentrations within living cells (40). The series of pictures in Fig.…”

Nanophotonics and supramolecular chemistry

“…In particular, development of a fluorescent probe for Ca 2+ ion detection fura-2 6 by Tsien and coworkers [43] promoted significant activity in the corresponding research field. , and Zn 2+ have also been developed [44][45][46][47][48] as well as probes for small molecules such as nitrogen monooxide (NO) [49,50]. Another important milestone in biological imaging is the application of green fluorescence protein (GFP) to cell media [51][52][53], which lead to the Nobel prize in Chemistry being awarded to Chalfie, Tsien, and Shimomura in 2008.…”