Synthesis and characterisation of Thio-H, a new excitation and emission ratioable fluorescent Ca2+/Mg2+indicator with high brightness

Abstract

A new fluorescent ratiometric indicator for Ca²⁺ and Mg²⁺, tricaesium salt of {2-[bis(carboxymethyl)amino]-5-(5-phenylthiophen-2-yl)phenoxy}acetic acid, Thio-H, has been synthesised and evaluated for its cation binding properties via fluorimetric titrations. Thio-H has the interesting property of being both excitation and emission ratioable, expanding the range of possible applications. The in vitro dissociation constant, K_d, measured at 20 °C in 100 mM KCl, pH 7.20 for the Ca²⁺–Thio-H complex is 45 ± 13 μM; for Mg²⁺–Thio-H a K_d value of 5.6 ± 0.6 mM is found. The free form of Thio-H has a high fluorescence quantum yield (0.74), which decreases to 0.50 and 0.65 upon binding to Ca²⁺ and Mg²⁺, respectively. Measurements under physiological conditions show that increasing the temperature from 20 to 37 °C decreases the affinity of Thio-H for Ca²⁺. Changing the pH from 7.05 to 7.40 does not affect the K_d value of the Mg²⁺ complex but it increases somewhat the affinity of the probe for Ca²⁺. In the presence of 1 mM Mg²⁺, the Ca²⁺ affinity of Thio-H decreases (K_d = 126 ± 46 μM at 20 °C, pH 7.05). Time-resolved fluorescence measurements confirm that the inflection point in the fluorimetric titration curve can be correctly assigned to K_d. Since the dissociation constant for the Mg²⁺–indicator complex falls within the intracellular [Mg²⁺] domain, while the K_d value for Ca²⁺ is well above the basal Ca²⁺ levels, Thio-H has potential applications as a Mg²⁺ indicator. The low affinity for Ca²⁺ can be exploited for detecting intracellular Ca²⁺ levels in the micromolar range, on condition that [Mg²⁺]_i remains practically constant during the measurements. Thio-H provides an excellent addition to the commercial ratiometric low-affinity Ca²⁺ indicators Mag-fura-2, Mag-fura-5, Mag-indo-1, Fura-FF, and BTC.