Design and properties of new 19F NMR Ca2+ indicators: modulation of the affinities of BAPTA derivatives via alkylation

Abstract

The effects of alkyl substitution in the aminodiacetic groups of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) have been examined systematically with the aim of increasing the affinity for Ca²⁺ of novel ¹⁹F NMR and fluorescent Ca²⁺ indicators based on the BAPTA structure into the range required to measure the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). Single methylation on the β-carbon of each aminodiacetic acid group of BAPTA derivatives gave increases in the association constant for Ca²⁺[K(Ca²⁺)] of up to 22-fold, depending on the structure of the parent BAPTA derivative. This was shown to be the most effective alkylation for BAPTA derivatives to raise K(Ca²⁺) with a minimal increase in pK_a and no loss of selectivity for Ca²⁺, so that the insensitivity of Ca²⁺ binding to pH and [Mg²⁺] over the physiological ranges was maintained.

(1) Single methylation on the β-carbon of each aminodiacetic acid group of BAPTA derivatives symmetrically substituted with single fluorines at the 4-position (4FBAPTA) or at the 5-position (5FBAPTA) raised log K(Ca²⁺) from 5.61 to 6.81 and 6.12 to 7.47, respectively. The corresponding pK_av values (average of the two highest pK_a, values in the molecule) were increased from 4.50 to 6.02 and 5.85 to 6.59. The ¹⁹F NMR spectroscopic properties of the methylated derivatives were unchanged compared with the parent compounds except for slower exchange rates. These new methylated derivatives of 4FBAPTA and 5FBAPTA therefore extend the range of K(Ca²⁺) available for ¹⁹F NMR indicators of [Ca²]_i.

(2) Single methylation on the β-carbon of each aminodiacetic acid group of BAPTA derivatives symmetrically substituted at the 5-position on the aromatic rings with trifluoromethyl groups increased log K(Ca²⁺) from 4.83 to 6.14 and the pK_av from 5.39 to 5.90, respectively. There is a Ca²⁺-induced chemical shift of the ¹⁹F NMR resonance of 0.81 ppm upfield with slow exchange at 376 MHz and 30 °C. This compound was selected as a prototype for ¹⁹F NMR indicators with enhanced sensitivity through the incorporation of two trifluoromethyl groups to replace the two single fluorine substituents in the current ¹⁹F NMR indicators.