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 N M R and fluorescent Ca2+ indicators based on the BAPTA structure into the range required to measure the cytosolic free Ca2+ concentration ( [Ca2+Ii). Single methylation on the j.3-carbon of each aminodiacetic acid group of BAPTA derivatives gave increases in the association constant for Ca2+ [K(Ca*+)] of up to 22-fold, depending o n 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,, and no loss of selectivity for Ca2+, so that the insensitivity of Ca2+ binding t o p H and [ Mg2+] over the physiological ranges was maintained.(1) Single methylation on the P-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, , values (average of the t w o highest pK, values in the molecule) were increased from 4.50 to 6.02 and 5.85 t o 6.59. The 19F N M R 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+Ii.(2) Single methylation o n the P-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, , from 5.39 to 5.90, respectively. There is a Ca2+-induced chemical shift of the 19F N M R resonance of 0.81 ppm upfield with slow exchange at 376 M H z and 30 "C. This compound was selected as a prototype for 19F N M R indicators with enhanced sensitivity through the incorporation of two trifluoromethyl groups to replace the t w o single fluorine substituents in the current 19F N M R indicators.

Polycarboxylate Fluorescent Indicators as Ion Concentration Probes in Biological Systems

“…The advantage therefore of the method is that it may be used without any interference from other ions present in the system, and that it may be applied in cells with strongly fluorescent intracellular components, where fluorescent dyes cannot be used. Disadvantages include low sensitivity and slow rate of response of the method [127][128][129][130]. Structural analogs of EDTA have been used to detect Mg 2+ concentrations [131].…”

Estimation of systolic and diastolic free intracellular Ca2+ by titration of Ca2+ buffering in the ferret heart

“…The new indicator, 4,5FBAPTA, was synthesized from 3,4difluorophenol by similar methods to those described [11,15]. In brief, 3,4-difluorophenol was nitrated with concentrated nitric acid in acetic acid at 5 mC until completion (assayed by TLC on silica developed in chloroform).…”

“…The basal [Ca# + ] and affinity were calculated by iteration [15]. Finally, K d was also determined by measuring the areas of bound and free NMR signals for various Ca# + concentrations using 100 µM indicator in citrate buffer [15]. All calculations of affinities were iterative and took account of free, bound and basal Ca# + concentrations.…”

“…upfield. NMR spectra for the other indicators have been published previously [11,15,16]. The chemical shifts of the free and bound resonances for all the indicators used are shown in Figure 2.…”

“…500 nM [8], which is significantly higher than the unperturbed diastolic [Ca# + ] i which is thought to be in the range 50-200 nM (reviewed in [1]). There are currently four "*F-NMR indicators that can be used to measure [Ca# + ] i in intact hearts : 5FBAPTA (K d 537 nM [11]), 1,2-bis-[2-(1-carboxyethyl)(carboxymethyl)amino-5-fluorophenoxy]ethane (DiMe-5FBAPTA) (K d 46 nM [15]), 1,2-bis-[2-(1-carboxyethyl)(carboxymethyl)amino-4-fluorophenoxy]ethane (DiMe-4FBAPTA) (K d 155 nM [15]) and 1,2-bis-[2-bis(carboxymethyl)amino-5-fluoro-4-methylphenoxy]ethane (MFBAPTA) (K d 270 nM [16]). We have previously shown that the observed [Ca# + ] i in the Langendorff-perfused ferret heart was significantly lower when measured with the high-affinity indicator DiMe-5FBAPTA than with 5FBAPTA [17].…”