Design and properties of a fluorescent indicator of intracellular free Na+ concentration

Abstract

We have recently described a cryptand structure, FCryp-1, with appropriate properties for an indicator of intracellular free Na+ concentration using the 19F-n.m.r. chemical shift of the incorporated 5FBAPTA [1,2-bis-(2-amino-5-fluorophenoxy)ethane-NNN'N'-tetra-acetic acid] reporter group to measure the free cytosolic Na+ concentration [( Na+]i) [Smith, Morris, Hesketh and Metcalfe (1986) Biochim. Biophys. Acta 889, 82-83]. FCryp-1 carries four carboxylate groups to confer aqueous solubility and the indicator is membrane-permeant when the carboxyls are esterified with acetoxymethyl ester groups. Here we describe the synthesis of FCryp-2 to provide a fluorescent indicator of [Na+]i. FCryp-2 retains the parent tribenzo (2:2:1) cryptand structure of FCryp-1, in which the benzenoid ring at C-21 in FCryp-1 is replaced by an indole derivative which acts as the fluorophor in FCryp-2. With excitation at 340 nm, FCryp-2 gives an emission maximum at 460 nm in the absence of Na+ which shifts to 395 nm when FCryp-2 is saturated with Na+, with an isosbestic point at 455 nm. The apparent dissociation constant of FCryp-2 in a buffer solution of 100 mM-KCl/20 mM-KH2PO4/K2HPO4, pH 7.0, at 37 degrees C is 6.0 mM and the free Na+ concentration can be measured either from the calibrated fluorescence intensity at 395 nm, which increases 25-fold when Na+ is bound to FCryp-2, or from the ratio of fluorescence intensities at 395 nm and 455 nm. The measurement of free [Na+] by either method is unaffected by K+, Ca2+ or Mg2+ in the normal intracellular concentration ranges. Free [Na+] measurements by the ratio method are unaffected by pH from 6.6 to 7.6.

Role of Na+and K+in Enzyme Function

“…Conceptual simplicity, broad applicability, and utility of these designed molecules led to a most deserved Nobel Prize in 1987 for these authors. Chemical coupling of these compounds yields fluorescent M ϩ sensors (136,208,302). Even larger macrocyclic compounds, such as calixarenes, have been developed more recently for use in M ϩ -selective electrodes (10,44).…”

6,7,9,10,17,18,20,21,28,29,31,32,39,40,42,43-Hexadecahydro-16,44:22,38-di(ethanooxyethano)-5,8,11,19,27,30,33,41-octaoxa-16,22,38,44-tetraazatetrabenzo[h,q,z,i 1]cyclohexatriacontine: a benzoannelated tricyclic cryptand

“…The crude tetralactam was purified by recrystallization from toluene. Reduction of the tetralactam using BH 3 in THF and subsequent work-up (Smith et al, 1986(Smith et al, , 1988 gave the title compound, (I), in 12% overall yield. ESI-MS: m/z = 429.2 (M + 2H + ).…”

Molecular Sensors for NMR-Based Detection

“…This is usually achieved by introducing multiple carboxylate acid groups onto the chelator, which exist in the ionic form at normal intracellular pH. In order to meet these criteria, structurally optimized 19 F NMR-based sensors for Na + continues to be developed. , …”