A Comprehensive Study of the Ca²⁺ Ion Binding of Fluorescently Labelled BAPTA Analogues

Abstract

Since its development, the ionophore BAPTA (1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N’,N’‐tetraacetic acid) has been used unchanged in calcium sensing applications. In this work we present a comprehensive experimental and theoretical study of novel alterations in the structure of BAPTA, with a focus on the systematic modification of the chain connecting the two aromatic rings of the molecule (denoted as “linker”). A bis‐(diethylamino)xantene fluorophore was also attached to the structures in a fixed position and the structure‐fluorescence response relationship of these molecules was investigated in addition. The effect of the linker's length, the number of oxygen atoms in this chain and even the removal of one of the rings was tested; these all proved to significantly alter the characteristics of the compounds. For example, it was found that the second aromatic ring of BAPTA is not essential for the turn‐on of the fluorescence. We also demonstrated that successful sensing can be realized even by replacing the chain with a single oxygen atom, which suggests the availability of a new calcium binding mode of the chelator. The reliable turn‐on characteristic, the steep Ca2+ fluorescence titration curve and the intense fluorescence emission combine to make this compound a prospective candidate as a calcium sensing molecular probe in diagnostic neurobiological applications.

Monitoring correlates of SARS-CoV-2 infection in cell culture using two-photon microscopy and a novel fluorescent calcium-sensitive dye

“…1a). Fluorescein-based fluorophores are well-known for their non-fluorescent lactone and fluorescent zwitterion tautomerism, which is heavily influenced by the substituents on the xanthene ring, pH and other factors 32 . At the same time, the pH and Ca 2+ dependent fluorescence of BAPTA-based Ca 2+ indicators is a key factor in their applicability.…”

“…NMR solvents were purchased from Eurisotop. Reactions were monitored by LC-MS (Shimazu MS2020, Supelco Ascentis, 2.0 × 50 mm, 2.1 pm C18 column; injection of 1 pi; 5-98% MeCN/H 2 O, linear gradient, with constant 0.1% v/v TFA additive; run of 6 min; flow of 0.8 ml min“ 1 ; ESI; positive ion mode). Reaction products were purified by gradient elution preparative HPLC (HPLC Gilson 333 instrument, UV detector 220 nm) on a Phenomenex Gemini C18,250×50.00 mm; 10 pm, 110A column using 0.2% v/v TFA in water and acetonitrile as the mobile phase components.…”

“…219 mg (0.363 mmol, 1.2 equiv.) of amino BAPTA derivative ( 4 ) 1 was added, the mixture was stirred overnight. The mixture was poured into distilled water (40 mL), the product was extracted with EtOAc (6 × 50 mL), the combined organic phases was washed with saturated sodium hydrogen carbonate (1 × 50 mL) and brine (1 × 100 mL).…”

“…The dye is aimed to be more sensitive and less disturbing to the cellular environments than previously applied ones. Most of the known fluorescent Ca 2+ ion sensors, especially those with BAPTA chelator functionality 32 , suffer from certain limitations in terms of their cellular applications. First, the widely used acetoxymethyl ester (AM) protected derivatives need to be applied at high concentrations (in 1 2 mM) to generate the active, deprotected form at suitable concentrations for the assay measurements.…”

An anthraimidazoledione-based charge transfer probe for dual mode sensing of calcium ions: role of the counter ion in signal improvement

“…3 Considering its huge biological relevance, numerous methods, such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry and ion-selective electrodes, have been developed to detect and monitor the activity of Ca 2+ ions. 4,5 In addition to high-cost instrumentation and extensive sample preparation, most of these techniques suffer from a lack of selectivity (significant interference from sodium, magnesium, and zinc ions, etc.) On the contrary, optical probes for calcium ions mostly consider the physicochemical properties of calcium ions, such as ionic radius, valency, and polarizability of its electron cloud, etc.…”