Dizinc Enzyme Model/Complexometric Indicator Pairs in Indicator Displacement Assays for Inorganic Phosphates under Physiological Conditions

Abstract

A dizinc phosphohydrolase enzyme model complex employing the dinucleating ligand 2,6-bis-[(bis-pyridin-2-ylmethyl-amino)methyl]-4-methylphenol (L1) was tested for binding to a series of 11 commercially available complexometric indicators in aqueous N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at pH 7.4, with the aim of determining the applicability of these indicators in indicator displacement assays (IDAs) under physiological conditions. Dissociation constants (Kd) were determined for 11 indicator-Zn2L1 complexes, spanning 2 orders of magnitude from 2.8 x 10(-4) M (alizarin red S) to 2.7 x 10(-6) M (bromo pyrogallol red). Phosphate and pyrophosphate were tested for their ability to displace bound indicator and produce a detectable colorimetric response. Three indicators (bromo pyrogallol red, mordant blue 9, and zincon) complex to Zn2L1 to form an indicator displacement assay selective for pyrophosphate over phosphate. Because selection of an indicator/analyte pair having appropriate relative Kd values is critical for their successful application in IDAs, the binding data for these 11 indicators should assist their extension to IDAs for other analytes.

Dizinc Phosphohydrolase Model Built on a m‐Terphenyl Scaffold and Its Use in Indicator Displacement Assays for Pyrophosphate Under Physiological Conditions

“…[46] The Zn 2 L2 complex used in absorption spectroscopic studies was prepared in situ in HEPES buffer (10 m, pH 7.4), following the procedure re-ported for L1. [30] The binding strength and optical changes of commercial indicators (Scheme 3) upon binding to Zn 2 L2 were probed to reveal any differences from m-xylylene scaffolded analogues. Data from absorption spectroscopic titrations were used to calculate dissociation constants (K d , Table 1) by a modification of the Benesi-Hildebrand method (Benesi-Hildebrand plots are provided in the Supporting Information).…”

“…Four commercial complexometric indicators previously shown [30] to exhibit notable differences in their absorption spectra between free and dizinc-bound states were tested ( Table 1). All of these indicators provide IDAs selective for PP i over P i and other simple anions (no displacement of indicator is observed upon addition of excess F -, Cl -, Br -, I -, AcO -, NO 3 -, CO 3 2-and HSO 4 -).…”

“…Our previous work on IDAs [30,31] included a study [30] which built on extensive work by others who established mxylylene scaffolded dizinc complexes as preeminent receptors for phosphate derivatives under physiologic conditions. [16,22,32,33] Zn 2 L1 (the product of Zn 2+ addition to aqueous L1, Scheme 3), for example, has a Zn-Zn distance (3.0 Å) [34] somewhat less than that in phosphotriesterase (3.5 Å), which binds with high specificity to P i derivatives, [35] whereas Zn 2 L1 and related complexes bind both P i and PP i .…”

“…[36,37] We tested Zn 2 L1 as an IDA receptor with various commercial indicators, but the similar binding affinity of P i and PP i to Zn 2 L1 made their differentiation difficult. [30] The Zn-Zn distance should play a key role in analyte preference. The dependence of phosphoester hydrolysis activity on Zn-Zn distance in phosphohydrolase models has been studied employing, for example, xylyl, naphthyl, anthryl, and biphenyl spacers between Zn-chelating moieties.…”

2-(4-Pyridyl)imino nitroxide-tetraphenylporphyrin zinc(II): A chemosensing ensemble for nicotine

“…Because the indicator and host are non-covalently bonded, chemosensing ensembles offer many advantages over traditional chemosensors, in which the receptor and reporter units are covalently linked. Using this chemosensing concept, new methods have been developed to detect analytes such as oxalate [6], amino acids [7][8][9], caffeine [10], cocaine [11], phosphate [12,13], chloride [14], carboxylated and phosphorylated sugars [15], glucose oxidase [16], and chiral carboxylic acids [17].…”