Formation
of Ternary Complexes with MgATP: Effects on the Detection of Mg<sup>2+</sup> in Biological Samples by Bidentate Fluorescent Sensors
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Abstract
Fluorescent indicators based on β-keto-acid
bidentate coordination motifs display superior metal selectivity profiles
compared to current <i>o</i>-aminophenol-<i>N</i>,<i>N</i>,<i>O</i>-triacetic acid (APTRA) based
chelators for the study of biological magnesium. These low denticity
chelators, however, may allow for the formation of ternary complexes
with Mg<sup>2+</sup> and common ligands present in the cellular milieu.
In this work, absorption, fluorescence, and NMR spectroscopy were
employed to study the interaction of turn-on and ratiometric fluorescent
indicators based on 4-oxo-4H-quinolizine-3-carboxylic acid with Mg<sup>2+</sup> and ATP, the most abundant chelator of biological magnesium,
thus revealing the formation of ternary complexes under conditions
relevant to fluorescence imaging. The formation of ternary species
elicits comparable or greater optical changes than those attributed
to the formation of binary complexes alone. Dissociation of the fluorescent
indicators from both ternary and binary species have apparent equilibrium
constants in the low millimolar range at pH 7 and 25 °C. These
results suggest that these bidentate sensors are incapable of distinguishing
between free Mg<sup>2+</sup> and MgATP based on ratio or intensity-based
steady-state fluorescence measurements, thus posing challenges in
the interpretation of results from fluorescence imaging of magnesium
in nucleotide-rich biological samples