Structural and spectroscopic insight into the metal binding properties of the o-aminophenol-N,N,O-triacetic acid (APTRA) chelator: implications for design of metal indicators

Abstract

The o-aminophenol-N,N,O-triacetic acid (APTRA) chelator is employed extensively as a metal-recognition moiety in fluorescent indicators for biological free Mg²⁺, as well as in low-affinity indicators for the detection of high levels of cellular Ca²⁺. Despite its widespread use in sensor design, the limited metal selectivity of this chelating moiety can lead to binding of competing cations that complicate the fluorescence-based detection of metals of interest in complex samples. Reported herein are the structural characterization of APTRA complexes with various biologically relevant cations, and the thermodynamic analysis of complex formation with Mg²⁺, Ca²⁺ and Zn²⁺. Our results indicate that the low affinity of APTRA for Mg²⁺, which makes it a suitable metal-recognition moiety for sensitive analysis of typical millimolar levels of this metal in cells, stems from a much higher enthalpic cost of Mg²⁺ binding compared to that of other cations. The results are discussed in the context of indicator design, highlighting the aspects that may aid the future development of fluorescent sensors with enhanced metal selectivity profiles.