New calcium-sensitive ligand for nuclear magnetic resonance spectroscopy.
Fluorinated calcium-sensitive indicators such as 5,5'-difluoro-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (difluoro-BAPTA) will often be less sensitive under in vivo conditions than gyromagnetic ratio considerations alone would have predicted. This is due to the very broad line widths displayed by these molecules within the living cell. In order to provide a spectroscopic alternative to these molecules, we have synthesized 13C-enriched 1-(2-aminophenoxy)-2-(2-aminoethoxy)ethane-N,N,N',N'-tetraac etic acid or AATA. The rationale for the design of this molecule was the increased signal to noise ratio available by selective detection of 13C-attached protons in AATA using proton-observe carbon-edited spectroscopy or multiple-quantum coherence. AATA has the advantage of increased number of detectable nuclei and narrow line widths. As such, it should provide a 6-10-fold improvement in the signal to noise ratio over existing fluorinated indicators. As a hybrid between EGTA and BAPTA, AATA should display intermediate pKa's, exchange rates, and KD values. We have measured pKa values of 5.94 +/- 0.05 and 9.03 +/- 0.05 for AATA. KD values of 350 +/- 80 nM and 6.6 +/- 2.0 mM were obtained for the AATA-Ca2+ and AATA-Mg2+ interactions, respectively, at 37 degrees C in 0.1 M KCl. As such, this new ligand displays the expected selectivity for Ca2+ over Mg2+. This new approach to detection of intracellular probes with NMR can be readily extended to other probes for intracellular ions, pH, and membrane potential. In addition, the move toward carbon-selected proton spectroscopy should also permit more flexibility in synthetic approaches since the strong electronegativity of fluorine often hampers synthetic design.