Synthesis and properties of Asante Calcium Red--a novel family of long excitation wavelength calcium indicators.

Abstract Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca 2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca 2+ -dependent (640 nm) and quasi-independent (530 nm) emission when using visible ( 2+ ] i measurement in GFP expressing cells. The indicators bind Ca 2+ with either high ( K d  = 0.49 ± 0.07 μM; ACR-1) or low affinity ( K d  = 6.65 ± 0.13 μM; ACR-1-LA). Chelating Zn 2+ ( K d  = 0.38 ± 0.02 nM) or Mg 2+ ( K d  ∼ 5 mM) slightly raises and binding Co 2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (p K a  = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.