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

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 (450-540nm) excitation with deep-red fluorescence (640nm). Upon Ca²⁺ 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 Ca²⁺-dependent (640nm) and quasi-independent (530nm) emission when using visible (<490nm) or multiphoton (~780nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca²⁺]_i measurement in GFP expressing cells. The indicators bind Ca²⁺ with either high (K_d=0.49±0.07μM; ACR-1) or low affinity (K_d=6.65±0.13μM; ACR-1-LA). Chelating Zn²⁺ (K_d=0.38±0.02nM) or Mg²⁺ (K_d~5mM) slightly raises and binding Co²⁺ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pK_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.