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 (450-540 nm) excitation with deep-red fluorescence (640 nm). 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 (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (similar to 780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]; measurement in GFP expressing cells. The indicators bind Ca2+ with either high (K-d = 0.49 +/- 0.07 mu M; ACR-1) or low affinity (K-d = 6.65 0.13 mu M; ACR-1-LA). Chelating Zn2+ (K-d = 0.38 +/- 0.02 nM) or Mg2+ (K-d similar to 5 mM) 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. (C) 2013 Elsevier Ltd. All rights reserved.