ICPBC and C12-ICPBC: Two new red emitting, fluorescent Ca2+ indicators excited with visible light

Two new, visible-excited and red-emitting fluorescent Ca2+ indicators were synthesized and the spectral profiles of their free and Ca2+ bound forms were studied. The fluorescent properties of these probes are due to the extended conjugation of the chromeno[3′,2′:3,4]pyrido[1,2a][1,3]benzimidazole chromophore incorporated in their BAPTA-type, Ca2+ chelating structure. The compounds, namely ICPBC and its N-dodecyl analog C12-ICPBC exhibit Ca2+ dissociation constants of 7.7 and 18.0 μM, respectively. The fluorescence spectra of the probes showed a clear shift in excitation wavelength maxima upon Ca2+ binding along with a large Stokes shift and changes in fluorescence intensity, indicating their potential use as Ca2+ indicators. The ability of ICPBC to trace high calcium spikes was tested in the human HepG2 cell line with positive results.