Imidazolyl-benzocoumarins as ratiometric fluorescence probes for biologically extreme acidity

A rational approach to develop a fluorescent probe for sensing biologically "extreme" acidity (pH <3) is disclosed. The probe, a push-full type 3-(imidazolyl)benzocoumarin dye, has the lowest pK_a = 1.3 among ratiometric probes known so far, which is ascribed due to a unique sensing mechanism. The probe has high quantum yields, high chemical stability and good aqueous solubility. The probe was successfully applied to ratiometric fluorescence imaging of intrabacterial acidity from pH 4.0-1.0, offering a practical means for studying biological systems under the extreme pH conditions.