A coumarin-indole-based near-infrared ratiometric pH probe for intracellular fluorescence imaging.

New pH-activatable ratiometric fluorescent probes have been developed that have a coumarin structure attached to benzothiazole or indole units with one or two conjugated bonds. They have optical responses in acidic conditions. Probes with benzothiazole (1) and indole (2, 3) units have opposite responses to changes in pH, in terms of shifts in absorption and emission. Quantum chemical calculations with the B3LYP exchange functional provide insights into the differences in optical properties between 1 and 2/3. Probe 3 (7-diethylamino-3-[4-(3,3-dimethyl-3H-indol-2-yl)-buta-1,3-dienyl]-chromen-2-one) displayed a significant 122 nm red shift of the emission maximum; the new peak is located in the near-infrared region. In vitro assays demonstrate that probe 3 can be used in quantitative fluorescence imaging for the detection of intracellular pH distributions. In addition to the large Stokes shift, it has rapid responsivity, excellent cell permeability, and high selectivity. This study reveals that the conjugation of coumarin with benzothiazole or indole to form an intramolecular charge transfer (ICT) platform is an effective method to construct new fluorescent pH probes for acidic microenvironments.