Three BODIPY-based near-infrared fluorescent probes have been synthesized, characterized, and evaluated for the sensing and imaging of lysosomal pH inside living cells.

Three uncommon morpholine-based fluorescent probes (A, B and C) for pH were prepared by introducing morpholine residues to BODIPY dyes at 4,4’- and 2,6-positions, respectively. In contrast to morpholine-based fluorescent probes for pH reported in literature, these fluorescent probes display high fluorescence in a basic condition while they exhibit very weak fluorescence in an acidic condition. The theoretical calculation confirmed that morpholine is unable to function as either an electron donor or an electron acceptor to quench the BODIPY fluorescence in the neutral and basic condition via photo-induced electron transfer (PET) mechanism because the LUMO energy of morpholine is higher than those of the BODIPY dyes while its HOMO energy is lower than those of the BODIPY dyes. However, the protonation of tertiary amines of the morpholine residues in an acidic environment leads to fluorescence quenching of the BODIPY dyes via d-PET mechanism. The fluorescence quenching is because the protonation effectively decreases the LUMO energy which locates between the HOMO and LUMO energies of the BODIPY dyes. Fluorescent probe C with deep-red emission has been successfully used to detect pH changes in mammalian cells.

Two water-soluble near-infrared luminescent probes, which possess both conventional intense Stokes fluorescence and unique single-photon frequency upconversion luminescence (FUCL), were developed for sensitive and selective detection of pH changes in live cells. The water solubility and biocompatibility of these probes were achieved by introducing mannose residues through 2,2′-(ethylenedioxy)diethylamine tethered spacers to a near-infrared conventional fluorescence (CF) and FUCL organic fluorophore. At a pH higher than 7.4, the probes have ring-closed spirocyclic lactam structures, thus are colorless and nonfluorescent. Nevertheless, they sensitively respond to acidic pH values, with a drastic structural change to ring-opened spirocyclic lactam forms, which cause significant absorbance increases at 714 nm. Correspondingly, their near-infrared CF and FUCL intensities at 740 nm are also significantly enhanced when excited by 690 and 808 nm, respectively. The probes hold a variety of advantages such as high sensitivity, excellent reversibility and selectivity to pH over metal ions, low cellular autofluorescence background interference, good cell membrane permeability and photostability, as well as low cytotoxicity. Our results have successfully proven that these probes can visualize intracellular lysosomal pH changes in live cells by monitoring both near-infrared CF and FUCL changes.