Abstract
Employing a computationally guided approach, this project will develop ratiometric fluorescent probes
operating in the red to the near-infrared region to accurately determine extracellular and intracellular pH
changes across cell membranes and in different organelles, such as lysosomes, mitochondria, the endoplasmic
reticulum, the Golgi apparatus, and to monitor mitochondrial delivery to the lysosomes during mitophagy
caused by nutrient starvation, hypoxia, and drug treatment. Spectrochemical attributes of probes will be
determined before synthesis in order to judge applicability in the desired spectral range. Probes will consist of
near-infrared fluorophores linked into near-infrared rhodol dyes to achieve ratiometric responses to pH
changes based on pH modulation resulting in p-conjugation changes of the probes. Red to near-infrared
fluorophores will include BODIPY dyes, traditional near-infrared xanthene dyes, and their derivatives, where
the central oxygen atom of xanthene cores will be replaced by other elements such as Si, S, and N. Spirolactone
and spirolactam molecular switches that can be tuned to appropriate pKa values matching the different
organelles under study will be used. Specific targeting to different organelles and cell membranes will be
achieved by introducing lysosome-specific morpholine, mitochondria-specific triphenylphosphonium, Golgi
apparatus-specific benzenesulfonamide, endoplasmic reticulum-specific p-toluene sulfonamide, or cell
membrane-specific amphilphilic zwitterion residues through oligo(ethylene glycol) spacers to the fluorescent
probes. The ratiometric red to near-infrared fluorescent probes based on spirolactam switches will have pKa
values from 4.5 to 6.2 and will only show fluorescence of near-infrared fluorophores under neutral and basic
pH conditions since rhodol moieties have closed spirolactam ring structures under the slightly basic conditions
within mitochondria. A lysosomal acidic environment will trigger the opening of the spirolactam switches on
the rhodol moieties, significantly enhancing p-conjugation between the fluorophore and rhodol moieties,
resulting in ratiometric fluorescence responses to pH changes. These processes will also be delineated via
theoretical calculations before syntheses are conducted. The probes will have the combined advantages of near-
infrared imaging (including deep tissue penetration, minimum cell damage, lack of interference from biological
autofluorescence), and ratiometric imaging with a self-calibration feature to overcome systematic errors of
intensity-only based fluorescent probes. In addition, the probes will possess good water solubility, high
stability, excellent cell permeability, good biocompatibility, excellent intracellular retention, high selectivity,
and sensitivity, as well as fast and reversible responses to pH changes. Seven undergraduate students will assist
in conducting these experiments for each year of the proposal which will allow them to learn valuable synthetic
and characterization research skills in this area. Undergraduate students will also conduct theoretical
calculations, using the program Gaussian, to determine the absorptive properties of the probes.
