Computationally Guided Approach to Produce Ratiometric Probes Operating in the Red to Near-infrared Region to Accurately Determine pH Levels within Organelles

摘要

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 π-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 π-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.