The project aims to develop water-soluble near-infrared fluorescent probes for the precise and quantitative
analysis of NAD(P)H concentration changes in mitochondria of live cells by incorporating the nicotinamide
moiety into different electron-withdrawing fluorophores as electron acceptors such as tetraphenylethylene
(TPE), dicyanomethylene-4H-chromene, coumarin, naphthalimide, rhodamine, rhodol, and BODIPY. We will
develop ratiometric near-infrared fluorescent probes to overcome systematic errors of intensity-based
fluorescent probes by introducing near-infrared BODIPY donors to near-infrared rhodamine and rhodol
acceptors, respectively. In order to significantly enhance water solubility and biocompatibility, we will introduce
branched oligo(ethylene glycol) residues to the probes. The strong electron-withdrawing properties of the
fluorophores such as dicyanomethylene-4H-chromene, coumarin, naphthalimide, rhodamine, rhodol, and
BODIPY dyes will facilitate a quick reduction of the nicotinamide moiety by NAD(P)H into an electron-donating
dihydronicotinamide moiety and significantly turn on the probe fluorescence. We will use computation
chemistry to guide how to design effective fluorescent probes and study the relationship of the optical
properties of the probes and their chemical structures such as HOMO and LUMO levels, pKa values,
absorption and emission wavelengths, fluorescence quantum yields, and redox reaction between the probes
and NAD(P)H, and assess the through-bond energy transfer efficiency from the donors to the acceptors of
ratiometric fluorescent probes in the presence of NAD(P)H. We will investigate the effects of substrate-specific
glucose and pyruvate/lactate imbalance on NADH levels in the glycolysis pathway in normal and cancer cells.
We will also investigate the co-relationship between GSH/GSSG (glutathione/glutathione disulfide) and the
cellular redox regulator NAD+/NADH in maintaining a critical redox balance for proper functioning in the
different metabolic processes. Finally, we will investigate the unique significant role of NADH in oxidative
phosphorylation in mitochondria, and monitor NADH concentration changes during the mitophagy process
caused by starvation and drug treatment.