Near-Infrared Visualization\nof NAD(P)H Dynamics in\nLive Cells and Drosophila melanogaster Larvae Using a Coumarin-Based Pyridinium Fluorescent Probe

A near-infrared fluorescent probe, <b>A</b>, was\ndesigned\nby substituting the carbonyl group of the coumarin dye’s lactone\nwith a 4-cyano-1-methylpyridinium methylene group and then attaching\nan electron-withdrawing NADH-sensing methylquinolinium acceptor via\na vinyl bond linkage to the coumarin dye at the 4-position. The probe\nexhibits primary absorption maxima at 603, 428, and 361 nm, and fluoresces\nweakly at 703 nm. The addition of NAD(P)H results in a significant\nblue shift in the fluorescence peak from 703 to 670 nm, accompanied\nby a substantial increase in fluorescence intensity. This spectral\nshift is attributed to the transformation from an A−π–A−π–D\nconfiguration to a D−π–A−π–D\npyridinium platform in probe <b>AH</b>, owing to the addition\nof a hydride from NADH to the electron-accepting quinolinium acceptor\nproducing the electron-contributing 1-methyl-1,4-dihydroquinoline\ndonor in probe <b>AH</b>. This conclusion is supported by theoretical\ncalculations. The probe was utilized to investigate NAD(P)H dynamics\nunder various conditions. In HeLa cells, treatment with glucose or\nmaltose resulted in a substantial elevation in near-infrared emission\nintensity, suggesting increased NAD(P)H levels. Chemotherapeutic agents\nincluding cisplatin and fludarabine at concentrations of 5, 10, and\n20 μM brought about a dose-dependent increase in emission intensity,\nreflecting heightened NAD(P)H levels due to drug-induced stress and\ncellular damage. In vivo experiments with hatched, starved Drosophila melanogaster larvae were also conducted.\nThe results showed a clear relationship between emission intensity\nand the levels of NADH, glucose, and oxaliplatin, confirming that\nthe probe can detect variations in NAD(P)H levels in a living organism.\nOur investigation also demonstrates that NAD(P)H levels are significantly\nelevated in the cystic kidneys of ADPKD mouse models and human patients,\nindicating substantial metabolic alterations associated with the disease.\nThis near-infrared emissive probe offers a highly sensitive and specific\nmethod for monitoring NAD(P)H levels across cellular, tissue and whole-organism\nsystems. The ability to detect NAD(P)H variations in reaction to varying\nstimuli, including nutrient availability and chemotherapeutic stress,\nunderscores its potential as a valuable resource for biomedical research\nand therapeutic monitoring.