Deep-Red and Near-Infrared\nCompact Cyanine Dyes for\nSensitive NAD(P)H Sensing in Live Cells and Kidney Disease Tissues

Cyanine\ndyes constructed for NAD(P)H near-infrared sensing utilize\nextended π-conjugation but often exhibit delayed fluorescence\nresponses to NAD(P)H due to reduced positive charge density in 3-quinolinium\nacceptors. This study introduces deep-red and near-infrared compact\ncyanine dyes represented by probes <b>A</b> and <b>B</b> for mitochondrial NAD(P)H detection in live cells. Probes <b>A</b> and <b>B</b> feature a unique structural design with\na double bond connection linking 3-quinolinium to strategically positioned\n1-methylquinolinium acceptor units at 2- and 4-positions, correspondingly.\nProbe <b>A</b> absorbs at 359 and 531 nm, while probe <b>B</b> absorbs at 324 and 370 nm, emitting subtle fluorescence\nat 587 and 628 nm, respectively, with no NADH present. Upon NADH exposure,\nprobes <b>A</b> and <b>B</b> exhibit significant emission\nenhancements at 612 and 656 nm, correspondingly, attributed to the\nefficient reduction of 3-quinolinium units to electron-donative 1-methyl-1,4-dihydroquinoline\nunits. Probe <b>B</b>, chosen for its near-infrared emission\nand fast response to NAD(P)H, effectively monitored dynamic intracellular\nNAD(P)H levels throughout diverse experimental conditions. In HeLa\ncells, minimal basal fluorescence increased upon NADH stimulation.\nIt also identified increased NAD(P)H levels following chemical treatments\nwith acesulfame potassium, cisplatin, carboplatin, and temozolomide,\nCoCl<sub>2</sub>-induced hypoxia, and TLR4 activation in macrophages\nand in disease models of kidney pathology, where diseased tissues\nexhibited higher fluorescence than normal tissues. In fruit fly larvae\nunder starvation conditions, probe <b>B</b> tracked NAD(P)H\nincreases triggered by exogenous NADH, demonstrating its in vivo applicability\nfor metabolic studies. These findings highlight probe <b>B</b>’s utility in elucidating dynamic NAD(P)H fluctuations in\ndiverse biological contexts, offering insights into mitochondrial\nfunction and cellular metabolism.