Deep-Red Cyanine-Based Fluorescent Probes with 6-Quinolinium Acceptors for Mitochondrial NAD(P)H Imaging in Live Cells and Human Diseased Kidney Tissues

We developed two deep-red cyanine chromophores, probes <b>A</b> and <b>B</b>, for selective mitochondrial NAD(P)H detection in live cells. Probe <b>A</b> features a 1,2,3,3-tetramethyl-3H-indolium core, while probe <b>B</b> incorporates a 1,1,2,3-tetramethyl-1H-benzo[e]indol-3-ium moiety, both linked to quinolinium via a vinyl bond to enable fluorescence modulation upon NAD(P)H reduction of probes <b>A</b> and <b>B</b>. To explore the role of electron-withdrawing groups in probe sensitivity, we synthesized three additional cyanine dyes (probes <b>C</b>, <b>D</b>, and <b>E</b>) via condensation of 6-quinolinecarboxaldehyde with 2,3-dimethyl-1,3-benzothiazolium acceptor and malononitrile derivatives, followed by methylation. Under NAD(P)H-deficient conditions, probe <b>A</b> showed absorption at 382 nm with weak fluorescence at 636 nm, while probe <b>B</b> absorbed at 443 nm with weak fluorescence at 618 nm. Upon NAD(P)H reduction, probe <b>A</b> exhibited red-shifted absorption at 520 nm with enhanced emission at 589 nm, and probe <b>B</b> at 550 nm with strong emission at 610 nm. Probe <b>C</b> showed absorption at 524 nm with enhanced emission at 586 nm, while probes <b>D</b> and <b>E</b> exhibited no detectable NAD(P)H response, highlighting the critical role of quinolinium acceptors. Probe <b>B</b> demonstrated superior sensitivity, successfully tracking NAD(P)H fluctuations in HeLa cells under glycolysis stimulation (glucose, lactate, pyruvate) and treatments with LPS and methotrexate. It also visualized NAD(P)H in <i>Drosophila</i> larvae, revealing increased levels after drug treatments. Notably, probe <b>B</b> distinguished between healthy and diseased human kidney tissues, detecting significantly elevated NADH levels in autosomal dominant polycystic kidney disease (ADPKD) samples, emphasizing its diagnostic potential. This study introduces probe <b>B</b> as a versatile and reliable NAD(P)H sensor for metabolic research and disease diagnostics, offering valuable insights into redox processes in live cells, organisms, and clinical samples.