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Deep-Red Cyanine-Based Fluorescent Probes with 6-Quinolinium Acceptors for Mitochondrial NAD(P)H Imaging in Live
Dilka Liyana Arachchige1,2, Sushil K Dwivedi1,2, Peter Agyemang1,2
1Department of Chemistry, Michigan Technological University, Houghton, Michigan 49931, United States.
Abstract:
We developed two deep-red cyanine chromophores, probes A and B, for selective mitochondrial NAD(P)H detection in live cells. Probe A features a 1,2,3,3-tetramethyl-3H-indolium core, while probe 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 A and B. To explore the role of electron-withdrawing groups in probe sensitivity, we synthesized three additional cyanine dyes (probes C, D, and E) 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 A showed absorption at 382 nm with weak fluorescence at 636 nm, while probe B absorbed at 443 nm with weak fluorescence at 618 nm. Upon NAD(P)H reduction, probe A exhibited red-shifted absorption at 520 nm with enhanced emission at 589 nm, and probe B at 550 nm with strong emission at 610 nm. Probe C showed absorption at 524 nm with enhanced emission at 586 nm, while probes D and E exhibited no detectable NAD(P)H response, highlighting the critical role of quinolinium acceptors. Probe 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 Drosophila larvae, revealing increased levels after drug treatments. Notably, probe 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 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.
