pH-Responsive Fluorescence Turn-On Copper(II)-Probe for Nitric Oxide Detection: A Reductive Nitrosylation Approach

Nitric oxide (NO) is one of the crucial biological signaling molecules, yet achieving its selective and spatiotemporal detection in in-situ/invitro or biological systems at specific pH remains a significant challenge. Hence, a probe capable of directly detecting NO would be immensely valuable in understanding its reactivity and biological functions. Here, to develop a Cu(II)-based probe for selective NO detection, we synthesized a Cu(II)-complex (1) using a N3-tridentate ligand having a pendant dansyl fluorophore (L₁) and evaluated it's NO reactivity under varying pH conditions. The binding of Cu(II) ion with ligand L₁ to prepare 1 showed > 96% fluorescence quenching (fluorescence turn-off) compared to highly fluorescent free L₁ (Φ = 59%). Complex 1 did not react with NO in neutral or acidic medium; however, in the presence of onefold base (pH ≥ 9), it showed the reductive nitrosylation reaction, which resulted in the reduction of Cu(II) to Cu(I) and ligand nitrosylation (L₁-NO) with fluorescence turn-on. Studies performed using different reactive nitrogen /or oxygen species (RNS/ROS) suggest that probe 1 is highly selective toward NO with a detection limit of 1 nM. For the first time, the base-catalyzed reductive nitrosylation of a Cu(II) complex resulted in the selective NO detection via fluorescence turn-on at pH ≥ 9 in methanol /or aqueous buffer medium. To understand the effect of secondary amine (─NH─) sites in the above reductive nitrosylation reaction, a Cu(II) complex (2) of the ligand (L₂), having two different (─NH sites, was reacted with NO under various pH conditions. Surprisingly, 2 showed selective mono-N-nitrosylation of the amine site coordinated to Cu(II) ion with the reduction of Cu(II) in the presence of onefold OH^-.