Molecular Engineering of a D–π–A–π–D Scaffold for Two-Photon Near-Infrared Imaging of Peroxynitrite in Vivo

Precise visualization of peroxynitrite (ONOO-) in living systems remains challenging due to its transient nature and the need for deep-tissue optical penetration. Here, we report XH-ONOO, a near-infrared fluorescent probe engineered with a donor-pi-acceptor-pi-donor (D-pi-A-pi-D) scaffold formed by coupling an electron-rich coumarin unit with a beta-diketonate-BF2 acceptor. Quantum-chemical analysis elucidates its optimized conjugation and charge-transfer characteristics. XH-ONOO exhibits an ultrafast and highly selective response to ONOO- , producing a >250 nm emission shift, which minimizes spectral crosstalk between the probe and its reaction product. Notably, the probe supports efficient two-photon excitation (740 nm), enabling high-contrast imaging of both exogenous and endogenous ONOO- in live cells. Furthermore, XH-ONOO achieves tissue, high-resolution ONOO- imaging in a mouse model, establishing its utility as a sensitive and versatile tool for tracking oxidativeinflammatory signaling in complex biological environments.