Universal Low-Background Fluorophore Platform via Structural Reprogramming of Oxazine 1 with Julolidine for Activatable Probe Design

Fluorescent probes play a vital role in biological detection and imaging; however, their application is often hampered by high background fluorescence, which compromises detection sensitivity and reduces the signal-to-noise ratio. Oxazine 1 is a modifiable near-infrared fluorophore that can be used to develop low-background probes. Nonetheless, it suffers from limitations such as insufficient red-shifted emission, low fluorescence quantum yield, and poor cellular uptake. To address these limitations, we designed and synthesized two new oxazine derivatives, JSO and JDO, by replacing the diethylamino donor group of oxazine 1 with a rigid julolidine moiety. This structural modification effectively enhanced electron-donating ability, suppressed nonradiative decay, and improved membrane permeability. Compared to oxazine 1, the emission wavelength of JDO was red-shifted (703 nm), and its fluorescence quantum yield increased 2-fold. JDO also had superior intracellular accumulation and excellent pH stability and photostability. By leveraging these advantages, we developed a novel peroxynitrite (ONOO–)-responsive probe, JDO-ONOO, as a proof-of-concept demonstration to validate the design platform. Through strategic disruption of the fluorophore's conjugated structure, JDO-ONOO emitted ultralow background fluorescence, and in the presence of ONOO–, the fluorescence switched to a "turn-on" mode, resulting in a 130-fold increase in intensity. The probe had high sensitivity (LOD = 3.7 nM), rapid response, and excellent selectivity under physiological conditions. It was successfully employed in the fluorescence imaging of ONOO– in living cells and in multiple mouse models of inflammation. Overall, this study presents a universal strategy for developing next-generation low-background fluorescent probes, demonstrated through ONOO– detection.