Design of Cell Membrane-Targeted Two-Photon Excited Bioorthogonal Near-Infrared Fluorogenic Probes

The cell membrane is the critical cell component that protects cell integrity and mediates intercellular communication. In this contribution, we develop a cell membrane-targeted bioorthogonally activatable fluorogenic probe, DCM-Tz3, by incorporating the tetrazine motif into the dicyanomethylene-4H-chromone scaffold. Upon inverse electron-demand Diels-Alder reaction, the resulting cycloadduct (DCM-Pz3) exhibits a high fluorescence turn-on ratio, near-infrared emission, and significant solvatochromic effects. Notably, DCM-Pz3 presents a two-photon excitation property with a high two-photon absorption cross-section of 113 GM. Density functional theory calculation reveals the fluorescence OFF-ON switching mechanism of the probe. Importantly, we successfully applied the combination of DCM-Tz3 and the cyclooctyne for NIR fluorescence labeling of the cell membrane in both one-photon and two-photon patterns in living cells under wash-free conditions. This work provides a valuable cell membrane imaging tool with high specificity, laying the foundation for exploring cell membrane-based biological processes.