Color-Tunable Light-up Bioorthogonal Probes for In Vivo Two-Photon Fluorescence Imaging

Light-up bioorthogonal probes have attracted increasing attention recently due to their capability to directly image diverse biomolecules in living cells without washing steps. The development of bioorthogonal probes with excellent fluorescent properties suitable for in vivo imaging, such as long excitation/emission wavelength, high fluorescence turn-on ratio, and deep penetration, has been rarely reported. Herein, a series of azide-based light-up bioorthogonal probes with tunable colors based on a weak fluorescent 8-aminoquinoline (AQ) scaffold were designed and synthesized. The azido quinoline derivatives are able to induce large fluorescence enhancement (up to 1352-fold) after click reaction with alkynes. In addition, the probes could be engineered to exhibit excellent two-photon properties (δ=542 GM at 780 nm) after further introducing different styryl groups into the AQ scaffold. Subsequent detailed bioimaging experiments demonstrated that these versatile probes can be successfully used for live cell/zebrafish imaging without washing steps. Further in vivo two-photon imaging experiments demonstrated that these light-up biorthogonal probe outperformed conventional fluorophores, for example, high signal-to-noise ratio and deep tissue penetration. The design strategy reported in this study is a useful approach to realize diverse high-performance biorthogonal light-up probes for in vivo studying.