Native Chemical Ligation Combined with Spirocyclization of Benzopyrylium Dyes for the Ratiometric and Selective Fluorescence Detection of Cysteine and Homocysteine

Spirocyclization of xanthene dyes has become a powerful technique for developing fluorescent probes. Herein, we extend this unique fluorescence switching mechanism to a near-infrared (NIR) dye, 2-(7-diethylamino-2-oxo-2H-1-benzopyran-3-yl)-4-(2-carboxyphenyl)-7-diethylamino-1-benzopyrylium (<b>CB</b>), and construct a ratiometric fluorescent probe <b>1</b> for cysteine (Cys)/homocysteine (Hcy). The ratiometric sensing of probe <b>1</b> toward Cys/Hcy is realized by utilizing a tandem native chemical ligation/spirocyclization reaction to interrupt the large π-conjugated system of <b>CB</b> fluorophore, thereby affording remarkable blue shifts in the spectra of sensing system (from 669 to 423 nm in absorption spectra and from 694 to 474 nm in emission spectra). Probe <b>1</b> shows a high sensitivity for Cys/Hcy, and the detection limits (3 δ) for Cys and Hcy are 1.6 × 10^–7 and 1.8 × 10^–7 M, respectively. Moreover, since both the sulfhydril and the adjacent amino groups are involved in the sensing process, probe <b>1</b> is selective toward Cys/Hcy over other thiols such as glutathione. All these unique features make it particularly favorable for ratiometric Cys/Hcy sensing and bioimaging applications. It has been preliminarily used for Cys detection in rabbit serum samples and the ratiometric fluorescent imaging of Cys in living HepG2 cells.