A new H2S-specific near-infrared fluorescence-enhanced probe that can visualize the H2S level in colorectal cancer cells in mice† †Electronic supplementary information (ESI) available: Experimental details, photophysical data, some fluorescence imaging figures, average fluorescence intensity figures

A highly sensitive H2S-specific near-infrared fluorescence-enhanced probe was developed for real-time imaging of endogenous H2S in colorectal cancer cells (HCT116 and HT29) in mice.


Experimental part
General chemicals and instruments. All chemicals and solvents used for synthesis were purchased from commercial suppliers and applied directly in the experiments without further purification. Merck silica gel 60 (100-200 mesh) was used for general column chromatography purification. 1 H NMR and 13 C NMR spectra were recorded on a Bruker 400 spectrometer, tetramethylsilane (TMS) or residual solvent peaks as internal standard (0 ppm) substances. High-resolution mass spectra (HRMS) were obtained on an Agilent 6540 UHD Accurate-Mass Q-TOFLC/MS or Varian 7.0 T FTICR-MS. The UV-visible spectra were recorded on a UV-3600 UV-VIS-NIR spectrophotometer (SHIMADZU, Japan). Fluorescence study was carried out using F-280 spectrophotometer (Tianjin GangdongSci& Tech., Development. Co., Ltd). For selective experiments, the persulfides were prepared according to a previous method (ACS Chem. Biol., 2013, 8, 1110-1116. 2,2'-Dithiodipyridine (1 mM) or cystamine (1 mM) in degassed PBS buffer (pH 7.4) was added freshly prepared sodium sulfide (100 μM) and the solution was incubated at room temperature for 3 h in sealed tube.

Synthesis
Cytotoxicity assay. The in vitro cytotoxicity was measured using standard methyl thiazolyltetrazolium (MTT, Sigma-Aldrich) assay in bEnd.3 cell lines. Briefly, cells growing in log phase were seeded into 96 well cell-culture plate at 1 × 10 4 /well. The For the exogenous H 2 S imaging, bEnd.3 cells were firstly treated with probe (10 µM) at 37 ℃ for 30 min, washed by PBS, and then incubated with Na 2 S (150 and 450 µM) for 30 min. Control cells were treated with just probe. After being washed with phosphate-buffered saline (PBS) twice, the cells were imaged by using a confocal laser scanning microscope (Leica, Wetzlar, Germany).
Control cells were treated with just probe. After being washed with PBS twice, the cells were imaged by using a confocal laser scanning microscope (Leica, Wetzlar, Germany).
For endogenous H 2 S imaging, FHC, HCT116, HT29 cells were incubated with probe 1 (10 µM) for 30 min, and then washed with PBS twice before imaging. In the control experiments, three cells lines were incubated with 1 mM ZnCl 2 or 200 µM AOAA inhibitor for 30 min, washed by PBS twice, and then incubated with probe 1 (10 µM) for 30 min. After being washed with PBS twice, the cells were imaged as description above. For exogenous H 2 S imaging, the female nude mice (6 weeks) were i.p. injected with probe 1 (150 µM, 200 µL) for 30 min. The mice were imaged on an IVIS Lumina II system (Xenogen/PerkinElmer), a small animal in vivo imaging system with a 710 nm excitation filter and an ICG emission filter. Next, Na 2 S (150 µM, 200 µL) was injected into the intraperitoneal cavity, and then images were collected within 30 min.
For endogenous H 2 S imaging, the peritoneal cavities of female nude mice (6 weeks S26. HRMS spectrum of probe 1 after treatment with H 2 S. 10 μL 10 mM 1 in DMSO and 10 μL 100 mM Na 2 S in H 2 O were co-incubated in 80 μL 50 mM PBS (pH 7.4, containing 10% DMSO ) for 1 h at room temperature. Then the reaction mixture was submitted into ESI-MS without purification.