Quantification of cysteine hydropersulfide with a ratiometric near-infrared fluorescent probe based on selenium–sulfur exchange reaction

A ratiometric near-infrared fluorescent probe based on a selenium–sulfur exchange reaction to quantify cysteine hydropersulfide in living cells and hepatic carcinoma rats.


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Analytical LC-MS/MS data for Cys-SSH in the cell lysis samples were acquired by using TSQ Quantum Access MAX LC-MS System (Thermo Fisher Scientific), with a linear 5% -90% methanol gradient for 14 min in 0.1% formic acid at 40 °C. A total flow rate of 0.2 mL/min and an injection volume of 20 μL were used. Ionization was achieved by using electrospray in the positive mode with a 3,500 V spray voltage. Nitrogen was the nebulizer gas, with the nebulizer pressure set at 50 psi. The desolvation gas (nitrogen) was heated to 350 °C and was delivered at the flow rate of 10 L/min. Collision-induced dissociation (CID) was achieved by using high-purity nitrogen as the collision gas at a pressure of 0.5 MPa. Mass spectra were recorded in the positive ion mode.
Animals: 5-weeks-old female BALB/c mice weighing 135 to 140 g, 2-week-old female SD rats weighing 60 to 80 g and 8-week-old female SD rats weighting 150 to 180 g were obtained from Binzhou Medical University. The animals were housed in cages and fed a standard laboratory diet and tap water ad libitum. Surgical procedures were carried out under anesthesia with ketamine hydrochloride (100 mg/kg, ip). All experiments were performed in accordance with the guidelines established by the Committee of Animal Research Policy of Binzhou Medical University.
Establish of the Walker-256 transplanted hepatoma SD Rats: Walker-256 carcinoma cells were purchased from Cell Preserve Center (Wuhan University, Wuhan, China). 2-week-old rats were subcutaneously inoculated 0.5 mL cell suspension (2 × 10 7 /mL). After a week, the 2-week-old subcutaneous tumor was sharply minced into small cubes about 1 mm × 1 mm × 1 mm (0.5 mg), corresponding to 1 × l0 5 tumor cells, and was prepared as tumor inocula. After depilation and disinfection, the 8-week-old rats underwent a small subxiphoid midline abdominal incision. The left lateral lobe of the liver was gently retracted out of the abdominal cavity. A small superficial incision on the liver was made using the tip of a surgical blade with the knife at a 30° -45° angle to the liver surface. A small piece of solidified gelfoam was inserted into the incision. Approximately l -2 min later, the bleeding had stopped and the gelfoam was removed. And a tumor fragment was gently placed into the pocket with an angled forceps so that the tumor was completely buried in hepatic parenchyma. The hepatic lobe was gently returned to the peritoneal cavity and the abdominal wall was closed. 20 H&E staining: Livers from normal rats and hepatic carcinoma rats were excised and fixed in 10% formaldehyde and embedded in paraffin. Then the treated livers were prepared to frozen sections and stained with hematoxylin and eosin (H&E) to confirm tumor histology.

Experiments of BALB/c Mice: A Bruker
In-vivo Imaging System was used for bio-imaging in this group of animal experiments. The excitation and emission wavelength were chosen as described in paper. Mice were anesthetized prior to injection and during imaging via inhalation of isoflurane.

Experiments of Hepatic Carcinoma Models:
Hepatic carcinoma models were built and then Xenogen IVIS Spectrum Pre-clinical In Vivo Imaging System was used for bio-imaging. Rats were anesthetized prior to injection and during imaging via inhalation of isoflurane. After in vivo imaging, the organs (such as brain, lung, heart, spleen, kidney and liver) were excised to perform ex vivo imaging with Xenogen IVIS Spectrum Pre-clinical In Vivo Imaging System. The excitation and emission wavelength were chosen as described in paper.

Synthesis of compound 1 (Cy-Dise).
Compound 2 (0.12 g, 0.1 mmol), triphosgene (0.09 g, 0.3 mmol) was dissolved in 50 mL anhydrous CH 2 Cl 2 under Ar atmosphere. 27 The mixture was suspended at 0 o C, then 1 mL DIPEA was added. The reaction was lasted for 30 min, and the color of the solution changed into green from blue. After removed solvent in vacuum, the obtained residue was dissolved in 50 mL anhydrous CH 2 Cl 2 , and added DIPEA (1 mL) and DMAP (20 mg). Compound 8 (0.050 g, 0.2 mmol) in CH 2 Cl 2 (2 mL) was added into above mixture, then the reaction mixture was stirred at 25°C, TLC monitored the reaction until the starting material was completely consumed. The obtained solid residue was purified through a silica gel chromatography (200 -300 mesh) with gradient eluent of CH 2 Cl 2 and CH 3 OH (100:0 to 85:15 v/v). The solvent was removed in vacuum, and the obtained solid was dissolved in CH 2 Cl 2 , filtered and evaporated to give a green solid (115 mg, yield 87 % and sodium methoxide (1.2 mL from a 0.5 M solution in MeOH) was added in anhydrous MeOH (5 mL). The reaction mixture was stirred at 25 °C for 1 h. After neutralized with diluted hydrochloric acid (5%), the solution was extracted with CH 2 Cl 2 (3 × 50 mL). The organic layer was dried with anhydrous Na 2 SO 4 . After concentrated under vacuum, the crude solid was purified by column chromatography (200 -300 mesh) using CH 2 Cl 2 : CH 3 OH (8:1 v/v) as eluent to obtain Cy-Dise as green solid (52 mg, 61 %). 1

F 797 nm and F 749 nm upon Treatment of Cys-SSH
The

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Standard fluorescence pH titrations were performed in 10 mM HEPES solution at a concentration of 10 μM (Cy-Dise and Cy). As shown in Fig. S3, the pH of the mediums hardly effected on fluorescence intensity within the range from 3.0 to 10.0. On this basis, we suggested that Cy-Dise would work well under physiological conditions (pH = 7.4).

Selectivity of Cy-Dise toward Various Reactive Substances
UV absorbance spectra of probe with various analytes were carried out to further verify the selectivity of the probe. Cy-Dise (10 μM) was treated with various analytes in HEPES buffer (10 mM, pH 7.4) at 37 o C. The emission and UV absorbance spectra were recorded after 60 min. As shown in Fig.S4, reactive oxygen species, reactive nitrogen species, anions and metal ions, as wells as reactive sulfur species and reactive selenium species could not induce interference. λ ex = 730 nm, λ em = 797 nm. The emission and UV absorbance spectra were recorded after 60 min.

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Considering the rapid metabolism unstable nature of Cys-SSH, the reaction kinetics of Cy-Dise towards Cys-SSH was performed to inspect whether the probe can fast respond to Cys-SSH. Cys-SSH was added at 10 s, and then the fluorescent intensity decreased at 797 nm and increased at 749 nm during 10 -70 s (Fig. S5a). The ratiofluorescence signal response to Cys-SSH changes would reach saturation within 60 s (Fig. S5b), indicating that our probe could be used as a unique real-time bioimaging tool for intracellular Cys-SSH.

Reaction Mechanism Discussion between Probe Cy-Dise and Cys-SSH
We had described the reaction mechanism in Fig. 1, the reduction of diselenide immediately formed an intermediate. Then a fast intramolecular cyclization occurred by cleavage of neighboring carbamate bond to release fluorophore. This proposed reaction mechanism was verified by HRMS, as shown in Fig. S6 However, there is a possibility which should be addressed, that is, the reduction of diselenide may formed an intermediate Cy-a (Fig. S7). However, we did not detect intermediate Cy-a by HRMR. Maybe intermediate Cy-a was high reactivity or unstable. Nonetheless, the intermediate selenylsulfides of Cy-a could be reduced by Cys-SSH straightway to form Cy-se. 28 Then a fast intramolecular cyclization occurs by cleavage of neighboring carbamate bond to release fluorophore. Even if our probe might follow the steps according to Fig. S7, there was also no extra consumption by the reaction.

MTT assay for Cy-Dise
To access the potential toxicity of Cy-Dise, MTT assays were carried out. HepG2 cells (10 6 cells/mL), HL-7702 cells (10 6 cells/mL) and primary mouse hepatocytes (10 6 cells/mL) were planted into 96-well microtiter plates in DMEM or RPMI 1640 with 10% fetal bovine serum (FBS). Plates were maintained at 37 °C in a 5% CO 2 /95% air incubator for 24 h. Then the cells were incubated for 24 h at 37 °C in a 5% CO 2 /95% air upon different concentrations probe of 0 μM to 100 μM respectively. MTT solution (5.0 mg/mL, PBS) was then added to each well. After 4 h, the remaining MTT solution was removed, and 200 μL of DMSO was added to each well, shaking 10 min to dissolve the formazan crystals at room temperature. Absorbance was measured at 570 nm and 630 nm in a TECAN infinite M200pro microplate reader.

Sublocation in Cells
To establish applicability of Cy-Dise in subcellular studies, we performed the co-localization experiments using Cy-Dise (10 μM), Calcein-AM, (1 μg/mL) (a commercial cytoplasm marker) and a DNA marker Hoechst 33342 (1 μg/mL) (a commercial nuclear dye) to determine the co-localization of fluorescence emission. The cells were incubated with Hoechst 33342 for 30 min, 1 μM Calcein-AM for 10 min and Cy-Dise for 5 min subsequently. With the help of Image-Pro Plus software, the respective spectra acquired from the three dyes were shown in Fig.  S9. The fluorescent images of Cy-Dise (red channel) and Calcein-AM (green channel) merged primely. Hoechst 33342 exhibited a perfect sublocation in nucles. The results confirmed that our probe could specifically localize in cytoplasm. And our probe also indicated a potentially powerful approach for real-time imaging cytoplasm Cys-SSH changes in cells and in vivo.  Fig. 4A and Fig. 4B   0s 15s 30s 45s 60s 75s 90s

Ratio Images of Time-dependent Cys-SSH Generation in NEM Treated Primary Hepatocytes
In order to verify that the probe could selectively image Cys-SSH generation in primary hepatocytes, NEM treated primary hepatocytes were used to performed as control. The primary hepatocytes were pretreated with Nethylmaleimide (NEM) to deplete all the endogenous Cys-SSH. As shown in Fig. S15, there was nearly no ratiometric fluorescence signal observed. This indicated that probe Cy-Dise possessed good selectivity towards Cys-SSH.

LC/MS Analysis for Cys-SSH in Cell Lysates
In order to get the accurate concentration of Cys-SSH in HL-7702, HepG2 cells and primary hepatocytes, we exploited Tag-Switch assay based on the LC/MS analysis. 19 Firstly, standard Cys-SS-bimane was synthesized by the reaction between 0.5 mM Cys, 0.5 mM P-NONOate and 0.5 mM NaHS in 10 mM Tris-HCl (pH 7.4) at room temperature for 30 min. Then 5 mM Br-bimane was added to the reaction mixture at room temperature for 30 min. The cultured HepG2, HL-7702 cells and primary hepatocytes were washed with PBS buffer twice. After centrifugation and removal of supernatant liquid, the resulting cells were stored at -80°C for 5 min. After thawing to room temperature, the cells were lysed by hyperacoustic in 1 mL lysis buffer (PBS with 0.1% Triton X-100, pH 7.4) and the supernatant liquid was collected after centrifugation (10,000 × g, 10 min, 4 °C). Then 100 μL cell lysates were mixture with 500 μL of a methanol solution containing 5 mM Br-bimane. The mixture was incubated at 37 °C for 30 min. After centrifugation (10,000 × g, 10 min, 4 °C), supernatants were collected to perform LC-MS/MS analysis with synthesized Cys-SS-bimane as standard.
Analytical LC-MS/MS data for Cys-SSH in the cell lysis samples were acquired by LC-ESI-MS/MS. Samples were separated on RP-HPLC with a YMC-Triart C18 column (50 × 2.0 mm inner diameter), with a linear 5% -90% methanol gradient for 14 min in 0.1% formic acid at 40 °C. Polysulfide derivatives were identified and quantified by means of MRM as shown in Fig. S16, with precursor ion (m/z): 344, product ion (m/z): 192, fragmentor voltage: 90 v, CID: 13. Quantification of Cys-SS-bimane in cell lysates was shown in Table 1.

H&E (hematoxylin and eosin) Staining of the Liver of Normal Rat and Hepatic Carcinoma Rat
Fig. S17 H&E staining of the liver of normal rats a) and hepatic carcinoma rats b). × 400

In vivo and ex vivo imaging of Cys-SSH in Walker-256 tumor SD rats by control probe.
In order to further verify the liver targeting ability of the probe, a control probe (Cy 3 -Dise) without liver targeting group was designed and synthesized. As shown in fig. S18, compound 3 (63.7 mg, 0.1 mmol) and triphosgene (0.09 g, 0.3 mmol) were dissolved in 50 mL anhydrous CH 2 Cl 2 under Ar atmosphere. 1 The mixture was suspended at 0 o C, then 1 mL DIPEA was added. The reaction was lasted for 30 min, and the color of the solution changed into green from blue. After removed solvent in vacuum, the obtained residue was dissolved in 50 mL anhydrous CH 2 Cl 2 , and added DIPEA (1 mL) and DMAP (20 mg). Compound 8, (0.050 g, 0.2 mmol) in CH 2 Cl 2 (2 mL) was added into above mixture, then the reaction mixture was stirred at 25°C, TLC monitored the reaction until the starting material was completely consumed. The obtained solid residue was purified through a silica gel chromatography (200 -300 mesh) with gradient eluent of CH 2 Cl 2 and CH 3 OH (100:0 to 85:15 v/v). The solvent was removed in vacuum, and the obtained solid was dissolved in CH 2 Cl 2 , filtered and evaporated to give a green solid (79 mg, yield 87 %). 1   Then the control probe was carried out to perform the in vivo and ex vivo imaging of Cys-SSH in Walker-256 tumor SD rats. The fluorescence images were obtained from two fluorescence collection windows. Channel 1: λ ex = 730 nm filter: 780 nm, and channel 2: λ ex = 610 nm filter: 710 nm. The Walker-256 tumor SD rats were given intravenous injection of the control probe (10 μM, 50 μL, in 1:99 DMSO/saline, v/v) for 15 min. As shown in Fig.  S19, the in vivo imaging of Walker-256 tumor SD rats in channel 1 by control probe showed almost whole body range fluorescence signal, because the control probe was widely distributed through the blood circulation. And from the ex vivo imaging, some other organs besides liver, such as spleen and kidney also gathered the control probe. The results illustrated that the control probe was poor liver targeting effect and the liver targeting group was indispensable. And in channel 2, the in vivo imaging showed fluorescence signal around liver position and the ex vivo imaging performed the fluorescence enhancement in liver, which may be due to the higher Cys-SSH concentration in liver than other organs. From the in vivo and ex vivo imaging of Cys-SSH in Walker-256 tumor SD rats by the control probe, we conclude that the liver targeting group was indispensable and absolutely necessary.