Highly efficient H2S scavengers via thiolysis of positively-charged NBD amines

H2S is a well-known toxic gas and also a gaseous signaling molecule involved in many biological processes. Advanced chemical tools that can regulate H2S levels in vivo are useful for understanding H2S biology as well as its potential therapeutic effects. To this end, we have developed a series of 7-nitro-1,2,3-benzoxadiazole (NBD) amines as potential H2S scavengers. The kinetic studies of thiolysis reactions revealed that incorporation of positively-charged groups onto the NBD amines greatly increased the rate of the H2S-specific thiolysis reaction. We demonstrate that these reactions proceed effectively, with second order rate constants (k2) of >116 M−1 s−1 at 37 °C for NBD-S8. Additionally, we demonstrate that NBD-S8 can effectively scavenge enzymatically-produced and endogenous H2S in live cells. Furthering the biological significance, we demonstrate NBD-S8 mediates scavenging of H2S in mice.


Synthesis
All chemicals and solvents used for organic synthesis were purchased from commercial suppliers and applied directly in the experiments without further purification. The progress of the reaction was monitored by TLC on pre-coated silica plates (Merck 60F-254, 250 µm in thickness), and spots were visualized by basic KMnO4, UV light or iodine. 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.
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 single-crystal X-ray diffraction data set was collected and tested on a Rigaku 007 Saturn 70 (Mo target) diffractometer.
After completion of reaction, the solvent was removed in vacuo, and the resulted residue was purified by column chromatography by eluting with methanol/dichloromethane to give the NBD-amines.

Synthesis of NBD-S5:
NBD-S5 was synthesized by the following three steps, according to a literature procedure. 2 Tert-butyl 4-methylpiperazine-1-carboxylate (BocMP): To the solution containing 1-methylpiperazine (0.5 g, 4.99 mmol) in tetrahydrofuran (6 mL) was added di-tert-butyl dicarbonate (1.72 mL, 7.49 mmol) under 0 °C, and the resulted mixture was stirred at room temperature for 2 h. The organic phase was removed under reduced pressure, and the resulted residue was purified by silica gel column chromatography to yield product BocMP (0.949 g, 95%). 1  Methyl triflate (0.409 g, 2.49 mmol) was dropwise added to the solution of 4-methylpiperazine-1-carboxylic acid tertbutyl ester (0.5 g, 2.49 mmol) in dry dichloromethane (10 mL) and stirred at room temperature over 1 h. Then the mixture was treated with triflic acid (0.599 g, 3.99 mmol) and allowed to stir for one hour. The organic phase was removed under reduced pressure, and the resulted residue was washed by 10 mL methanol to produce the white solid precipitate DMP (0.540 g, 82%). 1

Synthesis of S8-II:
2-Chloro-1-methylpyridin-1-ium iodide (0.2 g, 0.78 mmol) and DIPEA (269.5 μL, 1.56 mmol) were mixed in dichloromethane (8 mL), followed by dropwise addition of 1-bocpiprazine (0.16 g, 0.86 mmol) in dry dichloromethane (2 mL). The mixture was stirred at room temperature for 12 h. After completion of the reaction, the solvent was removed in vacuo and the residue was subjected for column chromatography by eluting with methanol/dichloromethane to provide a white solid (0.17 g, 53.6%). 1  Compound S8-I (0.1 g, 0.24 mmol) was dissolved in 5 mL dichloromethane and 2 mL trifluoroacetic acid and stirred for 4 h at room temperature. Then, the solvent was removed in vacuo and the resulted residue was purified by column chromatography to get a light yellow solid (0.056 g, 76%). 1

Spectra tests and HPLC analysis
General methods. 1-250 mM stock solutions of Na2S in degassed (or by bubbling N2 for 30 min) PBS buffer (pH 7.4) were used as H2S source. The DMSO stock solutions for NBD amines were 1-20 mM, which were diluted in PBS buffer to afford the final concentration of 1-200 µM, respectively. All measurements were performed in a 3 mL sealed cuvette. The UV-visible spectra were recorded on a UV-3600 UV-VIS-NIR spectrophotometer. Fluorescence studies were carried out using a F-280 spectrophotometer or Varian Cary Eclipse spectrophotometer. All reaction mixtures were shaken uniformly before spectra measurements.
10 μM NBD-based reagents in PBS buffer (pH 7.4) were mixed with different concentrations of Na2S in sealed cuvette, and the time-dependent absorbance spectra or intensity at 540 nm were recorded at 25 º C. The pseudo-first-order rate, kobs was determined by fitting the time-dependent intensity data with single exponential function.
The plots of log(kobs) vs log([H2S]) were fitted linearly to indicate the reaction order for H2S. The linear fitting between kobs and [H2S] gives the reaction rate (k2).
The water solubility was tested by concentration-dependent absorbance spectra of the scavengers in PBS (50 mM, pH = 7.4). 4 In all cases, the final concentration of DMSO in buffer was maintained to be 2%. The plots of absorbance intensity at 470 nm for NBD-S5 and at 490 nm for NBD-S8 against the reagent concentrations were linearly fitted ( Fig.   S14 and S15). The maximum concentration in the linear region was taken as the watersolubility.

X-ray crystallography studies
Crystals of NBD-S7 were obtained by recrystallization from dichloromethane and methanol (v:v, 1:1). The solution of NBD-S7 was heated at 60 °C for 20 minutes. After filtration, the resulted clear solution was isothermally evaporated at room temperature.
The red-transparent single crystals were emerged after several days.
Crystals of NBD-S8 were obtained by recrystallization from dichloromethanemethanol (v:v, 1:2) with traces of triethylamine. The solution of NBD-S8 was heated at 60 °C for 20 minutes. After filtration, the resulted clear solution was isothermally evaporated at room temperature. The red-transparent flat crystals were emerged within three days.

H2S scavenging tests
Normally, H2S concentration was determined via the Methylene Blue Assay (MBA). 7 The

Cell culture, MTT assay and bioimaging
The NBD-SH was prepared by a previous literature procedure. 9 HeLa cells were plated at a density of 100 k cells/well on a plastic 96 well plate in 10% FBS and 1% penicillin in phenol red-containing DMEM. The next day, the media was aspirated and cells were rinsed with FBS and phenol red-free DMEM. Cells then treated with different concentrations of NBD-SH (0-100 μM) in FBS and phenol red-free DMEM for 24 hours.
The media was removed, and the cells were rinsed again. The media was replaced with FBS and phenol red-free DMEM containing 10% CCK8 reagent (Dojindo) and further incubated until the assay was complete. Measurements were recorded using a BioTek Synergy 2 plate reader at 450 nm.
All bioimaging were based on our previous probe Cy7-NBD, 10 S27. Fluorescence images of exogenous H2S by probe Cy7-NBD and the H2S scavenging by NBD-S8 in mice. Mice were treated with different reagents via intraperitoneal injection: one group was injected with Cy7-NBD only; the second group was injected with Na2S first, then with Cy7-NBD; and the third group was injected with Na2S followed by NBD-S8, and then with Cy7-NBD.  Upon 5 h incubation, the results indicated that more than 90% p-toluenesulfonyl azide was decomposed under such conditions, but NBD-S8 was stable.

Supporting NMR and MS spectra
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