A bio-orthogonal based hydrogen polysulfide fluorescent probe and preparation and application thereof

Abstract

The application discloses a hydrogen polysulfide fluorescent probe based on biological orthogonality, and a preparation and application thereof. The hydrogen polysulfide detection probe based on the biological orthogonality of a cyclooctyne can detect hydrogen polysulfide in a living body in real time, fast, low toxicity and high selectivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicinal chemistry, in particular to a kind of based on biological ortho polysulfide hydrogen sulfide (H2S n , n>1) fluorescent probe and its preparation and application. BACKGROUND

[0002] Hydrogen sulfide (H2S) as a gaseous signal molecule plays a variety of functions in physiology and pathology. However, with the in-depth study of H2S biological effects in recent years, it is found that some of the physiological functions originally considered to be played by H2S are actually played by H2S n . In vivo, H2S n can be rapidly oxidized from H2S under the action of oxygen or enzyme, and has stronger reducing and nucleophilic properties. Compared with the classic signal molecule hydrogen sulfide (H2S), H2S n has higher protein sulfhydrylation efficiency, which makes it have greater advantages in regulating enzyme activity. For example, H2S n shows nearly 300 times higher efficiency than H2S in inducing Ca 2+ influx in astrocytes through TRAP1 channel S-thiolation (see Kimura Y, Mikami Y, Osumi K, Tsugane M, Oka J, Kimura H. Polysulfides are possible H2S-derived signaling molecules in rat brain. FASEB J. 2013 Jun;27(6):2451-7.). In addition, H2S nNuclear factor erythroid 2-related factor 2 (Nrf2) release is promoted and its negative regulator Kelch-like ECH-associated protein 1 (Keap1) is over-sulfurated to induce Nrf2 translocation to the nucleus, which subsequently leads to increased intracellular GSH levels and expression of HO-1, a Nrf2-regulated gene, to reduce intracellular reactive oxygen species (ROS) levels to play a role in protecting cells from oxidative stress (see Koike S, Ogasawara Y, Shibuya N, Kimura H, Ishii K. Polysulfide exerts a protective effect against cytotoxicity caused by t-buthylhydroperoxide through Nrf2 signaling in neuroblastoma cells. FEBS Lett. 2013 Nov 1; 587(21): 3548-55). In summary, there is growing research that H2S derived from H2S n may compensate for some physiological functions that H2S cannot perform, and research on H2S n has many problems to be solved. Therefore, in order to clarify the signaling or regulatory function of H2S n , it is urgent to develop corresponding recognition and tracking tools.

[0003] The current detection methods for H2S n include mass spectrometry, ultraviolet spectroscopy, indirect reduction release detection, detection after combination with cyanide or triphenylphosphine, and fluorescence probes. Mass spectrometry can directly detect H2S n in aqueous solution or monobromodibenzene derivative, but this method is not suitable for detection at near neutral pH, and the derivative is unstable during measurement. H2S n can also be detected using spectroscopic methods, and the absorption peaks at 300 and 372 nm can be directly used for the determination of H2S n , or the in situ ultraviolet-visible spectroelectrochemistry with GaAs electrodes can be used to detect the H2S n oxidation / regeneration process. This method is simple and clear, but is limited by low sensitivity. In addition, H2S n can react with the reducing agent dithiothreitol (DTT) to generate H2S, combine with cyanide to generate stable mercapto compounds, combine with triphenylphosphine to generate sulfide triphenylphosphine oxide, and form sulfides or disulfides with iodoacetamide, and then be indirectly detected by chromatography, mass spectrometry, etc. However, these methods have poor specificity, and other sulfane sulfur compounds can also give positive results, leading to errors in sample detection. The above methods have many limitations and are not suitable for detecting H2S n.

[0004] In contrast, fluorescent probes have been widely used in real-time detection of H2S in living cells and tissues due to their high sensitivity, good selectivity, fast operation, real-time monitoring in vivo, and low damage to biological samples. n In 2014, Xian et al. synthesized 2-mercaptobenzoate fluorescent probes for the first time to detect polysulfides in living systems. In the past decade, 2-fluoro-5-nitrobenzoate, aziridine, reductive, thioester, and cinnamic acid ester fluorescent probes have been designed and synthesized for detecting polysulfides in living systems due to the high nucleophilicity and reducing property of H2S. n However, these fluorescent probes still have poor water solubility and require the addition of cetyltrimethylammonium bromide (CTAB) or acetonitrile as a cosolvent during detection, which limits their application in vivo. SUMMARY

[0005] To overcome the poor water solubility of current fluorescent probes and improve the detection efficiency and specificity of the probes for polysulfides, while maintaining low toxicity to living organisms, a fluorescent probe for polysulfides based on cyclooctyne with ring strain is designed and synthesized.

[0006] Therefore, the present application aims to provide a fluorescent probe for polysulfides based on biological orthogonality. Another object of the present application is to provide a preparation method for the compound. A further object of the present application is to provide the application of the compound in the detection of polysulfides.

[0007] The present application provides a method for detecting polysulfides, wherein the fluorescent probe used in the method has one or more alkynes with ring strain. The method specifically includes: due to the photo-induced electron transfer effect (PET effect), the fluorescent probe is normally in a fluorescence quenching state; when the fluorescent probe contacts with polysulfides, a ring strain-induced click reaction occurs rapidly, the PET effect is eliminated, and a fluorescent group is released, thereby achieving the purpose of real-time detection and tracking of polysulfides.

[0008] The present application provides a fluorescent probe for polysulfides based on biological orthogonality, which has the structure shown in formula I:

[0009]

[0010] wherein: R1 represents a fluorescent group; and R2 represents a carbon 2 or higher straight-chain quaternary amine group.

[0011] In some specific examples, the fluorescent group represented by R1 includes rhodamine, coumarin, and cyanine.

[0012] In some embodiments, R2 is a C2-C6 linear quaternary amine group, more preferably a C2-C4 linear quaternary amine group.

[0013] In some embodiments, the present application provides a specific bio-orthogonal based H2S probe, Flu-CT, as shown in the structure.

[0014]

[0015] The present application also provides a method for preparing the H2S probe.

[0016] In some embodiments, the present application also provides a method for preparing the H2S probe Flu-CT.

[0017]

[0018] The present application also provides the use of the H2S probe in the detection of H2S.

[0019] The present application also provides the use of the H2S probe in the preparation of a H2S detection reagent.

[0020] In some embodiments, the present application provides a mechanism for detecting H2S using the H2S probe of Formula I as follows:

[0021]

[0022] In some embodiments, the present application provides a mechanism for detecting H2S using the H2S probe of Formula Flu-CT as follows:

[0023]

[0024] Unless otherwise specified, the H2S referred to in the present application is H2S n wherein n > 1.

[0025] The present application provides a class of H2S detection probes based on the bio-orthogonal reaction of cyclooctyne, which can detect H2S in vivo in real time, rapidly, with low toxicity and high selectivity. Compared with the prior art, the present application has the following beneficial effects:

[0026] The compound Flu-CT can be used for detecting trace hydrogen polysulfides in vivo with high efficiency and rapidness, and can produce relatively strong fluorescently labeled hydrogen polysulfides within 10 minutes. Stability experiments show that the probe Flu-CT can exist stably in vivo (PBS, 37℃), and the fluorescence intensity does not change within 7 minutes. When hydrogen polysulfides exist, the fluorescence intensity changes with time in a dependent manner, and reaches the maximum fluorescence intensity at 17 minutes. The probe has high fluorescence efficiency, and 20 μM of the compound can produce relatively strong fluorescence, and has little influence on the organism. Moreover, for lower concentration of hydrogen polysulfides, the fluorescence intensity of the probe is linear with the concentration of sodium persulfate, and the probe can be used for quantitative detection of hydrogen polysulfides. Meanwhile, the probe has high selectivity, and other sulfane sulfur compounds will not produce interference. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the fluorescence response experimental result of the probe Flu-CT to hydrogen polysulfides;

[0028] Figure 2 is the influence of hydrogen polysulfide concentration on Flu-CT detection;

[0029] Figure 3 is the selectivity experimental result of the probe Flu-CT;

[0030] Figure 4 is the toxicity experimental result of the compound Flu-CT;

[0031] Figure 5 is the in vivo fluorescence imaging result of the compound Flu-CT. DETAILED DESCRIPTION

[0032] The following examples facilitate better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods, and if no special description is given, the experimental materials are obtained from conventional biochemical reagent stores.

[0033] Example 1 Preparation of cyclooctyne fluorescent probe Flu-CT

[0034]

[0035] F-1 : Synthesis of (9H-fluoren-9-yl)methyl (3-hydroxyphenyl)carbamate. 3- Aminophenol (3 g, 27.49 mmol), fluorenylmethoxycarbonylsuccinimid (Fmoc- OSu) (11.13 g, 32.99 mmol) were dissolved in 30 ml of absolute ethanol, and the reaction solution was stirred well, and N,N-diisopropylethylamine (DIEA, 4.26 g, 32.99 mmol) was slowly added dropwise thereto, and stirring was continued at room temperature for 10 h. The reaction solution was poured into water (200 ml), and extracted with dichloromethane (3 x 200 ml), and the combined extract was dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered, and the filtrate was concentrated under reduced pressure to obtain an oily crude product, and petroleum ether (20 ml) was added thereto, and sonicated until white precipitates appeared. The filtrate was filtered off, and the precipitates were washed with petroleum ether (3 x 20 ml) to obtain a white powdery product (7.25 g, 80%).

[0036] 1 H NMR (300 MHz, DMSO-d6) δ 9.62 (s, 1H), 9.37 (s, 1H), 7.96 - 7.90 (m, 2H), 7.78 (d, J = 7.4 Hz, 2H), 7.49 - 7.41 (m, 2H), 7.37 (td, J = 7.4, 1.3 Hz, 2H), 7.12 - 6.98 (m, 2H), 6.87 (d, J = 8.2 Hz, 1H), 6.41 (ddd, J = 8.0, 2.4, 1.0 Hz, 1H), 4.47 (d, J = 6.8 Hz, 2H), 4.32 (t, J = 6.7 Hz, 1H).

[0037] 13 C NMR (75 MHz, DMSO-d6) δ 158.26, 153.84, 144.31, 141.32, 140.66, 139.94, 137.95, 129.92, 129.44, 128.21, 127.80, 127.64, 125.68, 121.89, 120.68, 120.52, 110.18, 109.69, 106.00, 66.02, 47.16.

[0038] F-2: Synthesis of compound Flu-A1. Compound (9H-fluoro-9-yl)methyl (3- hydroxyphenyl)carbamate (7.25 g, 21.88 mmol), phthalic anhydride (1.67 g, 11.27 mmol), zinc chloride (6.05 g, 44.41 mmol) were dissolved in methylsulfonic acid (30 ml) and stirred at room temperature for 0.5 h until complete dissolution. Then heated to 45 °C and stirred at 45 °C for 15 h. The reaction was cooled under ice-salt bath and ice water (20 ml) was added until orange precipitate appeared. The filtrate was filtered off and the precipitate was washed with anhydrous diethyl ether (3 x 20 ml). The crude product was dissolved in methanol (15 ml) and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product was purified by silica gel column chromatography (eluted with ethyl acetate and petroleum ether gradient) to isolate the product as a red powder (330 mg, 2.7%).

[0039] 1 H NMR (500 MHz, Methanol-d4) δ 8.06 (d, J = 7.7 Hz, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.79 (t, J = 7.4 Hz, 1H), 7.72 (t, J = 6.9 Hz, 3H), 7.61 (d, J = 16.4 Hz, 1H), 7.42 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H), 7.24 (d, J = 7.6 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H), 6.71 (d, J = 8.7 Hz, 1H), 6.63 (d, J = 2.2 Hz, 1H), 6.57 (d, J = 8.6 Hz, 1H), 6.50 (dd, J = 8.7, 2.2 Hz, 1H), 4.56 (d, J = 6.5 Hz, 2H), 4.31 (t, J = 6.6 Hz, 1H).

[0040] 13 C NMR (126 MHz, DMSO-d6) δ 169.29, 153.83, 153.75, 152.97, 152.49, 151.82, 144.20, 141.62, 141.33, 135.97, 130.48, 129.39, 128.98, 128.85, 128.22, 127.65, 126.86, 125.61, 125.05, 124.51, 120.70, 114.69, 113.58, 111.77, 105.78, 99.66, 83.99, 66.27, 47.11.

[0041] HRMS: Calculated for C 35 H 24 N2O5[M+H] +553.1758, found 553.1755

[0042] F-3: Synthesis of Flu-A2. Compound Flu-A1 (100 mg, 0.18 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 10 ml), and then bis(trichloromethyl) carbonate (BTC, 24 mg, 0.08 mmol) was dissolved in 1 ml of anhydrous THF and added dropwise to the above reaction solution, followed by the addition of triethylamine (TEA, 18 mg, 0.18 mmol). The reaction solution was stirred at room temperature for 0.5 h, and the color changed from red to light red. 2-Cyclooctyn-1-ol (112 mg, 0.9 mmol) was added, and triethylamine (TEA, 18 mg, 0.18 mmol) was added dropwise. White smoke appeared, and the color of the reaction solution became lighter. The solution was stirred at room temperature for 6 h. After concentration under reduced pressure, the product was separated as a white powder (50 mg, 39%) by silica gel column chromatography (eluted with a gradient of ethyl acetate and petroleum ether). (See Kang D, Kim J. Bioorthogonal Retro-Cope Elimination Reaction of N,N-Dialkylhydroxylamines and Strained Alkynes. J Am Chem Soc. 2021 Apr 21;143(15):5616-5621. for the synthesis of 2-cyclooctyn-1-ol.)

[0043] 1 H NMR (500 MHz, Chloroform-d) δ 8.05 (dd, J = 7.5, 1.2 Hz, 1H), 7.82 (d, J = 7.5 Hz, 2H), 7.67 (ddd, J = 15.5, 7.4, 1.2 Hz, 4H), 7.53 (d, J = 29.8 Hz, 1H), 7.45 (t, J = 7.5 Hz, 2H), 7.37 (tt, J = 7.5, 1.3 Hz, 2H), 7.18 - 7.13 (m, 1H), 7.01 - 6.92 (m, 2H), 6.80 - 6.72 (m, 4H), 5.43 (s, 1H), 4.62 (d, J = 6.5 Hz, 2H), 4.31 (t, J = 6.4 Hz, 1H), 2.37 - 2.29 (m, 1H), 2.29 - 2.20 (m, 2H), 2.16 - 2.08 (m, 1H), 1.96 (d, J = 6.9 Hz, 2H), 1.85 (dt, J = 16.9, 8.9 Hz, 1H), 1.76 (q, J = 8.5, 7.4 Hz, 1H), 1.67 (dq, J = 24.2, 7.9 Hz, 2H), 0.95 - 0.85 (m, 1H).

[0044] 13C NMR (75 MHz, Chloroform-d) δ 168.85, 168.59, 152.14, 151.23, 150.77, 142.57, 140.32, 138.93, 134.14, 128.78, 127.61, 126.80, 126.13, 125.36, 124.04, 123.91, 122.91, 119.03, 113.24, 112.50, 105.31, 101.45, 89.36, 81.67, 66.86, 65.98, 63.88, 46.00, 42.30, 40.73, 33.16, 30.48, 29.11, 28.67, 28.54, 25.09, 24.58, 19.67, 18.02, 12.64.

[0045] HRMS: Calculated for C 44 H 34 N2O7[M+H] + 703.2439, found 703.2443

[0046] F-4: Synthesis of Flu-A3. Compound Flu-A2 (50 mg, 7.11 mmol) was dissolved in dichloromethane (DCM, 5 ml), piperidine (1 ml) was added dropwise, and stirred at room temperature for 3 h. The reaction changed from colorless to orange red after the reaction was completed, and the product was isolated as a red powder (31 mg, 91%) after concentration under reduced pressure and purification by silica gel column chromatography (eluted with dichloromethane and methanol gradient).

[0047] 1 H NMR (500 MHz, Chloroform-d) δ 8.04 (d, J = 7.5 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.18 (dd, J = 11.8, 8.2 Hz, 1H), 6.91 (dd, J = 28.2, 8.9 Hz, 1H), 6.77 - 6.69 (m, 2H), 6.61 - 6.53 (m, 2H), 6.38 (d, J = 8.6 Hz, 1H), 5.43 (s, 1H), 2.38 - 2.31 (m, 1H), 2.27 - 2.21 (m, 2H), 2.13 (q, J = 7.4, 6.5 Hz, 1H), 1.96 (m, 2H), 1.90 - 1.83 (m, 1H), 1.76 (d, J = 8.8 Hz, 1H), 1.69 (m, 2H).

[0048] 13C NMR (126 MHz, Chloroform-d) δ 169.61, 168.46, 152.62, 152.25, 139.84, 134.91, 129.62, 129.20, 128.78, 126.97, 125.07, 124.11, 114.19, 114.01, 111.84, 108.70, 108.48, 106.32, 102.52, 101.50, 90.45, 67.92, 44.63, 41.81, 34.23, 29.63, 26.17, 22.59, 20.74.

[0049] HRMS: Calculated for C 29 H 24 N2O5[M+H] + : 481.1758, found 481.1763

[0050] F-5: Synthesis of Flu-CT. Compound Flu-A3 (31 mg, 0.065 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 3 ml), and then dropwise added to the above reaction solution was a solution of triphosgene (BTC, 19 mg, 0.065 mmol) in 1 ml of anhydrous THF, followed by dropwise addition of triethylamine (TEA, 6.6 mg, 0.065 mmol), and the reaction solution was stirred at room temperature for 0.5 h, during which time the solution changed from red to light red. Flu-B (75 mg, 0.325 mmol) was added, and dropwise addition of triethylamine (TEA, 6.6 mg, 0.065 mmol) was performed, and the solution became even lighter in color, and was stirred at room temperature for 7 h. The excess Flu-B was removed by filtration, and the solid was washed with THF (3 x 5 ml). After concentration under reduced pressure, the product was isolated as a light red powder (40 mg, 80%) by purification on a C-18 reverse-phase column chromatography (eluted with a gradient of acetonitrile and water). (For the synthesis of Flu-B, see Saha A, Panda S, Paul S, Manna D. Phosphate bioisostere containing amphiphiles: a novel class of squaramide-based lipids. Chem Commun (Camb). 2016 Jul 19;52(60):9438-41.)

[0051] 1H NMR (300 MHz, Methanol-d4) δ 8.11 (d, J = 7.4 Hz, 1H), 7.83 (dd, J = 12.7, 7.4 Hz, 2H), 7.74 - 7.66 (m, 2H), 7.29 (d, J = 7.5 Hz, 1H), 7.10 (t, J = 10.0 Hz, 2H), 6.74 (dd, J = 8.6, 4.6 Hz, 2H), 5.38 (s, 1H), 3.82 (d, J = 16.6 Hz, 2H), 3.62 (d, J = 6.5 Hz, 2H), 3.32 (s, 9H), 2.32 - 2.11 (m, 4H), 1.97 (d, J = 27.1 Hz, 3H), 1.82 - 1.70 (m, 3H).

[0052] 13 C NMR (126 MHz, Chloroform-d) δ 169.79, 155.75, 153.36, 152.78, 151.68, 142.13, 140.65, 129.94, 128.74, 128.17, 126.44, 125.03, 123.97, 114.66, 113.84, 113.32, 111.96, 106.30, 102.20, 96.84, 90.90, 89.04, 83.54, 67.67, 54.06, 41.65, 34.23, 29.74, 29.61, 29.57, 26.18, 20.70, 14.14.

[0053] HRMS: Calcd for C 35 H 37 N4O6[M] + : 609.2708, found 609.2699.

[0054] Example 2 Fluorescence response of probe Flu-CT to polysulfide hydrogen

[0055] In 3 ml of phosphate buffer (PBS) added 20 μΜ of Flu-CT, 37 ℃ reaction for 7 min, every 1 min with fluorescence spectrophotometer to detect the change of fluorescence intensity (excitation wavelength: 334 nm, maximum emission wavelength 524 nm), fluorescence intensity has no obvious change. Subsequently added 50 μΜ Na2S2, 37 ℃ reaction for 17 min, every 1 min with fluorescence spectrophotometer to detect the change of fluorescence intensity (excitation wavelength: 334 nm, maximum emission wavelength 524 nm), fluorescence intensity with time dependent growth, at 17 min, the fluorescence intensity reached the maximum value, basically unchanged Figure 1 ).

[0056] Example 3 The effect of hydrogen polysulfide concentration on detection

[0057] In 3ml of phosphate buffer solution (PBS) added 20 μM Flu-CT, respectively added different concentrations of Na2S2(0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 120 μM), 37°C for 5 min, respectively, using fluorescence spectrophotometer to detect the fluorescence intensity changes (excitation wavelength: 334 nm, maximum emission wavelength 524 nm). At lower concentrations, the fluorescence intensity and sodium sulfide concentration showed a linear relationship, which can be used for quantitative detection of hydrogen polysulfide Figure 2 ).

[0058] Example 4 Probe Flu-CT selectivity experiment

[0059] In 3ml of phosphate buffer solution (PBS) added 20 μM Flu-CT, respectively added 50 μM Na2S2O3, Na2SO3, S, Na2S, glutathione (GSH), cysteine (Cys), Na2S2, NaOH, 37°C for 5 min, respectively, using fluorescence spectrophotometer to detect the fluorescence intensity (excitation wavelength: 334 nm, maximum emission wavelength 524 nm). 50 μM Na2S2 significantly enhanced the fluorescence intensity, other sulfane sulfur compounds and alkali had no significant effect on the fluorescence intensity, indicating that the probe has high selectivity Figure 3 ).

[0060] Example 5 Compound Flu-CT toxicity experiment

[0061] MTT method to determine the toxicity of compound Flu-CT on cells. In 96-well plates, PC12 cells were seeded at a density of 6 x 10 4 After 24 h of cell seeding, the culture medium was discarded and the cells were washed with PBS, and then 0 μM (control), 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM Flu-CT were added to the culture medium, respectively, and incubated for 24 h. After incubation, the culture medium was discarded and the cells were washed with PBS, and then 10 μl of MTT (5 mg / ml) and 90 μl of liquid medium were added to each well. The final results were read using a SpectraMax iD3 multifunctional enzyme marker. The experimental results showed that each concentration of Flu-CT did not produce significant toxicity to the cells (P>0.05) Figure 4 ).

[0062] Example 6 In vivo fluorescence imaging of compound Flu-CT

[0063] Nikon inverted fluorescence microscope in vivo fluorescence imaging. In 24-well plates, PC12 cells were seeded at a density of 5 x 10 4The density of PC12 cells was seeded. After 24 h of cell seeding, the culture medium was discarded, the cells were washed with PBS, and the cells were incubated with 40 μM of Fluc-CT for 30 min. After the incubation, the culture medium was discarded, the cells were washed with PBS. The culture medium containing only DMEM (control), 50 μM Na2S2, 100 μM Na2S2, 50 μM S, and 50 μM Na2S was added, respectively, and incubated for 20 min. Live fluorescence imaging was performed using a Nikon inverted fluorescence microscope (channel: 475 nm). Compared with the control group, the fluorescence intensity of the group treated with 50 μM Na2S2increased significantly, and the fluorescence intensity of the group treated with 100 μM Na2S2increased strongly. However, the fluorescence intensity of the groups treated with 50 μM S and 50 μM Na2S did not change significantly ( Figure 5 ).​

Claims

1. A specific bioorthogonal-based fluorescent probe for hydrogen polysulfide, the structure of which is shown in Flu-CT:

2. The method for preparing the polysulfide fluorescent probe according to claim 1:

3. The application of the hydrogen polysulfide fluorescent probe according to claim 1 in the detection of hydrogen polysulfide.

4. The application of the hydrogen polysulfide fluorescent probe according to claim 1 in the preparation of hydrogen polysulfide detection reagents.

Images