A fluorescent probe for specific detection of H2S and its synthesis method

Abstract

The present invention belongs to the field of fluorescent probe technology and specifically discloses a fluorescent probe for the specific detection of H2S. The present invention also discloses a method for synthesizing the fluorescent probe. The fluorescent probe disclosed in the present invention has advantages such as simple design, ease of synthesis, high fluorescence intensity, excellent selectivity, high sensitivity, and a low detection limit. It can be applied to image endogenous H2S in cells, has excellent biological application potential, and can become a useful tool for understanding complex intracellular regulatory systems.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe for specifically detecting H2S and a synthesis method thereof. Background Art

[0002] Hydrogen sulfide (H2S) is considered a toxic gas with a foul odor. Excessive inhalation can cause cardiac dysfunction, myocardial damage, and even be life-threatening. However, in terms of its signaling capacity and the biodistribution of endogenous H2S-producing enzymes, H2S is a gaseous transmitter similar to nitric oxide (NO) and carbon monoxide (CO), participating in important physiological and pathological processes. Recent studies have shown that H2S affects multiple physiological functions in the human body and is involved in the regulation of diseases such as neurodegenerative diseases, cardiovascular diseases, cancer, and inflammation. Unfortunately, tracking and accurately quantifying H2S in biological systems remains difficult, and the regulatory mechanisms of H2S remain unclear. Therefore, it is necessary to develop a highly selective method for the detection of endogenous H2S in organisms.

[0003] With the development and progress of scientific research, a variety of methods for detecting H2S have been developed, such as the methylene blue method, the H2S ion-selective electrode method, and gas chromatography. However, these methods all have certain drawbacks. For example, the methylene blue method has relatively low detection sensitivity and is not suitable for detecting trace substances (in the micromolar range). Since sulfide easily escapes from acid-labile containers, its concentration may be misestimated. The H2S ion-selective electrode method requires a long time, and the gas chromatography method also requires the airtightness of the equipment. In order to more sensitively and conveniently detect H2S in complex biological systems, small molecule fluorescent probes have come into the sight of researchers. Their advantages such as low cost, high biocompatibility, sensitive response, easy synthesis, and high selectivity have attracted widespread attention. Summary of the Invention

[0004] The purpose of the present invention is to provide a fluorescent probe for specific detection of H2S and a synthesis method thereof to solve the above technical problems.

[0005] One aspect of the present invention relates to a fluorescent probe for specific detection of H2S, wherein the chemical structure of the fluorescent probe is:

[0006]

[0007] The fluorescent probe is named "DSNP", and its Chinese name is: (Z)-4-(2-(6-(((2,4-difluorophenyl)sulfonyl)oxy)naphthalen-2-yl)vinyl)-1-methylpyridine iodide.

[0008] The principle of the fluorescent probe DSNP provided by the present invention is as follows: the probe uses iodinated 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridine (Hnmp-1) as a fluorescent group and a 2,4-difluorobenzenesulfonyl group as an H2S reaction site. The cleavage of the sulfonyl ester bond generates fluorescence with a wavelength of 568 nm, which allows the H2S to react with DSNP to generate Hnmp-1 with high fluorescence intensity.

[0009]

[0010] Another aspect of the present invention relates to a method for synthesizing the fluorescent probe. The raw materials of the fluorescent probe include a first raw material I and a second raw material II. The structural formula of the first raw material I is The structural formula of the second raw material II is

[0011] Preferably, the first raw material I reacts with 6-hydroxy-2-naphthaldehyde to obtain the second raw material II.

[0012] Preferably, the synthesis method comprises the following steps:

[0013] S1. Synthesis of the first raw material I:

[0014] 0.5-2 mmol 4-methylpyridine, 1-3 mmol iodomethane and 3-6 mL acetonitrile were added to a flask, reacted at 77-86°C for 11-14 hours, cooled to room temperature, recrystallized with ethanol, filtered under reduced pressure, washed the filter residue with ethanol, and dried to obtain the product, the first raw material I: 1,4-dimethylpyridine iodide;

[0015] S2. Synthesis of the second raw material II:

[0016] 0.7-2 mmol of the first raw material I, 1-3.5 mmol of 6-hydroxy-2-naphthaldehyde, 0.3-0.8 mL of piperidine catalyst, and 4-8 mL of methanol were reacted at 45-55°C for 1-1.5 hours; after cooling to room temperature, the mixture was extracted with water and distilled under reduced pressure to obtain the product, the second raw material II: 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridine iodide (II);

[0017] Synthetic fluorescent probe III:

[0018] Under N2 environment, 0.5-1.8mmol of the second raw material II, 0.5-3.2mmol of 2,4-difluorobenzenesulfonyl chloride, 0.5-3mL of N,N-dimethylformamide, 0.5-3mL of triethylamine and 3-7mL of dichloromethane were mixed and stirred at room temperature for 2-6h. After evaporating the solvent using a rotary evaporator, silica gel column chromatography was used to obtain a light yellow powdery solid, which is the fluorescent probe (Z)-4-(2-(6-(((2,4-difluorophenyl)sulfonyl)oxy)naphthalen-2-yl)vinyl)-1-methylpyridine iodide (III).

[0019] The third aspect of the present invention relates to the use of the above-mentioned fluorescent probe for specific detection of H2S in H2S detection.

[0020] Preferably, the detection limit of the fluorescent probe for H2S is 30.7nm. The linear relationship between fluorescence intensity and H2S concentration is y=40.812x+72.512, R 2 =0.9945.

[0021] Preferably, the excitation wavelength required for detection is in the range of 200-400 nm, and the fluorescence emission wavelength is 568 nm.

[0022] Preferably, the fluorescent probe is used for H2S detection in cells, for non-disease diagnosis and treatment purposes.

[0023] The present invention provides a novel mitochondrial-targeted fluorescent probe capable of detecting H2S with high sensitivity and selectivity. This probe can be applied to the detection of H2S in living cells. The fluorescent probe DSNP features simple design, easy synthesis, excellent selectivity, low toxicity, and good biocompatibility. This invention provides a useful targeted fluorescent probe for studying intracellular H2S, offering new insights and tools for studying the cellular microenvironment and possessing significant value in environmental monitoring and biomedical research.

[0024] The fluorescent probe provided by the present invention uses iodinated 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridine (Hnmp-1) as a fluorescent group and 2,4-difluorobenzenesulfonyl as an H2S reaction site, and generates fluorescence with a wavelength of 568nm by breaking the sulfonyl ester bond.

[0025] The molecular structure of the fluorescent probe provided by the present invention contains an asymmetric electronic structure. DSNP can be used for endogenous H2S imaging in cells and has excellent biological application potential, which can make it a useful tool for detecting H2S and better understanding the complex regulatory system in cells.

[0026] The fluorescent probe provided by the present invention exhibits enhanced fluorescence after efficient reaction with H2S. It has the characteristics of simple design, easy synthesis, high fluorescence intensity, good selectivity, high sensitivity, low detection limit, and low cytotoxicity. It has excellent biological application potential and is expected to become a useful tool for detecting H2S and better understanding the complex regulatory systems within cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the synthetic route of DSNP fluorescent probe.

[0028] Figure 2A For the probe DSNP (III) 1 HNMR spectrum.

[0029] Figure 2B For the probe DSNP (III) 13 CNMR spectrum.

[0030] Figure 3A For Hnmp-1(II) 1 HNMR spectrum.

[0031] Figure 3B For Hnmp-1(II) 13 CNMR spectrum.

[0032] Figure 4 Figure 2 is the fluorescence emission spectrum of the mixed solution of DSNP and each target at 568 nm.

[0033] Figure 5 The fluorescence intensity of the DSNP experimental group and the control group at 568 nm at different times (5-120 min).

[0034] Figure 6 The fluorescence intensity of the experimental group (DNSP+H2S) and the control group (DSNP) under different pH conditions (3-12) at 568 nm.

[0035] Figure 7 is the fluorescence emission wavelength of DSNP and different concentrations (0-10 μM) of H2S at 568 nm.

[0036] Figure 8 Cell viability at different concentrations (from 10 to 50 μM) of DSNP.

[0037] Figure 9 Cell imaging of the experimental group (DSNP+H2S) and the control group (DSNP). DETAILED DESCRIPTION

[0038] Example: Synthesis of fluorescent probe DSNP

[0039] Synthesis steps such as Figure 1 As shown, the following steps are involved:

[0040] S1. Synthesis of 1,4-dimethylpyridine (I) iodide

[0041] 0.5-2 mmol 4-methylpyridine (M=93.13, CAS: 108-89-4, Beijing Bailingwei Technology Co., Ltd., product number: P815734-100 ml), 1-3 mmol iodomethane (M=141.95, CAS: 74-88-4, Shanghai Aladdin Biochemical Technology Co., Ltd., product number: I573480-25 ml) and 3-6 mL acetonitrile were added to a flask, reacted at 77-86°C for 11-14 hours, cooled to room temperature, recrystallized with ethanol, filtered under reduced pressure, washed the filter residue with a small amount of ethanol, and dried to obtain the product 1,4-dimethylpyridine iodide (M=108.16).

[0042] Specifically, the product is a white powdery solid.

[0043] In a specific embodiment of the present invention, the input amount of 4-methylpyridine is 0.1863 mg (2 mmol), the input amount of iodomethane is 0.4258 g (3 mmol), and the acetonitrile is 5 mL. The reaction conditions are 85° C. for 12 h, and the final yield of iodinated 1,4-dimethylpyridine (I) is 94%.

[0044] S2. Synthesis of 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridine iodide (Hnmp-1, II)

[0045] Add 0.7-2 mmol of 1,4-dimethylpyridine iodide (M=108.16), 1-3.5 mmol of 6-hydroxy-2-naphthaldehyde (M=108.16, CAS: 78119-82-1, Beijing Bailingwei Technology Co., Ltd., Cat. No.: H834750-5g), 0.3-0.8 mL of piperidine catalyst, and 4-8 mL of methanol to a flask. React at 45-55°C for 1-1.5 hours. After cooling to room temperature, extract with water three times and evaporate under reduced pressure to obtain the product, 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridine iodide (II). The product is a reddish-brown powdery solid.

[0046] In a specific embodiment of the present invention, the input amount of 1,4-dimethylpyridine iodide is 0.2163 mg (2 mmol), the input amount of 6-hydroxy-2-naphthaldehyde is 0.3444 g (2 mmol), the volume of piperidine is 0.5 mL, and the volume of methanol is 5 mL. The yield of Hnmp-1(II) is 60%. Characterization diagram Figure 3A and Figure 3B As shown:

[0047] 1 H NMR (400MHz, DMSO) δ8.79(d,J=6.3Hz,2H),8.17(d,J=6.2Hz,2H),8.04(d,J=13.4Hz,2H),7.7 8(d,J=8.8Hz,2H),7.73–7.54(m,2H),7.48(d,J=16.2Hz,1H),7.15–7.08(m,2H),4.23(s,3H). 13 C NMR(101MHz,DMSO)δ159.91,153.22,145.23,141.85,136.62,130.77,130.7 5,129.37,127.20,127.13,124.14,123.47,121.62,121.02,109.70,47.23.

[0048] S3. Synthesis of fluorescent probes (III)

[0049] Under N2 environment, 0.5-1.8 mmol Hnmp-1 (M = 262), 0.5-3.2 mmol 2,4-difluorobenzenesulfonyl chloride (M = 212.6, CAS: 13918-92-8, Beijing J&K Technology Co., Ltd., product number: D829371-25g), 0.5-3 mL N, N-dimethylformamide (M = 73.09, CAS: 68-12-2, Beijing J&K Technology Co., Ltd., N807505), 0.5-3 mL triethylamine (M = 101.19, CAS: 121-44-8, Beijing J&K Technology Co., Ltd., product number: T818774) and 3-7 mL dichloromethane were mixed and stirred at room temperature for 2-6 h. After evaporating the solvent using a rotary evaporator, the product was purified by silica gel column chromatography (200 mesh) using CH2Cl2 / MeOH (volume ratio = 50-20:1) as eluent to obtain a light yellow powdery solid (III), which is the fluorescent probe (Z)-4-(2-(6-(((2,4-difluorophenyl)sulfonyl)oxy)naphthalen-2-yl)vinyl)-1-methylpyridine iodide (III) (DSNP).

[0050] In a specific embodiment of the present invention, 0.2623 mg (1 mmol) of Hnmp-1, 0.3189 g (1.5 mmol) of 2,4-difluorobenzenesulfonyl chloride, 0.5 mL of N,N-dimethylformamide, 0.5 mL of triethylamine, and 5 mL of dichloromethane were mixed and stirred at room temperature for 3 hours under a nitrogen atmosphere. The molar ratio of Hnmp-1 to 2,4-difluorobenzenesulfonyl chloride was 1:1.5. The molar ratio of N,N-dimethylformamide, triethylamine, and dichloromethane was 1:1:10.

[0051] After evaporating the solvent using a rotary evaporator, the product was purified by 200-mesh silica gel column chromatography using eluents ranging from CH2Cl2 / MeOH (volume ratio = 49:1) to CH2Cl2 / MeOH (volume ratio = 19:1) to obtain a pale yellow powdery solid (III), the fluorescent probe (Z)-4-(2-(6-(((2,4-difluorophenyl)sulfonyl)oxy)naphthalen-2-yl)vinyl)-1-methylpyridine iodide (DSNP), in a 46% yield. The molar ratio of N,N-dimethylformamide, triethylamine, and dichloromethane was 1:1:10.

[0052] Examples 1-5

[0053] The main differences between the synthesis methods of Examples 1-5 are shown in Table 1 below:

[0054] Table 1: Main differences between Examples 1-5

[0055]

[0056] The specific synthetic route of the fluorescent probe DSNP provided in Example 1 is as follows: Figure 1 shown.

[0057] Characterization Figure 2A and Figure 2B As shown:

[0058] 1 H NMR (400MHz, DMSO) δ8.89(d,J=6.3Hz,2H),8.25(d,J=5.8Hz,2H),8.15(d,J=16.3Hz,1H),8.10–8.00(m,3H),7.98–7.92(m,1H),7 .81(d,J=2.5Hz,1H),7.68(d,J=16.3Hz,1H),7.44(t,J=8.9Hz,2H),7.34(dd,J=9.0,2.3Hz,1H),7.04–6.83(m,1H),4.28(s,3H).

[0059] 13 C NMR (101MHz, DMSO) δ152.66,147.60,145.67,140.43,134.30,134.17,133.97,133.85,132.15,131.63,129.7 8,129.33,125.46,124.90,124.18,121.90,120.10,113.81,113.62,113.58,107.78,107.53,104.75,63.25.

[0060] By comparing the above synthesis methods, it can be seen that the yield of the fluorescent probe DSNP synthesized by the method of Example 5 is the highest, which is 46.44% (86%×72%×75%).

[0061] Fluorescence spectroscopy experiments

[0062] 1. Experimental methods

[0063] Aqueous solutions used for fluorescence measurements were prepared with deionized water. The solution preparation steps were as follows: solid DSNP was prepared with DMSO / PBS (2:8) to a 2 mmol / L stock solution, which was then diluted to 20 μmol / L with PBS to obtain the solution used for spectral measurement. Target solutions were diluted with deionized water to the corresponding concentrations. For optical measurements, 0.35 mL of DSNP and 0.35 mL of target solution were mixed and poured into a quartz optical cell, and the fluorescence emission spectrum was recorded.

[0064] The DSNP solution with a concentration of 0.02mmol / L was mixed with Arg, His, Cys, Na + , Ag + , Cu 2+ , Ca 2+ , Mg 2+ , NH4 + , PO4 3- , I - , NO 2- ,HCO3 - , CO3 2- , SO4 2- , SO3 2- , HSO3 - 350 μL of each of NH3, HAC and H2S solutions were mixed and reacted at 60°C for 1 h, and then the fluorescence emission spectrum of the mixed solution was measured.

[0065] 2. Experimental results

[0066] The selectivity experiment of DSNP fluorescence response to interfering substances showed that traditional anions, cations and biothiols, including Arg, His, Cys, Na + , Ag + , Cu 2+ , Ca 2+ , Mg 2+ , NH4 + , PO4 3- ,,I - , NO 2- ,HCO3 - , CO3 2- , SO4 2- , SO32- , HSO3 - , NH3, HAC and H2S, the experimental results are as follows Figure 4 As shown in the figure, even at a fairly high level (10eqv), no significant interference will be caused, while H2S can respond to DSNP and emit strong fluorescence at 568nm.

[0067] Time control experiment

[0068] 1. Experimental methods

[0069] 350 μL of a 20 μmol / L DSNP solution and a 350 μL H2S solution of the same concentration were mixed and maintained at 60°C. The fluorescence emission spectra of the mixed solution were measured at 30 s, 1 min, 2 min, 5 min, 10 min, 30 min, 1 h, 1.5 h, and 2 h. In the control group, PBS solution was used to replace the H2S solution and the fluorescence emission spectra were measured at 30 s, 1 min, 2 min, 5 min, 10 min, 30 min, 1 h, 1.5 h, and 2 h.

[0070] 2. Experimental results

[0071] like Figure 5 As shown in Figure 3, the fluorescence intensity of the solution at 568 nm gradually increased with the increase of reaction time and reached a stable state after 45 min.

[0072] pH control experiment

[0073] 1. Experimental methods

[0074] The pH of the PBS solution was adjusted to 3-12 using 0.1 mol / L NaOH solution or HCl solution. The 2 mmol / L DSNP stock solution was diluted to 20 μmol / L using PBS solutions of different pH values.

[0075] Experimental group: 350 μL of 20 μmol / L DSNP solution and 350 μL of H2S solution of the same concentration were mixed.

[0076] Control group: 20 μmol / L DSNP solution was mixed with 350 μL PBS solution. After the mixture was reacted at 60°C for 1 hour, the fluorescence emission spectra of the mixture were measured.

[0077] 2. Experimental results

[0078] The experimental results are as follows Figure 6As shown in the figure, the fluorescence emission spectra under different pH environments show that the fluorescence intensity of the DSNP-H2S mixture at 568 nm is low in an acidic environment, but it can emit strong fluorescence in neutral and alkaline environments. The pH of the human body environment is about 7.4, so the probe is suitable for physiological pH.

[0079] Concentration gradient experiments

[0080] 1. Experimental methods

[0081] Prepare H2S solutions with concentrations of 0-10 μmol / L, take DSNP solution with a concentration of 20 μmol / L and mix evenly with 350 μL of H2S solutions of various concentrations, react at 60°C for 1 hour, and then measure the fluorescence emission spectrum of the mixed solution.

[0082] 2. Experimental results

[0083] The experimental results are as follows Figure 7 As shown, the fluorescence intensity at 568 nm of a mixed solution of probe DSNP (20 μmol / L) and various concentrations of HS increased linearly with increasing HS concentrations (1-10 μmol / L). Furthermore, the fluorescence intensity showed a linear relationship with HS concentration. The detection limit of probe DSNP for HS was calculated to be only 30.7 nm.

[0084] Cytotoxicity assay

[0085] In order to investigate the cytotoxicity of the probe DSNP, the present invention conducted an MTT test. The steps are as follows: First, T24 cells were placed in a humidified incubator containing 10% fetal bovine serum (FBS, Invitrogen), aerated with 5% CO2, and incubated at 37°C for 24 hours; then, the cells were treated with different concentrations of DSNP (10μM, 20μM, 30μM, 40μM, 50μM) for 24 hours. Next, 25μL of methylthiozolyl tetrazolium (MTT) (5mgmL) was added to each well. -1 ) and incubated for 4 hours, and the cytotoxicity test was performed using MTT, as follows:

[0086] Discard the MTT solution, add 150 μL of DMSO, shake for 15 minutes, and measure the OD value at 490 nm on a microplate reader. The toxicity of MOCP is calculated as follows.

[0087] The cell viability(%)=(AB) / (BC)×100%

[0088] A represents cells incubated with different concentrations of probes, B represents cells without probes, and C represents wells containing culture medium only.

[0089] Since DSNP is highly selective for H2S, its application in intracellular H2S imaging can be explored. Before performing cell imaging, the MTT assay is essential to evaluate the cytotoxicity of DSNP.

[0090] The experimental results are as follows Figure 8 As shown, under the experimental conditions (20 μM), more than 85% of the cells were viable, indicating the low cytotoxicity of DSNP. Therefore, it can be inferred that DSNP can be used for intracellular imaging in living cells.

[0091] Cell imaging

[0092] 1. Experimental methods

[0093] T24 cells (Wuhan Sean Biotechnology Co., Ltd.) were cultured in glass culture dishes and incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24-36 h.

[0094] The experimental group was pretreated with H2S (10 μM) and then incubated with DSNP (10 μM) for 1 h.

[0095] The control group was directly incubated with DSNP (10 μM) for 1 hour. After incubation, the cells were washed three times with PBS and then imaged using an inverted fluorescence microscope.

[0096] 2. Experimental results

[0097] The experimental results are as follows Figure 9 As shown, the cells in the experimental group showed yellow fluorescence in the dark field, while the cells in the control group showed blue fluorescence in the dark field. Therefore, it can be judged that the probe DSNP can be applied to cell imaging.

[0098] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A fluorescent probe for specific detection of H2S, characterized in that: The chemical structural formula of the fluorescent probe is:

2. A method for synthesizing a fluorescent probe for specific detection of H2S according to claim 1, characterized in that: The raw materials of the fluorescent probe include a first raw material I and a second raw material II. The first raw material I reacts with 6-hydroxy-2-naphthaldehyde to obtain the second raw material II. The structural formula of the first raw material I is The structural formula of the second raw material II is 3. The synthesis method according to claim 2, characterized in that The following steps are involved: S1. Synthesis of the first raw material I: 0.5-2 mmol 4-methylpyridine, 1-3 mmol iodomethane and 3-6 mL acetonitrile were added to a flask, reacted at 77-86°C for 11-14 hours, cooled to room temperature, recrystallized with ethanol, filtered under reduced pressure, washed the filter residue with ethanol, and dried to obtain the product, the first raw material I: 1,4-dimethylpyridine iodide; S2. Synthesis of the second raw material II: 0.7-2 mmol of the first raw material I, 1-3.5 mmol of 6-hydroxy-2-naphthaldehyde, 0.3-0.8 mL of piperidine catalyst, and 4-8 mL of methanol were reacted at 45-55°C for 1-1.5 hours; after cooling to room temperature, the mixture was extracted with water and distilled under reduced pressure to obtain the product, the second raw material II: 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridine iodide (II); S3. Synthesis of fluorescent probe III: Under N2 environment, 0.5-1.8mmol of the second raw material II, 0.5-3.2mmol of 2,4-difluorobenzenesulfonyl chloride, 0.5-3mL of N,N-dimethylformamide, 0.5-3mL of triethylamine and 3-7mL of dichloromethane were mixed and stirred at room temperature for 2-6h. After evaporating the solvent using a rotary evaporator, silica gel column chromatography was used to obtain a light yellow powdery solid, which is the fluorescent probe (Z)-4-(2-(6-(((2,4-difluorophenyl)sulfonyl)oxy)naphthalen-2-yl)vinyl)-1-methylpyridine iodide (III).

4. Use of the fluorescent probe for specific detection of H2S according to claim 1 in the detection of H2S for non-disease diagnosis and treatment purposes.

5. The use according to claim 4, characterized in that The detection limit of the fluorescent probe for H2S is 30.7 nm, and the linear relationship between fluorescence intensity and H2S concentration is y=40.812x+72.512, R 2 =0.9945.

6. The use according to claim 4, characterized in that The excitation wavelength required for detection is in the range of 200-400 nm, and the fluorescence emission wavelength is 568 nm.

7. The use according to any one of claims 4 to 6, characterized in that The fluorescent probe is used for detecting H2S in cells.

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