Fluorescent probe for specifically detecting H2S and synthesis method

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly discloses a fluorescent probe for specific detection of H2S. The invention also discloses a synthesis method of the fluorescent probe. The fluorescent probe disclosed by the invention has the advantages of being simple in design, easy to synthesize, high in fluorescence intensity, good in selectivity, high in sensitivity, low in detection limit and the like, can be applied to endogenous H2S imaging in cells, has excellent biological application potential, and can become a useful tool for understanding a complex regulation and control system in the cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes and particularly relates to a method for specifically detecting H 2 Fluorescent probe and synthesis method of S. Background Art

[0002] Hydrogen sulfide (H 2 H) is considered to be a toxic gas with a foul odor. Excessive inhalation may cause cardiac dysfunction, myocardial damage, and even life-threatening. However, its signal transduction ability and endogenous H 2 In terms of the biodistribution of S-generating enzymes, H 2 S is a gaseous transmitter similar to nitric oxide (NO) and carbon monoxide (CO), and is involved in important physiological and pathological processes. 2 Sulfur affects a variety of physiological functions in the human body and is involved in the regulation of a variety of diseases such as neurodegenerative diseases, cardiovascular diseases, cancer and inflammation. Unfortunately, in biological systems, H 2 Tracking and accurate quantification of S remains difficult, and H 2 The regulatory mechanism of S is also unclear. Therefore, it is necessary to develop a highly selective method for detecting endogenous H 2 S's method.

[0003] With the development and progress of scientific research, a variety of methods for detecting H 2 S method, such as methylene blue method, H 2 S ion selective electrode method, gas chromatography, etc. However, these methods have certain defects. For example, the detection sensitivity of the methylene blue method is relatively low and is not suitable for detecting trace substances (in the micromolar range). Since sulfide can easily escape from acid-unstable containers, its concentration may be misestimated. 2 The S ion selective electrode method takes a long time, and the gas chromatography method also requires the airtightness of the equipment. In order to more sensitively and conveniently detect H in complex biological systems 2 S, small molecule fluorescent probes have come into the sight of researchers, and have attracted widespread attention due to their advantages such as low cost, high biocompatibility, sensitive response, easy synthesis and high selectivity. Summary of the invention

[0004] The present invention aims to provide a method for specifically detecting H 2 A fluorescent probe and a synthesis method of S are provided to solve the above technical problems.

[0005] One aspect of the present invention relates to a method for specifically detecting H 2 The fluorescent probe of S, the chemical structural formula 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-methylpyridinium salt.

[0008] The principle of the fluorescent probe DSNP provided by the present invention is as follows: the probe uses 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridinium salt (Hnmp-1) as a fluorescent group and 2,4-difluorobenzenesulfonyl as a H 2 The S reaction site produces fluorescence at a wavelength of 568 nm by breaking the sulfonyl ester bond, which makes H 2 S reacts 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 and 6-hydroxy-2-naphthaldehyde are reacted 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] 0.5-2mmol 4-methylpyridine, 1-3mmol iodomethane and 3-6mL acetonitrile were added into a flask, reacted at 77-86°C for 11-14h, cooled to room temperature, recrystallized with ethanol, filtered under reduced pressure, washed the filter residue with ethanol, and dried to obtain the product first raw material I: 1,4-dimethylpyridine;

[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, water was used for extraction, and the product second raw material II was obtained by vacuum distillation: 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridine (II);

[0017] Synthetic fluorescent probe III:

[0018] In N2 Under the 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, and the solvent was evaporated by a rotary evaporator, and then a light yellow powdery solid was obtained by silica gel column chromatography, which is the fluorescent probe (Z)-4-(2-(6-(((2,4-difluorophenyl)sulfonyl)oxy)naphthalene-2-yl)vinyl)-1-methylpyridine salt (III).

[0019] The third aspect of the present invention relates to the method for specifically detecting H 2 Fluorescent probe of S in H 2 Application in S detection.

[0020] Preferably, the fluorescent probe is 2 The detection limit of S is 30.7 nm. The fluorescence intensity is related to H 2 The linear relationship between S concentration and R is y=40.812x+72.512. 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 applied to H 2 S testing is not for disease diagnosis or treatment purposes.

[0023] The present invention provides a novel mitochondrial targeted fluorescent probe, which can detect H 2 S. This probe can be applied to H 2 The fluorescent probe DSNP has the characteristics of simple design, easy synthesis, good selectivity, low toxicity and good biocompatibility. 2 S provides a useful targeted fluorescent probe, offering new perspectives and tools for the study of cellular microenvironment, and has important value in environmental monitoring and biomedical research.

[0024] The fluorescent probe provided by the present invention uses 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridinium salt (Hnmp-1) as a fluorescent group and 2,4-difluorobenzenesulfonyl as a H 2 The S reaction site generates fluorescence at a wavelength of 568 nm through the cleavage of 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 applied to endogenous H 2S imaging has excellent biological application potential, which can make it a promising target for detecting H 2 S and useful tools to better understand complex regulatory systems within cells.

[0026] The fluorescent probe provided by the present invention is 2 The fluorescence of S shows enhanced characteristics after the efficient reaction. 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 new method for detecting H 2 S and useful tools to better understand 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 C NMR spectrum.

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

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

[0032] Figure 4 The fluorescence emission spectra of the mixed solutions of DSNP and each target at 568 nm.

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

[0034] Figure 6 The experimental group (DNSP+H 2 S) and the fluorescence intensity of the control group (DSNP).

[0035] Figure 7 DSNP and different concentrations (0-10 μM) of H 2 The fluorescence emission wavelength of S is at 568 nm.

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

[0037] Fig. 9 The experimental group (DSNP+H 2 S) Cell imaging of the control group (DSNP). DETAILED DESCRIPTION

[0038] Example: Synthesis of fluorescent probe DSNP

[0039] The synthesis steps are as follows Figure 1 As shown, the following steps are specifically involved:

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

[0041] 0.5-2mmol 4-methylpyridine (M=93.13, CAS: 108-89-4, Beijing Bailingwei Technology Co., Ltd., product number: P815734-100ml), 1-3mmol iodomethane (M=141.95, CAS: 74-88-4, Shanghai Aladdin Biochemical Technology Co., Ltd., product number: I573480-25ml) and 3-6mL acetonitrile were added into a flask, reacted at 77-86°C for 11-14h, cooled to room temperature, recrystallized with ethanol, filtered under reduced pressure, washed the residue with a small amount of ethanol, and dried to obtain the product 1,4-dimethylpyridine (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), acetonitrile is 5 mL, and the reaction conditions are 85° C. for 12 h. The final yield of 1,4-dimethylpyridine (I) is 94%.

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

[0045] 0.7-2mmol 1,4-dimethylpyridine (M=108.16), 1-3.5mmol 6-hydroxy-2-naphthaldehyde (M=108.16, CAS: 78119-82-1, Beijing Bailingwei Technology Co., Ltd., item number: H834750-5g), 0.3-0.8mL piperidine catalyst and 4-8mL methanol were added to a flask and reacted at 45-55°C for 1-1.5h. After cooling to room temperature, water was used for extraction three times, and the product 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridine (II) was obtained by vacuum distillation. The product was a reddish brown powdery solid.

[0046] In a specific embodiment of the present invention, the input amount of 1,4-dimethylpyridine 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. Synthetic fluorescent probe (III)

[0049] In N 2 Under the environment, 0.5-1.8mmol Hnmp-1 (M=262), 0.5-3.2mmol 2,4-difluorobenzenesulfonyl chloride (M=212.6, CAS: 13918-92-8, Beijing Bailingwei Technology Co., Ltd., product number: D829371-25g), 0.5-3mL N, N-dimethylformamide (M=73.09, CAS: 68-12-2, Beijing Bailingwei Technology Co., Ltd., N807505), 0.5-3mL triethylamine (M=101.19, CAS: 121-44-8, Beijing Bailingwei Technology Co., Ltd., product number: T818774) and 3-7mL dichloromethane were mixed and stirred at room temperature for 2-6h. After the solvent was evaporated by rotary evaporator, silica gel column chromatography (200 mesh) was used, and the eluent was CH 2 Cl 2 / MeOH (volume ratio = 50-20:1) 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-methylpyridinium salt (DSNP).

[0050] In a specific embodiment of the present invention, specifically in N 2 Under the environment, 0.2623mg (1mmol) of Hnmp-1, 0.3189g (1.5mmol) of 2,4-difluorobenzenesulfonyl chloride, 0.5mL of N,N-dimethylformamide, 0.5mL of triethylamine and 5mL of dichloromethane were mixed and stirred at room temperature for 3h. 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 mixture was chromatographed on a 200 mesh silica gel column using CH 2 Cl 2 / MeOH (volume ratio = 49:1) to CH 2 Cl 2 / MeOH (volume ratio = 19:1) to obtain a light yellow powder solid (III), which is the fluorescent probe (Z)-4-(2-(6-(((2,4-difluorophenyl)sulfonyl)oxy)naphthalen-2-yl)vinyl)-1-methylpyridinium salt (DSNP), with a yield of 46%. The molar ratio of N,N-dimethylformamide, triethylamine and dichloromethane is 1:1:10.

[0052] Examples 1-5

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

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

[0055]

[0056]

[0057] The specific synthetic route of the fluorescent probe DSNP provided in Example 1 is as follows Figure 1 shown.

[0058] Characterization Figure 2A and Figure 2B As shown:

[0059] 1H 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).

[0060] 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.

[0061] 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%).

[0062] Fluorescence spectroscopy experiment

[0063] 1. Experimental methods

[0064] The aqueous solutions used for fluorescence measurement were all prepared with deionized water. The solution preparation steps were as follows: solid DSNP was prepared with DMSO / PBS (2:8) to a stock solution with a concentration of 2 mmol / L, and then diluted to 20 μmol / L with PBS to obtain a solution for spectral measurement. The target solutions were all diluted to the corresponding concentrations with deionized water. During optical measurement, 0.35 mL of DSNP and 0.35 mL of target solution were mixed and poured into a quartz optical cell each time, and the fluorescence emission spectrum was recorded.

[0065] The DSNP solution with a concentration of 0.02 mmol / L was mixed with 0.2 mmol / L Arg, His, Cys, Na + , Ag + , Cu 2+ , Ca 2+ ,Mg 2+ , NH 4 + , P.O. 4 3- , I- , NO 2- , HCO 3 - , CO 3 2- , SO 4 2- , SO 3 2- , HSO 3 - , NH 3 , HAC and H 2 350 μL of each S solution was mixed and reacted at 60° C. for 1 h, and then the fluorescence emission spectrum of the mixed solution was measured.

[0066] 2. Experimental results

[0067] The selectivity of DSNP fluorescence response to interferents showed that traditional anions, cations and biothiols, including Arg, His, Cys, Na + , Ag + , Cu 2+ , Ca 2+ ,Mg 2+ , NH 4 + , P.O. 4 3- ,,I - , NO 2- , HCO 3 - , CO 3 2- , SO 4 2- , SO 3 2- , HSO 3 - , NH 3 , HAC and H 2 S, the experimental results are as follows Figure 4 As shown, even at a fairly high level (10eqv), no significant interference will occur, while H 2 S can respond to DSNP and emit strong fluorescence at 568nm.

[0068] Time control experiment

[0069] 1. Experimental methods

[0070] A 20 μmol / L DSNP solution was mixed with H 2Take 350 μL of each H S solution and mix them, keep them at 60°C, and measure the fluorescence emission spectra of the mixed solution at 30s, 1min, 2min, 5min, 10min, 30min, 1h, 1.5h, and 2h. 2 S solution and fluorescence emission spectra at 30s, 1min, 2min, 5min, 10min, 30min, 1h, 1.5h, and 2h.

[0071] 2. Experimental results

[0072] like Figure 5 As shown, with the increase of reaction time, the fluorescence intensity of the solution at 568 nm gradually increased and reached a stable state after 45 min.

[0073] pH control experiment

[0074] 1. Experimental methods

[0075] 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.

[0076] Experimental group: DSNP solution with a concentration of 20 μmol / L and H 2 Take 350 μL of each S solution and mix them.

[0077] 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.

[0078] 2. Experimental results

[0079] The experimental results are as follows Figure 6 As shown in the figure, the fluorescence emission spectra under different pH environments show that: DSNP-H 2 The fluorescence intensity of the S mixed solution at 568 nm is low in an acidic environment, but it can emit strong fluorescence in a neutral and alkaline environment. The pH of the human body environment is about 7.4, so the probe is suitable for physiological pH.

[0080] Concentration gradient experiment

[0081] 1. Experimental methods

[0082] Prepare H with a concentration of 0-10 μmol / L 2 S solution, take DSNP solution with a concentration of 20 μmol / L and mix it with H 2350 μL of S solution was mixed evenly, reacted at 60° C. for 1 h, and then the fluorescence emission spectrum of the mixed solution was measured.

[0083] 2. Experimental results

[0084] The experimental results are as follows Figure 7 As shown, with H 2 With the increase of S concentration (1-10 μmol / L), the probe DSNP (20 μmol / L) and various concentrations of H 2 The fluorescence intensity of the H-S mixed solution at 568 nm increased linearly. 2 The probe DSNP has a linear relationship with H 2 The detection limit of S is only 30.7 nm.

[0085] Cytotoxicity assay

[0086] 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) and 5% CO 2 gas and incubated at 37°C for 24 h; then, the cells were treated with different concentrations of DSNP (10 μM, 20 μM, 30 μM, 40 μM, 50 μM) for 24 h. Next, 25 μL of methylthiozolyl tetrazolium (MTT) (5 mg mL -1 ) and incubated for 4 hours, and the cytotoxicity test was performed using MTT, the specific steps are as follows:

[0087] The MTT solution was discarded, 150 μL DMSO was added, and the mixture was shaken for 15 min. The OD value was measured at 490 nm using a microplate reader. The toxicity of MOCP was calculated as follows.

[0088] The cell viability(%)=(AB) / (BC)×100%

[0089] A represents cells incubated with different concentrations of probe, B represents cells without probe, and C represents wells containing culture medium only.

[0090] Since DSNP has a 2 S is highly selective, so its intracellular H 2 Before cell imaging, the MTT assay is essential to evaluate the cytotoxicity of DSNPs.

[0091] The experimental results are as follows Figure 8As shown, under the experimental conditions (20 μM), more than 85% of the cells were viable, indicating the low cytotoxicity of DSNPs, and thus it can be inferred that DSNPs can be used for imaging in living cells.

[0092] Cell imaging

[0093] 1. Experimental methods

[0094] T24 cells (Wuhan Sean Biotechnology Co., Ltd.) were cultured in glass culture dishes and incubated at 37°C with 5% CO 2 Culture in a humid atmosphere for 24-36h.

[0095] The experimental group used H 2 S (10 μM) pretreatment, and then incubated with DSNP (10 μM) for 1 h.

[0096] The control group was directly incubated with DSNP (10 μM) for 1 hour. After incubation, the cells were washed 3 times with PBS and then the experimental group (DSNP+H 2 S) and control group (DSNP) cell imaging.

[0097] 2. Experimental results

[0098] The experimental results are as follows Fig. 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.

[0099] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined 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 as claimed in 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 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 first raw material I reacts with 6-hydroxy-2-naphthaldehyde to obtain the second raw material II.

4. The synthesis method according to claim 3, characterized in that The following steps are involved: S1. Synthesis of the first raw material I: 0.5-2mmol 4-methylpyridine, 1-3mmol iodomethane and 3-6mL acetonitrile were added into a flask, reacted at 77-86°C for 11-14h, cooled to room temperature, recrystallized with ethanol, filtered under reduced pressure, washed the filter residue with ethanol, and dried to obtain the product first raw material I: 1,4-dimethylpyridine; 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, water was used for extraction, and the product second raw material II was obtained by vacuum distillation: 4-(2-(6-hydroxy-2-naphthyl)ethyl)1-methylpyridine (II); S3. Synthetic 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, a light yellow powdery solid was obtained by silica gel column chromatography, which is the fluorescent probe (Z)-4-(2-(6-(((2,4-difluorophenyl)sulfonyl)oxy)naphthalene-2-yl)vinyl)-1-methylpyridine salt (III).

5. Use of the fluorescent probe for specific detection of H2S according to claim 1 in H2S detection.

6. The use according to claim 5, characterized in that: 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.

7. The use according to claim 5, characterized in that: The excitation wavelength required for detection is in the range of 200-400nm, and the fluorescence emission wavelength is 568nm.

8. The use according to any one of claims 5 to 7, characterized in that: The fluorescent probe is used for H2S detection in cells, non-disease diagnosis and treatment purposes.

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