Flavonoid fluorescent probe with large Stokes displacement for detecting biological mercaptan

Abstract

The invention relates to a flavonoid fluorescent probe with large Stokes displacement for detecting biological mercaptan, and belongs to the technical field of chemical analysis and detection. The probe molecule provided by the invention has no fluorescence, and after the probe molecule reacts with biological mercaptan, obvious fluorescence appears at 540nm. The probe molecule has good linearity with biological mercaptan in a certain concentration range, has strong recognition capability on biological mercaptan, can detect Cys, Hcy, GSH and H2S in a distinguished manner in the aspects of fluorescence intensity and reaction speed under naked eyes, has large Stokes displacement (greater than 100 nm), has strong selectivity and anti-interference capability, has good performance in a wide pH range, and can be applied to detection of Cys, Hcy, GSH and H2S. Therefore, the probe has important application value in the fields of biochemistry and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis and detection, and specifically relates to a preparation method based on flavonoid fluorescent dyes and application of the method in detecting biological thiols. Background Art

[0002] Amino acids with functional sulfhydryl groups in organisms are called biothiols, including the following: cysteine ​​(Cys), homocysteine ​​(Hcy), glutathione (GSH), N-acetylcysteine, γ-glutamylcysteine, coenzyme A and mercaptoethylamine. Biothiols have strong redox properties and nucleophilicity, and can act as antioxidants to protect cells and tissues from oxidation by endogenous reactive oxygen species (ROS) and free radicals.

[0003] Biothiols play an important role in maintaining normal physiological activities of the human body and are related to many diseases. For example, abnormal Cys content can cause diseases such as hair loss, slow development, edema, liver damage, cardiovascular damage and skin damage; excessive Hcy content in the body can cause damage to cardiovascular and bone tissues; GSH is the most abundant biothiol in human cells and is closely related to diseases such as aging, inflammation and tumors. Therefore, accurate and efficient detection of biothiols and data analysis are conducive to regulating physiological activities and detecting and preventing related diseases. At present, the detection methods of biothiols include mass spectrometry, high performance liquid chromatography, gas chromatography, Fourier transform infrared spectroscopy, enzyme method, electrochemical analysis and ultraviolet / visible spectroscopy. Compared with the above methods, fluorescence analysis has the advantages of convenient operation, rapidity, good sensitivity and high selectivity. Fluorescent probes can not only detect in real time and efficiently, but also have the advantages of high sensitivity, high specificity, low detection limit, in situ detection and direct application to cells and living bodies, which has attracted extensive attention and research from researchers.

[0004] Flavonoid fluorescent probes are widely used to detect biothiols, but how to find a flavonoid fluorescent probe with good selectivity and strong anti-interference ability is a unanimous pursuit of those skilled in the art. Summary of the invention

[0005] One object of the present invention is to provide a simple and efficient method for synthesizing a fluorescent probe; another object of the present invention is to provide a fluorescent probe with good selectivity, strong anti-interference ability, and capable of distinguishing and detecting biological thiols.

[0006] The technical solution adopted by the present invention to solve the problem is a new type of fluorescent probe for distinguishing and detecting biothiols by fluorescence method, and its molecular structure is as follows:

[0007] The preparation method comprises the following steps:

[0008] S1: dissolving 2-(2-bromophenyl)-3-hydroxyflavone and 4-chloro-7-nitrophenylfuroxan in acetonitrile solution;

[0009] S2: under a protective atmosphere, add triethylamine to the solution and react at 40-60°C;

[0010] S3: After the reaction is complete, cool to room temperature;

[0011] S4: filtering the solution after the reaction to obtain a filter cake, washing the filter cake with anhydrous ethanol, and then purifying it by column chromatography to obtain the flavonoid compound.

[0012] Preferably, in the preparation method of the present invention, the eluent used in the column chromatography purification is a mixture of dichloromethane and methanol.

[0013] Preferably, in the preparation method of the present invention, the volume ratio of dichloromethane to methanol in the mixture is 1:10-20.

[0014] Preferably, in the preparation method of the present invention, TLC is used in step S3 to track and detect whether the reaction is complete.

[0015] Preferably, in the preparation method of the present invention, the molar ratio of 2-(2-bromophenyl)-3-hydroxyflavone to 4-chloro-7-nitrophenylfuroxan is 1:1-1.5.

[0016] Preferably, in the preparation method of the present invention, the concentration of the triethylamine solution is 3-5 mM.

[0017] Preferably, in the preparation method of the present invention, the protective atmosphere is an oxygen-free environment, a nitrogen atmosphere or an inert gas atmosphere.

[0018] The mechanism of action of the fluorescent probe of the present invention is as follows: the fluorescence of the probe molecule itself is in a quenched state. The biothiol can replace the phenyl ether part in the probe molecule through a nucleophilic substitution reaction, hydrolyze to obtain a phenolic hydroxyl group, and generate the original fluorescent dye compound. Thus, the purpose of specifically distinguishing and detecting biothiols (Cys, Hcy and GSH) is achieved. The response process of the probe molecule is as follows: Fig.14 shown.

[0019] The fluorescent probe of the present invention has good selectivity. The test system of the probe molecule is a 10mM PBS buffer solution with a pH of 7.4 containing different volumes of dimethyl sulfoxide, and the measurement is performed at room temperature. After adding 10 times the equivalent of biological thiols (Cys, Hcy and GSH), the fluorescence intensity of the probe molecule changes at a maximum emission wavelength of about 550nm. When some anions, cations or thiol amino acids (Cys, Hcy, GSH, H2S, Tyr, Ile, Glu, His, Ala, Thr, Ieu, Lys, Try, Asp, Val, Gln, Asn, Pro, Met, Phe, Na + , K + , Ca 2+ 、Zn 2+ Mg 2+ , Fe 2+ , Cl - , ClO - 、SCN - 、NO2 - 、HCO3 - 、H2PO4 - 、S2O3 2- 、SO3 2- 、SO4 2- , glucose and H2O2), the change of fluorescence intensity was negligible.

[0020] The fluorescent probe of the present invention has strong anti-interference ability, and other anions, cations or amino acids (Cys, Hcy, GSH, H2S, Tyr, Ile, Glu, His, Ala, Thr, Ieu, Lys, Try, Asp, Val, Gln, Asn, Pro, Met, Phe, Na + , K + , Ca 2+ 、Zn 2 + Mg 2+ , Fe 2+ , Cl - , ClO - 、SCN - 、NO2 - 、HCO3 - 、H2PO4 - 、S2O3 2- 、SO3 2- 、SO4 2- , glucose and H2O2) does not affect the interaction between the probe molecule and the biothiol.

[0021] The probe molecule of the present invention has no obvious fluorescence intensity change under different pH, but has a relatively obvious fluorescence intensity change after adding biological thiols (Cys, Hcy and GSH) and has a wide application range. The fluorescent probe has practical application value in the fields of biology and chemistry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The synthetic route of the fluorescent probe of the present invention is shown in FIG.

[0023] Figure 2a and 2b The selectivity effect diagrams of the fluorescent probe of the present invention are shown in Figure 1. -6 mol / L) in PBS (10 mM, pH=7.4) / acetonitrile 40% solution, after reacting with biological thiols (Cys, Hcy and GSH) and hydrogen sulfide, the horizontal axis is the wavelength and the vertical axis is the fluorescence intensity.

[0024] Figure 3 The fluorescent probe (5.0×10 -6 Figure 2. Images of 504 nm and 504 nm after the reaction of 247 nm (40 mol / L) with biological thiols (Cys, Hcy and GSH) and hydrogen sulfide in PBS (10 mM, pH = 7.4) / acetonitrile 40% solution.

[0025] Figure 4 The fluorescent probe (5.0×10 -6 mol / L) in different solvents (MeOH, EtOH, ACN, DMSO, DMF) after reacting with 10 equivalents of biothiols (Cys, Hcy and GSH). The horizontal axis is the solvent type and the vertical axis is the fluorescence intensity ratio.

[0026] Figure 5 The fluorescent probe (5.0×10 -6 mol / L) in PBS (10 mM, pH=7.4) / different volumes of acetonitrile, after reacting with Cys, the horizontal axis is the solvent ratio, and the vertical axis is the fluorescence intensity.

[0027] Figure 6 The fluorescent probe (5.0×10 -6 mol / L) in PBS (10 mM, pH = 7.4) / acetonitrile 40%, and the changes in the fluorescence intensity spectrum at different pH values ​​before and after the reaction with biological thiols (Cys, Hcy and GSH). The abscissa is the wavelength and the ordinate is the fluorescence intensity.

[0028] Figure 7To demonstrate the selectivity of the fluorescent probe of the present invention, the fluorescent probe (5.0×10 -6 mol / L) in PBS (10 mM, pH = 7.4) acetonitrile 40% after the reaction with biological thiols (Cys, Hcy and GSH).

[0029] Figure 8 The anti-interference ability of the fluorescent probe of the present invention is that when biological thiols (Cys, Hcy and GSH) coexist with other anions and cations, they interact with the fluorescent probe (5.0×10 -6 mol / L) in PBS (10 mM, pH = 7.4) and 40% acetonitrile after the reaction with biological thiols (Cys, Hcy and GSH).

[0030] Figure 9a The fluorescent probe (5.0×10 -6 mol / L) in PBS (10 mM, pH = 7.4) / acetonitrile 40%, and the fluorescence spectra after responding to different concentrations of Cys;

[0031] Figure 9b It is a curve diagram of the change of fluorescence intensity of the probe and different concentrations of Cys. The inset is a linear relationship diagram when the Cys concentration is 0-50 μM. The horizontal axis is the wavelength and the vertical axis is the fluorescence intensity.

[0032] Fig.10a The fluorescent probe (5.0×10 -6 mol / L) in PBS (10 mM, pH = 7.4) / acetonitrile 40%, and the fluorescence spectra after responding to different concentrations of Hcy;

[0033] Fig.10b It is a curve diagram of the change of fluorescence intensity of the probe and different concentrations of Cys. The inset is a linear relationship diagram when the Hcy concentration is 0-50 μM. The horizontal axis is the wavelength and the vertical axis is the fluorescence intensity.

[0034] Fig.11a The fluorescent probe (5.0×10 -6 mol / L) in PBS (10 mM, pH = 7.4) / acetonitrile 40%, and the fluorescence spectra after responding to different concentrations of GSH;

[0035] Fig.11b It is a curve diagram of the change of fluorescence intensity of the probe and different concentrations of Cys. The inset is a linear relationship diagram when the GSH concentration is 0-50 μM. The horizontal axis is the wavelength and the vertical axis is the fluorescence intensity.

[0036] Fig.12 The fluorescent probe (5.0×10 -6mol / L) to biological thiols (Cys, Hcy and GSH) and hydrogen sulfide, the abscissa is time and the ordinate is fluorescence intensity.

[0037] Fig.13 It is the structural formula of Cys, Hcy and GSH.

[0038] Fig.14 is the response process of the probe molecule.

[0039] Specific implementation examples

[0040] In Example 1 of the present application

[0041] Synthesis of compound 1

[0042] Weigh 1.36g (10mmol) of o-hydroxyacetophenone, dissolve it in 15mL of anhydrous ethanol, add it to a 50mL round-bottom flask, add 0.28g of potassium hydroxide, and stir for 40min under nitrogen protection and ice bath conditions. Dissolve 1.24g (10mmol) of 2-bromobenzaldehyde in 15mL of anhydrous ethanol and add it to a round-bottom flask. React at room temperature for 2h, place it in a water bath and heat it to 40℃ for 12h, and monitor it by TLC tracking. After the reaction is completed, add 10% hydrochloric acid (20mL) to it, and a large amount of yellow solid material precipitates. Filter it to obtain a yellow solid, recrystallize it with anhydrous ethanol, and then place the product in a vacuum drying oven to dry for 2h, and finally obtain 1.987g of yellow solid, with a yield of 81.8%.

[0043] Synthesis of 2-(2-bromophenyl)-3-hydroxyflavone (Compound 2)

[0044] 3.03g (10mmol) of compound 1 was added to 25mL of anhydrous ethanol to dissolve, and then 0.28g of potassium hydroxide was weighed and all added to a 100mL round-bottom flask. 30mL of H2O2 was added dropwise with a constant pressure dropping funnel in 1h under ice bath conditions while stirring, and then reacted at 40°C for 8h, and TLC tracking monitoring was performed. After the reaction was completed, the system was added to ice water, and the pH was adjusted to about 8 with 10% hydrochloric acid, and a gray solid was precipitated, filtered, and the crude product was column chromatographed (petroleum ether: ethyl acetate = 20: 1, v: v) and the product was spin-dried. The dried product was added to a 5mL round-bottom flask, 2mL of petroleum ether was measured for pulping for 30min, repeated 3 times, the product was filtered, and dried to obtain a light gray solid 2.041g (2-(2-bromophenyl)-3-hydroxyflavone), with a yield of 58.6%. The results of spectral detection were: 1H NMR (400MHz, CDCl3) δ8.49(s,2H),8.47(s,2H),7.67(d,J=1.1Hz,2H),7.66(d,J=1.1Hz,2H),3.25(s,1H). 13 C NMR (101MHz, CDCl3) δ149.50,142.91,135.93,131.33,130.47,130.44,128.54,126.10,46.52.

[0045] Embodiment 1:

[0046] This embodiment provides a method for preparing a probe molecule-flavonoid compound:

[0047] Weigh 1.58g (5mmol) of 2-(2-bromophenyl)-3-hydroxyflavone and 995mg (5mmol) of NBD-Cl (4-chloro-7-nitrophenylfurazan), dissolve in 20mL of anhydrous acetonitrile, and add to a 50mL round-bottom flask. Add triethylamine to the reaction mixture to make the concentration of triethylamine reach 4mM, then heat to 45°C in a water bath for 10h, track and monitor by TLC, and cool to room temperature after the reaction is complete. After filtration, the obtained solid was washed three times with 3×10mL of anhydrous ethanol to obtain a black solid, which was then purified by column chromatography (methanol: dichloromethane = 15:1, v:v), and dried to obtain 1.368g of brown-black solid (flavonoid compound), with a yield of 57.1%. The results of spectral detection are: 1 H NMR (400MHz, CDCl3) δ8.30 (d, J=8.8Hz, 1H), 7.73 (dd, J=20.5, 8.2Hz, 2H), 7.64 (d, J=7. 6Hz,1H),7.54(d,J=8.4Hz,1H),7.51–7.43(m,2H),7.38(t,J=7.7Hz,1H),6.58(s,1H). 13 C NMR (101MHz, CDCl3) δ173.53,155.90,146.21,138.66,133.80,133.58,131.78,131.71,131.62,127.35,125.61,124.69,123.07,121.34,118.53.

[0050] Embodiment 2:

[0051] This embodiment provides a method for preparing a probe molecule-flavonoid compound:

[0052] Weigh 1.58g (5mmol) of 2-(2-bromophenyl)-3-hydroxyflavone and 1194mg (6mmol) of NBD-Cl (4-chloro-7-nitrophenylfurazan), dissolve in 22mL of anhydrous acetonitrile, and add to a 50mL round-bottom flask. Add triethylamine to the reaction mixture to make the concentration of triethylamine reach 3mM, then heat to 40°C in a water bath for 10h, track and monitor by TLC, and cool to room temperature after the reaction is complete. After filtration, the obtained solid is washed three times with 3×10mL of anhydrous ethanol to obtain a black solid, which is then purified by column chromatography (methanol: dichloromethane = 10:1, v:v), and spin-dried to obtain a brown-black solid-flavonoid compound with a yield of 55.8%.

[0053] Embodiment 3:

[0054] This embodiment provides a method for preparing a probe molecule-flavonoid compound:

[0055] Weigh 1.58g (5mmol) of 2-(2-bromophenyl)-3-hydroxyflavone and 1492.5mg (7.5mmol) of NBD-Cl (4-chloro-7-nitrophenylfurazan), dissolve in 25mL of anhydrous acetonitrile, and add to a 50mL round-bottom flask. Add triethylamine to the reaction mixture to make the concentration of triethylamine reach 5mM, then heat to 60°C in a water bath for 10h, track and monitor by TLC, and cool to room temperature after the reaction is complete. After suction filtration, the obtained solid is washed three times with 3×10mL of anhydrous ethanol to obtain a black solid, which is then purified by column chromatography (methanol: dichloromethane = 20:1, v:v), and spin-dried to obtain a brown-black solid-flavonoid compound with a yield of 54.2%.

[0056] Effect Example

[0057] This embodiment is the application of fluorescent probe.

[0058] The probe molecule prepared in Example 1 was dissolved in PBS (10 mM, pH = 7.4) / acetonitrile 40% and prepared into 5.0 × 10 -6 mol / L solution, the fluorescence spectrum changes when biothiols (Cys, Hcy, GSH) and H2S are added to the solution, but the fluorescence spectrum changes when other anions and cations (Tyr, Ile, Glu, His, Ala, Thr, Ieu, Lys, Try, Asp, Val, Gln, Asn, Pro, Met, Phe, Na + , K + , Ca 2+ 、Zn 2+ Mg 2+ , Fe 2+ , Cl - , ClO - 、SCN -、NO2 - 、HCO3 - 、H2PO4 - 、S2O3 2- 、SO3 2- 、SO4 2- , glucose and H2O2) did not cause obvious changes in fluorescence. When biothiols (Cys, Hcy and GSH) and interfering substances (H2S, Tyr, Ile, Glu, His, Ala, Thr, Ieu, Lys, Try, Asp, Val, Gln, Asn, Pro, Met, Phe, Na + , K + , Ca 2+ 、Zn 2 + Mg 2+ , Fe 2+ , Cl - , ClO - 、SCN - 、NO2 - 、HCO3 - 、H2PO4 - 、S2O3 2- 、SO3 2- 、SO4 2- When the probe coexists with 2,000 mmol / l, glucose and H2O2, it is not affected by interference factors and shows strong anti-interference ability. The probe molecule has a good linear relationship with the biological thiol (Cys, Hcy and GSH) response within a certain time and concentration range. This shows that the probe has a good application prospect in vivo.

[0059] Based on the above ideal embodiments of this application, the relevant staff can make various changes and modifications without departing from the technical concept of this application through the above description. The technical scope of this application is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A fluorescent probe for identifying biothiols, characterized in that: The structure of the fluorescent probe is:

2. A method for preparing flavonoid compounds, characterized in that: The structural formula of flavonoids is: The preparation method comprises the following steps: S1: dissolving 2-(2-bromophenyl)-3-hydroxyflavone and 4-chloro-7-nitrophenylfuroxan in acetonitrile solution; S2: under a protective atmosphere, add triethylamine to the solution and react at 40-60°C; S3: After the reaction is complete, cool to room temperature; S4: filtering the solution after the reaction to obtain a filter cake, washing the filter cake with anhydrous ethanol, and then purifying it by column chromatography to obtain the flavonoid compound.

3. The preparation method according to claim 2, characterized in that: The eluent used for column chromatography purification was a mixture of dichloromethane and methanol.

4. The preparation method according to claim 3, characterized in that: The volume ratio of dichloromethane:methanol in the mixture is 1:10-20.

5. The preparation method according to any one of claims 2 to 4, characterized in that: In step S3, TLC was used to monitor the completion of the reaction.

6. The preparation method according to any one of claims 2 to 5, characterized in that: The molar ratio of 2-(2-bromophenyl)-3-hydroxyflavone to 4-chloro-7-nitrophenylfurazan is 1:1-1.

5.

7. The preparation method according to any one of claims 2 to 6, characterized in that: The concentration of the triethylamine solution is 3-5 mM.

8. The preparation method according to any one of claims 2 to 7, characterized in that: The protective atmosphere is an oxygen-free environment.

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