A small molecule fluorescent probe of hydrogen peroxide designed based on quinoline and its preparation method

Abstract

The present invention discloses a quinoline-based small-molecule fluorescent probe for hydrogen peroxide and its preparation method. This invention belongs to the field of fluorescent probe technology. The specific structural formula of the fluorescent probe is shown in Formula (I): #imgabs0# The present invention uses 2-methyl-7-hydroxyquinoline as the fluorescent group and 4-bromomethylphenylboronic acid pinacol ester as the recognition group. The prepared fluorescent probe exhibits high sensitivity and selectivity for H2O2 in solution. The preparation method of this fluorescent probe is simple, the raw materials are readily available, and the resulting product is a solid powder that is easy to store. It has broad application prospects and can be used in large-scale production.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes and relates to a method for preparing a small molecule fluorescent probe of hydrogen peroxide designed based on quinoline; specifically, it relates to the optical properties of a small molecule fluorescent probe of hydrogen peroxide designed based on quinoline. Background Art

[0002] Reactive oxygen species (ROS) refer to active substances containing oxygen. Each ROS has different chemical reactivity and lifespan. Among them, hydrogen peroxide (H2O2) is one of the more active ROS, which has attracted much attention due to its relatively mild reactivity and long lifespan. According to relevant research, hydrogen peroxide in the body is closely related to cell signal transduction. Abnormal hydrogen peroxide concentrations in living cells may cause a series of organ dysfunctions, which will further aggravate the oxidative damage of nucleic acids and changes in protein structure, and ultimately lead to human aging and various age-related diseases such as cardiovascular disease, neurodegenerative diseases, diabetes and cancer.

[0003] Among the many existing detection methods, fluorescent probes offer unparalleled advantages due to their rapid response and high sensitivity. Near-infrared (NIR) fluorescent probes are particularly popular for in vivo imaging due to their deep tissue penetration and excellent signal-to-visibility ratio. Therefore, developing a low-toxic, highly sensitive, and highly selective hydrogen peroxide probe is of paramount importance. Summary of the Invention

[0004] In response to the above problems, the present invention provides a small molecule fluorescent probe of hydrogen peroxide designed based on quinoline. The fluorescent probe compound specifically recognizes hydrogen peroxide and performs ratiometric detection with high sensitivity.

[0005] The present invention also provides a preparation method and performance of a hydrogen peroxide small molecule fluorescent probe designed based on quinoline.

[0006] The technical solution of the present invention is: the present invention is a small molecule fluorescent probe of hydrogen peroxide designed based on quinoline, the structural formula of the fluorescent probe is shown in formula (I):

[0007]

[0008] Furthermore, a method for preparing a small molecule fluorescent probe of hydrogen peroxide based on quinoline design is provided, wherein the specific preparation steps are as follows:

[0009] Step (1), the prepared m-aminophenol is subjected to a cyclization reaction to obtain the intermediate (II);

[0010] Step (2): intermediate (II) and prepared 4-bromomethylphenylboronic acid pinacol ester are used as raw materials to react in an organic solvent to finally obtain fluorescent probe (I), i.e., hydrogen peroxide small molecule fluorescent probe.

[0011] Furthermore, in step (1), the specific preparation steps of the intermediate (II) are as follows:

[0012] First, the prepared m-aminophenol is dissolved in hydrochloric acid to prepare a reaction reagent containing m-aminophenol;

[0013] Next, a pre-prepared chloranil n-butanol solution is added to the above-mentioned reaction reagents under stirring at room temperature to prepare a mixed solution. The obtained reaction mixture is heated to reflux while stirring, and then the pre-prepared crotonaldehyde n-butanol solution is slowly added dropwise to the stirred reflux solution for at least 30 minutes. After the addition is complete, the reaction mixture is refluxed for an additional 2-4 hours.

[0014] After the reaction is completed, the reaction solution is cooled to room temperature, and then the organic solvent n-butanol is removed by distillation under reduced pressure. The residue is then dissolved in water and extracted with ether to remove residual organic matter in the residue.

[0015] Finally, the aqueous layer was neutralized with 10% NaOH solution and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous Na2SO4, and the organic solvent was removed under reduced pressure to obtain a crude product. The crude product was then purified by thin layer chromatography to obtain pure intermediate (II).

[0016] Furthermore, the molar ratio of m-aminophenol to hydrochloric acid is: 1:3-4;

[0017] The molar ratio of chloranil to n-butanol in the chloranil n-butanol solution is 1:250-350;

[0018] The molar ratio of crotonaldehyde to n-butanol in the crotonaldehyde n-butanol solution is 1:1-5.

[0019] Furthermore, in step (2), the specific preparation steps of the fluorescent probe (I) are as follows:

[0020] First, the prepared intermediate (II), the prepared 4-bromomethylphenylboronic acid pinacol ester and potassium carbonate are dissolved in acetonitrile in sequence, and the mixture is refluxed and stirred; after the reaction is completed, a reaction solution is obtained;

[0021] Then, the obtained reaction solution was filtered, and the filter residue was washed with acetonitrile and dried in a vacuum drying oven to obtain the fluorescent probe (I).

[0022] Furthermore, the molar ratio of the intermediate (II), 4-bromomethylphenylboronic acid pinacol ester and potassium carbonate is: 1:1.2:1.2.

[0023] Furthermore, a small molecule fluorescent probe of hydrogen peroxide designed based on quinoline is used to detect hydrogen peroxide in solution.

[0024] Specifically, 1. m-Aminophenol is an organic compound with the chemical formula C6H7NO. It is an important fine chemical intermediate and is widely used in the pharmaceutical, dye, pesticide and other industries.

[0025] 2. Tetrachlorobenzoquinone, also known as tetrachloro-p-benzoquinone, tetrachloroquinone, and chloran; molecular formula C6Cl4O2, molecular weight 245.88; precipitated from acetic acid or acetone as golden flaky crystals; precipitated from benzene or toluene or obtained by sublimation as monoclinic yellow columnar crystals; has an unpleasant, persistent odor; and is sublimable. It is insoluble in water and poorly soluble in cold ethanol and cold petroleum ether, but soluble in ether, chloroform, carbon tetrachloride, and carbon disulfide. It dissolves slowly in alkaline solutions and forms a reddish-brown to purple precipitate (potassium tetrachloro-p-benzoquinone) in the presence of air. It can be used as a non-systemic fungicide for sterilizing seeds of grains, peanuts, vegetables, cotton, and beans. It can also be used as a dye intermediate, pesticide raw material, and in the leather industry.

[0026] The beneficial effects of the present invention are as follows: the probe connects quinoline and borate through an ether bond, thereby avoiding the formation of quaternary ammonium salts that leads to difficulty in separation and purification of probe molecules and poor membrane permeability; the obtained product is a solid powder that is easy to store and has good stability; the probe molecule shows a good linear relationship with the concentration of hydrogen peroxide, and R 2 =0.996, with a detection limit as low as 1.447 μmol / L, a value lower than that of most quinoline-based fluorescent probes for hydrogen peroxide. Compared with other related species, including reactive oxygen species, amino acids, and inorganic salts, the probe exhibits excellent selectivity for hydrogen peroxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is a graph showing the ultraviolet absorption spectra of the hydrogen peroxide fluorescent probe prepared in the embodiment of the present invention in response to different concentrations of hydrogen peroxide (H2O2) in 0.1% DMSO PBS buffer (10 mM, pH=7.4);

[0028] Figure 2 Graph showing the fluorescence spectrum response of the hydrogen peroxide fluorescent probe prepared in an embodiment of the present invention to different concentrations of hydrogen peroxide (H2O2) in 0.1% DMSO-containing PBS buffer (10 mM, pH = 7.4);

[0029] Figure 3 This is a response time diagram of the fluorescent probe prepared in an embodiment of the present invention when detecting hydrogen peroxide;

[0030] Figure 4 : This is a fluorescence spectrum response diagram of the hydrogen peroxide fluorescent probe prepared in an embodiment of the present invention at different pH values ​​in a 0.1% DMSO PBS buffer (10 mM, pH = 7.4);

[0031] Figure 5 This is a fluorescence spectrum response diagram of the hydrogen peroxide fluorescent probe prepared in an embodiment of the present invention selectively to related species in 0.1% DMSO PBS buffer (10 mM, pH = 7.4);

[0032] Figure 6 is a mass spectrum MS spectrum of the fluorescent probe prepared in the embodiment of the present invention;

[0033] Figure 7 The nuclear magnetic resonance of the fluorescent probe prepared in the embodiment of the present invention is 1 H-NMR spectrum;

[0034] Figure 8 The nuclear magnetic resonance of the fluorescent probe prepared in the embodiment of the present invention is 13 C-NMR spectrum;

[0035] Figure 9 It is the specific preparation reaction route diagram of the present invention. DETAILED DESCRIPTION

[0036] In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention is further described in detail below with reference to the accompanying drawings:

[0037] The synthetic route of the present invention is as follows:

[0038]

[0039] Among them, the intermediate (II) is 2-methyl-7-hydroxyquinoline, and the compound (I) is the fluorescent probe for detecting hydrogen peroxide of the present invention.

[0040] Furthermore, the preparation process includes:

[0041] (1) Preparation of intermediate (II):

[0042] Dissolve m-aminophenol in 12N hydrochloric acid to prepare a reaction reagent containing m-aminophenol;

[0043] Then, the tetrachlorobenzoquinone n-butanol solution is added to the reagent in sequence. After the addition is completed, the mixture is refluxed at 105° C. Subsequently, the pre-prepared crotonaldehyde n-butanol solution is slowly added dropwise to the stirred reflux solution at this temperature. After the addition is completed, the mixture is refluxed again. After the reaction is completed, the organic solvent n-butanol is removed by vacuum rotary evaporation, and the residue is dissolved in water and washed with ether. The aqueous layer is neutralized with 10% NaOH solution, extracted with ethyl acetate, and purified by thin layer chromatography to obtain intermediate (II).

[0044] (2) Preparation of fluorescent probe (I), namely, quinoline-based hydrogen peroxide fluorescent probe compound I:

[0045] The prepared intermediate (II), the prepared 4-bromomethylphenylboronic acid pinacol ester and potassium carbonate are dissolved in the organic solvent acetonitrile in sequence and refluxed with stirring at 80°C; after the reaction is completed, the reaction liquid is filtered, the filter residue is washed with acetonitrile, and then placed in a vacuum drying oven for drying to obtain the prepared fluorescent probe (I), i.e., quinoline-based hydrogen peroxide fluorescent probe compound I.

[0046] The invention discloses an application of a quinoline-based small molecule fluorescent probe for hydrogen peroxide in detecting hydrogen peroxide in a solution.

[0047] Unless otherwise specified, the experimental methods used in this invention are conventional methods. The materials and reagents used in the experiments, unless otherwise specified, can be obtained from commercial sources. All the following reagents used in the examples were commercially available of analytical or chemical purity.

[0048] Example 1

[0049] A method for preparing a small molecule fluorescent probe of hydrogen peroxide designed based on quinoline, wherein the preparation method includes:

[0050] (1) Preparation of intermediate (II) 2-methyl-7-hydroxyquinoline

[0051] First, m-aminophenol (775.0 mg, 7.1 mmol) was dissolved in 12N HCl, and then chloranil (1.81 g, 7.1 mmol) and n-butanol (2.5 mL) were added to the reaction mixture in sequence. After the addition was completed, the reaction mixture was refluxed at 105° C.; then, at this temperature, a mixture of crotonaldehyde (0.2 mL) and n-butanol (0.2 mL) was added dropwise to the reaction mixture over 20 minutes, and after the addition was completed, the mixture was refluxed for 30 minutes; after the reaction was completed, the n-butanol in the reaction mixture was removed by vacuum rotary evaporation, and the residue was dissolved in water and washed with ether. The aqueous layer was neutralized with 10% NaOH solution and extracted with ethyl acetate (50 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by thin layer chromatography (V EA :V PE=3:2), spin-dried, and dried in vacuo to give 2-methyl-7-hydroxyquinoline (31.8 mg, yield 20%).

[0052] The structural formula of the intermediate (II) obtained is:

[0053]

[0054] (2) Preparation of hydrogen peroxide fluorescent probe compounds based on quinoline design:

[0055] 2-Methyl-7-hydroxyquinoline (159.2 mg, 0.2 mmol), 4-bromomethylphenylboronic acid pinacol ester (71.3 mg, 0.24 mmol) and potassium carbonate (33.2 mg, 0.24 mmol) were dissolved in acetonitrile in sequence and refluxed at 80°C for 12 h. After the reaction was completed, the reaction solution was filtered, and the residue was washed with acetonitrile (5 mL × 2) and dried in a vacuum drying oven to obtain a quinoline-based hydrogen peroxide fluorescent probe compound I as a white powder (225.2 mg, yield 60%).

[0056] The obtained fluorescent probe compound has the structural formula:

[0057]

[0058] Hydrogen peroxide fluorescent probe compound prepared by the present invention 1 H NMR(400MHz,Chloroform-d)δ7.94(d,J=8.3Hz,1H),7.84(d,J=8.1Hz,2H),7.64(d,J=8.9Hz,1H),7.48(d,J=8.2Hz,2 H),7.42(d,J=2.5Hz,1H),7.21(dd,J=8.9,2.5Hz,1H),7.13(d,J=8.3Hz,1H),5.21(s,2H),2.70(s,3H),1.34(s,12H). 13 C NMR(101MHz,Chloroform-d)δ159.9,159.3,149.4,139.7,136.0,135.2(2C),128.7,126.8(2C ),121.8,120.0,119.2,108.0,83.9(2C),77.4,70.0,25.4,25.0(4C).MS(API),m / z(%):Calcd for C 23 H 26 BNO3([M+H] + ):376.3,Found:376.2.

[0059] The mass spectrometry MS spectrum and nuclear magnetic resonance spectroscopy of the fluorescent probe prepared in Example 1 1 H-NMR spectrum, nuclear magnetic resonance 13 The C-NMR spectra are as follows Figure 6 、 Figure 7 and Figure 8 As shown, it shows that the fluorescent probe of the present invention was successfully synthesized.

[0060] Example 2

[0061] The hydrogen peroxide detection fluorescent probe prepared in Example 1 was prepared into a 1 mM probe stock solution with PBS buffer, and hydrogen peroxide and related comparison species were prepared into a 3 mM stock solution with deionized water. 30 μL of the probe stock solution and 100 μL of the hydrogen peroxide stock solution were added to 3 mL of 0.1% DMSO in PBS buffer, and the samples were tested using a fluorescence spectrometer and an ultraviolet spectrophotometer. The test results showed that the maximum excitation wavelength of the fluorescent probe was 242 nm, and the maximum emission wavelength was 390 nm. The specific test results are as follows:

[0062] Take two cuvettes, add 3 mL of 0.1% DMSO in PBS buffer and 30 μL of probe stock solution respectively, add 0-100 μL of hydrogen peroxide stock solution to one of the cuvettes, and do not add hydrogen peroxide stock solution to the other cuvette, and perform UV spectrum test; Figure 1 As shown in the figure, the fluorescent probe itself has strong ultraviolet absorption at wavelength λ=311nm. When hydrogen peroxide is added to the solution, the ultraviolet absorption peak gradually weakens. In addition, the fluorescent probe has almost no ultraviolet absorption at wavelength λ=390nm. When the concentration of hydrogen peroxide is increased in the solution, the ultraviolet absorption peak gradually strengthens. The results show that the probe has a high sensitivity to H2O2.

[0063] like Figure 2 The probe has a fluorescence spectrum response diagram to different concentrations of hydrogen peroxide (H2O2); 30 μL of the probe stock solution and 0 to 100 μL (0, 10, 20, 30 ... 90, 100 μL) of hydrogen peroxide stock solution are added to 3 mL of 0.1% DMSO in PBS buffer. The fluorescent probe itself is fluorescent in the solution. As the concentration of hydrogen peroxide increases, the fluorescence at 390 nm continuously decreases and the fluorescence at 506 nm continuously increases. The detection limit is as low as 1.447 μmol / L, and R 2 =0.996, indicating that the probe is capable of ratiometric detection and exhibits a good linear relationship with the hydrogen peroxide concentration.

[0064] like Figure 3As shown, when 30 μL of the probe stock solution and 100 μL of the hydrogen peroxide stock solution were added to 3 mL of 0.1% DMSO in PBS buffer, the fluorescence intensity of the probe at 390 nm gradually decreased and reached a stable value within 40 minutes.

[0065] like Figure 4 As shown, 3 mL of PBS buffer adjusted with HCl and NaOH to pH 4, 5, 6, 7, 8, 9, and 10 was added, and then 30 μL of the probe and 100 μL of hydrogen peroxide stock solution were added; after shaking, the solution was allowed to stand for 1 hour, and its fluorescence intensity was measured; after the probe reacted with hydrogen peroxide, the fluorescence intensity decreased significantly in the pH range of 4-8, and then as the pH continued to increase, it was found that the fluorescence intensity reached a minimum at pH = 8, and then the fluorescence intensity rebounded within the pH range of 9-10, indicating that the probe has the ability to detect hydrogen peroxide in a weakly alkaline environment.

[0066] like Figure 5 As shown, the fluorescence spectra of the probe after adding various comparison species are shown; 30 μL of the probe stock solution and 100 μL of various comparison species stock solutions were added to 3 mL of 0.1% DMSO in PBS buffer. The results show that when hydrogen peroxide is added, the fluorescence intensity at 390 nm of the fluorescence spectrum decreases significantly, while when other comparison species are added, the fluorescence does not change significantly, that is, the fluorescent probe of the present invention has good selectivity for hydrogen peroxide.

[0067] Finally, it should be understood that the embodiments described in the present invention are only used to illustrate the principles of the embodiments of the present invention; other variations may also fall within the scope of the present invention; therefore, as examples rather than limitations, alternative configurations of the embodiments of the present invention may be considered consistent with the teachings of the present invention; accordingly, the embodiments of the present invention are not limited to the embodiments explicitly introduced and described herein.

Claims

1. A small molecule fluorescent probe of hydrogen peroxide designed based on quinoline, characterized in that: The fluorescence The structural formula of the probe is shown in formula (I): 。 2. Preparation of a small molecule fluorescent probe of hydrogen peroxide based on quinoline design as claimed in claim 1 Preparation method, characterized in that, The specific preparation steps are as follows: Step (1), the prepared m-aminophenol is subjected to a cyclization reaction to obtain an intermediate (II); the structure of the intermediate (II) is ; Step (2), using intermediate (II) and prepared 4-bromomethylphenylboronic acid pinacol ester as raw materials, The reaction is carried out in an organic solvent to finally prepare a fluorescent probe (I), namely a hydrogen peroxide small molecule fluorescent probe.

3. A small molecule fluorescent probe of hydrogen peroxide based on quinoline according to claim 2 The preparation method is characterized in that In step (1), the specific preparation steps of the intermediate (II) are as follows: First, the prepared m-aminophenol is dissolved in hydrochloric acid to prepare a reaction mixture containing m-aminophenol. Reagents; Secondly, the pre-prepared chloranil n-butanol solution was added to the above reaction reagents under stirring at room temperature; The reaction mixture thus obtained is stirred and heated to reflux, and then the prepared crotonaldehyde n-butanol solution is added dropwise to the stirred reflux solution for not less than 30 minutes. After the addition is completed, the reaction mixture is refluxed for another 2-4 hours; After the reaction is completed, the reaction solution is cooled to room temperature and then distilled under reduced pressure to remove the The organic solvent is n-butanol, and then the remaining residue is dissolved in water and extracted with ether to remove the remaining organic matter in the residue; Finally, the aqueous layer was neutralized with 10% NaOH solution and extracted with ethyl acetate. The combined organic phases were After drying with anhydrous Na2SO4, the organic solvent was removed under reduced pressure to obtain a crude product; the crude product was then passed through a thin layer The intermediate (II) was purified by chromatography.

4. A small molecule fluorescent probe of hydrogen peroxide based on quinoline according to claim 3 The preparation method is characterized in that The molar ratio of m-aminophenol to hydrochloric acid is 1:3-4; the molar ratio of chloranil to n-butanol in the chloranil n-butanol solution is 1:250-350; and the molar ratio of crotonaldehyde to n-butanol in the crotonaldehyde n-butanol solution is 1:1-5.

5. A small molecule fluorescent probe of hydrogen peroxide based on quinoline design according to claim 2 The preparation method is characterized in that In step (2), the specific preparation steps of the fluorescent probe (I) are as follows: First, the prepared intermediate (II), the prepared 4-bromomethylphenylboronic acid pinacol ester and potassium carbonate were mixed. The mixture was dissolved in an organic solvent, acetonitrile, and refluxed with stirring until the reaction was complete to obtain a reaction mixture; Then, the obtained reaction solution was filtered, the filter residue was washed with acetonitrile, and dried in a vacuum drying oven. The fluorescent probe (I) was obtained.

6. A small molecule fluorescent probe of hydrogen peroxide designed based on quinoline according to claim 5 The preparation method is characterized in that The molar ratio of the intermediate (II), 4-bromomethylphenylboronic acid pinacol ester and potassium carbonate is 1:1.2:1.

2.

7. Use of a quinoline-based small molecule fluorescent probe for hydrogen peroxide prepared by the method according to any one of claims 2 to 6 in preparing a reagent for detecting hydrogen peroxide in a solution.

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