CN113004216A

CN113004216A - Preparation method and application of novel benzoxazine hypochlorous acid fluorescent molecular probe

Info

Publication number: CN113004216A
Application number: CN201911322539.8A
Authority: CN
Inventors: 不公告发明人
Original assignee: Hunan Chaoji Chemical Technology Co ltd
Current assignee: Hunan Chaoji Testing Technology Co ltd
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2021-06-22
Anticipated expiration: 2039-12-20
Also published as: CN113004216B

Abstract

The invention discloses a preparation method and application of a novel benzoxazine hypochlorous acid fluorescent molecular probe, wherein the chemical structural formula of the benzoxazine hypochlorous acid fluorescent molecular probe is as follows:

Description

Preparation method and application of novel benzoxazine hypochlorous acid fluorescent molecular probe

Technical Field

The invention belongs to the technical field of analytical chemistry, and relates to a preparation method and application of a novel benzoxazine hypochlorous acid fluorescent molecular probe.

Background

Hypochlorous acid (HOCl), which is catalytically produced from hydrogen peroxide and chloride ions by Myeloperoxidase (MPO), is an important member of the active oxygen family in vivo and plays an important role in physiological and pathological processes. HOCl is strongly oxidative, killing pathogens, thus contributing to host defense, and endogenous HClO can also serve as a signal to activate caspases and mediate apoptosis. However, excessive amounts of HClO in the body can cause oxidative damage to biomolecules such as nucleic acids, proteins and lipids, which can lead to various diseases such as cardiovascular disease, atherosclerosis, osteoarthritis, rheumatoid arthritis and lung injury. Therefore, monitoring the concentration level of HClO in cells is of great importance to further understand its biological role and to diagnose related diseases early.

In recent years, electrochemical, colorimetric, chemiluminescent, and fluorescence imaging methods have been used for detecting HClO. Among them, the fluorescence imaging method is widely used in detection and imaging of HClO due to its advantages of high sensitivity, strong specificity, short response time, real-time monitoring, etc. CN 110357869A reports a fluorescent molecular probe based on a naphthalimide mother nucleus and capable of being used for HClO specific detection, and the fluorescent molecular probe has good chemical and light stability. However, after the probe responds to HClO, the fluorescence at 510 nm gradually weakens, and the probe presents a 'Turn-OFF' type fluorescence response, is easily influenced by environment and has larger detection error. CN 109400563A reported a fluorescent probe CoPh-ClO that achieved rapid detection of HClO specificity, which could be used as a specific indicator of the presence of HClO in aqueous solutions and in biological cells. However, the probe has the problem of short emission wavelength (-510 nm) and is easily interfered by background fluorescence when in intracellular imaging. CN 109942504A reports that HClO response probe molecules are constructed by using near-infrared fluorophore basic blue as a fluorescent molecular framework, fluorescence turn-on is realized by using the conversion of a reduction state and an oxidation state, and when HClO is added, the probe shows fluorescence enhancement at 670 nm, so that HClO detection can be realized. However, the probe has poor water solubility, so that the biocompatibility is poor, and the further application of the probe in detecting HClO in organisms is limited. Therefore, the development of a red light emitting fluorescent molecular probe with high sensitivity, high specificity and good water solubility has very important significance for realizing the detection of HClO in environment and organisms.

Disclosure of Invention

Aiming at the defects of the existing HClO fluorescent molecular probe, the invention aims to provide the HClO fluorescent molecular probe which has good water solubility, high specificity and red light emission.

The second purpose of the invention is to provide a high-efficiency preparation method of the fluorescent molecular probe.

The third purpose of the invention is to provide the application of the fluorescent molecular probe in the detection of HClO in aqueous solution and organisms.

In order to achieve the above object, the present invention provides a fluorescent molecular probe having a structure of formula I:

formula I

The preparation method of the fluorescent probe is preferably as follows:

dissolving benzoxazine in anhydrous DMF solution, 0^oAdding 60% NaH solution and p-bromobenzyl bromide in batches at the temperature of C, stirring at room temperature to react, and cooling to 0^oC, dropwise adding water to quench the reaction, and respectively using water and saturated NaHCO₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and purifying by column chromatography to obtain a white product. Dissolving the purified product in CHCl₃The solution was added NBS in portions and the reaction was stirred at room temperature to completion. Taking off the reaction, adding water dropwise to quench the reaction, and respectively using water and saturated NaHCO₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and recrystallizing with ethyl acetate to obtain white solid. The purified product, 4-nitrophenol, potassium acetate and Pd (dppf) Cl₂DCM was dissolved in anhydrous DMF solution and heated under reflux under argon to completion. The reaction was cooled to room temperature, and then water and saturated NaHCO were added to the reaction mixture₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and purifying by column chromatography to obtain a white product. The purified product and SnCl₂·2H₂O addition to CH₃OH, concentrated HCl and H₂Stirring the mixture at room temperature until the reaction is completed, and adding saturated Na₂CO₃Adjusting pH of the solution to alkaline, and respectively adding water and saturated NaHCO₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and purifying by column chromatography to obtain the target molecular probe.

The synthesis of the invention is as follows:

the invention provides application of the fluorescent probe, which can be applied to detection of HClO. The detection mechanism of the probe is shown as the following figure:

compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:

the fluorescent molecular probe for detecting HClO has the following advantages:

(1) the fluorescent probe has the advantages of stable and high chemical property and optical stability, and has no autoxidation and no photobleaching phenomenon;

(2) the fluorescent probe has the advantages of good water solubility, quick response and high specificity, can avoid the interference of other objects to be detected, is favorable for quickly detecting HClO in the environment, and has stronger practical application value in the field of environmental science;

(3) the probe has stronger red light emission, can effectively avoid the interference of biological autofluorescence, has good cell membrane permeability and low cytotoxicity, can be used for biological imaging of HClO, and has stronger practical application value in the field of life science.

Drawings

FIG. 1 is a graph showing the emission spectrum of the fluorescence intensity of the fluorescent probe varying with the HClO concentration in the practice of the present invention;

FIG. 2 is a graph showing the selectivity of fluorescent probes for HClO in the practice of the present invention;

FIG. 3 is a confocal image of fluorescence of fluorescent probe in HeLa cell in the practice of the present invention.

Detailed Description

The following embodiments are intended to further illustrate the present invention and are not intended to limit the present invention.

Example 1

Synthesis of Compound 1:

benzoxazine (1830 mg, 10 mmol) was dissolved in 25 mL anhydrous DMF at 0^oC, adding 60% NaH solution (4727 mg, 12 mmol) in portions, stirring for 10 minutes, adding p-bromobenzyl bromide (3000 mg, 12 mmol) in portions, and stirring at room temperature until the reaction is finished. Cooling to 0 deg.C after reaction, adding water dropwise to quench the reaction, and adding water and saturated NaHCO respectively₃Extracting the solution with saturated saline solution, combining organic layers, drying with anhydrous magnesium sulfate, concentrating, and purifying by column chromatography to obtain 2899 mg of white product with the yield of 82.3%.

Synthesis of Compound 2:

compound 1 (2817.6 mg, 8 mmol) was dissolved in 25 mL HCl₃To the solution, NBS (2854.6 mg, 9.08 mmol) was added in portions, and the reaction was stirred at room temperature until completion. Taking off the reaction, adding water dropwise to quench the reaction, and respectively using water and saturated NaHCO₃The solution and saturated brine were extracted, the organic layers were combined, dried over anhydrous magnesium sulfate, concentrated and dried to give a crude product, which was recrystallized from ethyl acetate to give 2044.1 mg of a white solid with a yield of 50.1%.

Synthesis of Compound 3:

compound 2 (2040 mg, 4 mmol), 4-nitrophenol (2225.6 mg, 16 mmol), potassium acetate (2348.7 mg, 24 mmol) and Pd (dppf) Cl₂DCM (2817.7 mg, 0.4 mmol) was dissolved in 7 mL of anhydrous DMF and heated under reflux under argon to completion. The reaction was cooled to room temperature, and then water and saturated NaHCO were added to the reaction mixture₃Extracting the solution with saturated saline, combining organic layers, drying with anhydrous magnesium sulfate, concentrating, drying, and purifying by column chromatography to obtain white product 1128.2 mg with yield 41.2%.¹H NMR (300 MHz, CDCl3) δ 8.82 (d, 6H, J=7.8 Hz), 7.43 (d, 2H, J=8.3 Hz), 7.28 (d, 2H, J=8.3 Hz), 7.21 (d, 6H, J=7.8 Hz), 6.66 (s, 2 H), 6.52-6.51(m, 4H), 4.32 (s, 2H)。

Synthesis of target molecular probe:

to compound 3 (684.6 mg, 1 mmol) and SnCl₂·2H₂O (3371.4 mg, 15 mmol) was added to 16 mL CH₃OH: concentrated HCl: h₂A mixed solution of O =3:2:3 was stirred at room temperature until the reaction was completed. After the reaction is finished, saturated Na is used₂CO₃Adjusting pH of the solution to alkaline, and respectively adding water and saturated NaHCO₃The solution was extracted with saturated brine, and the organic layers were combined, dried over anhydrous magnesium sulfate, concentrated and dried. After column chromatography purification, 319.3 mg of the target molecular probe is obtained, and the yield is 53.7%.¹H NMR (300 MHz, CDCl3) δ 7.35 (d, 2H, J=8.4 Hz), 7.30 (d, 2H, J=8.4 Hz), 7.21 (d, 6H, J=7.8 Hz), 6.76-6.73 (m, 12H), 6.65 (s, 2H), 6.53-6.50 (m, 4H), 6.27 (s, 6H), 4.30 (s, 2 H)。HRMS (ESI): calculated [M+H]⁺: 595.22670, found [M+H]⁺: 595.21371。

Example 2

Preparation of fluorescent probe mother liquor

The product isolated above and having a purity of 99% is accurately weighed at 5.94 mg and carefully transferred into a 50 mL volumetric flask, into which CH is added at room temperature₃CN solution is dissolved completely, and the volume is determined to the scale mark, thus obtaining the probe mother liquor with the concentration of 1 mM. During the test, 20. mu.L of the above solution was taken out by a micro-injector each time, and dissolved in the test system so that the total volume per test was 2 mL, at which time the concentration of the fluorescent probe was 10. mu.M.

Example 3

Preparation of HClO mother liquor

HClO was prepared as 5 mL stock solutions in different concentration gradients (0.1 mM, 0.3 mM, 0.6 mM, 1.0 mM, 1.5 mM, 2.0 mM, 3.0 mM, 4.0 mM) in PBS buffer. The other substances to be tested required by the test are respectively prepared into mother liquor with the concentration of 3 mM by using PBS buffer solution.

Example 4

Relationship between fluorescence intensity of fluorescent probe and HClO concentration

4.900 mL of PBS buffer solution was measured, 50. mu.L of 1 mM probe stock solution was dissolved therein, and 50. mu.L of different HClO stock solutions were transferred so that the concentration of the probe in the entire detection system was 10. mu.M and Hg was finally obtained²⁺The concentrations of (A) were 1. mu.M, 3. mu.M, 6. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 30. mu.M and 40. mu.M, respectively. After incubation at room temperature for 20 min, the fluorescence spectra of the different systems were tested in 10 mm cuvettes, respectively (FIG. 1). The results show that the fluorescence emission intensity of the system at 590 nm is gradually increased with the gradual increase of the HClO concentration.

Example 5

Selectivity of fluorescent probes for HClO detection

The concentration of the probe master is 1 mMmu.L of the solution was dissolved in 4.900 mL of PBS buffer solution, and 50. mu.L of 3 mM O was transferred₂-、NO、H₂O₂Tert-butyl peroxy alcohol, NO₃-, L-cysteine, L-glutathione, Fe³⁺The mother solutions were added to the system, incubated at room temperature for 20 min, the fluorescence spectra were measured, and the fluorescence intensity at 590 nm was recorded (FIG. 2). As shown in the figure, the results show that the fluorescence of the fluorescent probe is obviously enhanced only by adding HClO, and no or only weak fluorescence change is generated when other test metal ions or molecules are added. The fluorescent probe is shown to have good selectivity.

Example 6

Response of fluorescent probes to HClO in cells

Adding 10 μ M fluorescent probe solution into HeLa medium, and placing at 37^oC, 5% CO₂After incubation in the incubator for 30 minutes, the cells were washed three times with 0.1M PBS buffer (10 mM, pH = 7.4) to remove probe molecules that have not entered the cells, the medium was then replaced, the cells were incubated with hcl buffer solution (25 μ M) for 30 minutes, washed three times with 0.1M PBS buffer (10 mM, pH = 7.4), and the change in fluorescence was observed under a fluorescence microscope, as shown in fig. 3. Experiments show that the probe molecules entering the cell body react with HClO to emit strong red fluorescence, so that the fluorescent probe has a good imaging effect on HClO in the cell and can be used for detecting HClO in the organism.

Although the present invention has been described with reference to the specific embodiments shown in the drawings, it is not intended to limit the scope of the present invention, and various modifications or variations can be made by those skilled in the art from the disclosure of the present invention without inventive efforts.

Claims

1. A novel benzoxazine hypochlorous acid fluorescent molecular probe is characterized by having a structure shown in a formula (I):

formula I.

2. The method for preparing the novel fluorescent probe for hypochlorous acid detection according to claim 1, wherein the method comprises the following steps: dissolving benzoxazine in anhydrous DMF solution, 0^oAdding 60% NaH solution and benzyl bromide 4-BnBr in batches at the temperature of C, stirring at room temperature to react, cooling to 0^oC, dropwise adding water to quench the reaction, and respectively using water and saturated NaHCO₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and purifying by column chromatography to obtain a white product;

dissolving the purified product in CHCl₃Adding NBS into the solution in batches, and stirring and reacting at room temperature until the reaction is finished;

taking off the reaction, adding water dropwise to quench the reaction, and respectively using water and saturated NaHCO₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and recrystallizing with ethyl acetate to obtain white solid;

the purified product, 4-nitrophenol, potassium acetate and Pd (dppf) Cl₂DCM was dissolved in anhydrous DMF solution and heated under reflux under argon protection until the reaction was complete;

the reaction was cooled to room temperature, and then water and saturated NaHCO were added to the reaction mixture₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and purifying by column chromatography to obtain a white product;

the purified product and SnCl₂·2H₂O addition to CH₃OH, concentrated HCl and H₂Stirring the mixture at room temperature until the reaction is completed, and adding saturated Na₂CO₃Adjusting pH of the solution to alkaline, and respectively adding water and saturated NaHCO₃Extracting the solution with saturated saline water, and respectively extracting with water and saturated NaHCO₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and purifying by column chromatography to obtain the target molecular probe.

3. Use of the fluorescent probes according to claims 1 and 2 for the detection of HOCl in the environment and in cells.

4. Use of a fluorescent probe according to any of claims 1-3, characterized in that the detection conditions in the environment and the cells are: the excitation wavelength is 365 nm, the fluorescence emission spectrum detection is carried out between 400-700 nm, the solvent of the detection system is PBS (10 mM, pH = 7.4) buffer solution, and the pH is 5.8-8.4.

5. The use according to claims 1 to 4, wherein the solution to be tested is added to the fluorescent probe detection solution, and the fluorescence intensity of the detection solution is measured as an evaluation index for the presence or absence of hypochlorous acid and the quantitative determination of hypochlorous acid concentration.