Single-molecule fluorescent probe based on BODIPY derivative BP-BNO2 as well as synthesis method and application of single-molecule fluorescent probe

Abstract

The invention provides a single-molecule fluorescent probe based on a BODIPY derivative BP-BNO2 as well as a synthesis method and application of the single-molecule fluorescent probe, and the specific Chinese name of the derivative is 5, 5-difluoro-1, 3, 7, 9-tetramethyl-10-(4-nitrophenyl)-5H-4 lambda 4, 5 lambda 4-dipyrrolo [1, 2-c: 2 ', 1'-f] [1, 3, 2] diazaborane. The invention also provides a synthesis method of the derivative and application of the derivative in detection of hypobromous acid in vitro, cells and nasal cavities of mice with allergic rhinitis. According to the method, on the basis of a derivative BP-BNO2 of BODIPY, the concentration change of hypobromous acid is detected through a fluorospectro photometer in a mixed solution of a phosphate buffer solution and acetonitrile (the volume ratio is 7: 3). The method also realizes noninvasive in-situ imaging at cell and mouse levels, and can be used for in-situ monitoring of hypobromous acid level in the nasal cavity of the mouse with allergic rhinitis and auxiliary diagnosis of allergic rhinitis.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescent probes, and particularly relates to a single-molecule fluorescent probe based on BODIPY derivative BP-BNO2, its synthesis method and application. Background Art

[0002] Eosinophils are an important part of the human immune system. On the one hand, they maintain the health and homeostasis of the human immune system, and on the other hand, they play a role in the pathophysiology of various allergic and non-allergic diseases. There is a close relationship between airway allergic diseases and eosinophils. In airway allergic diseases (including allergic rhinitis, asthma, chronic rhinosinusitis), the increase in eosinophils often indicates the polarization of the inflammatory response of the airway mucosa towards Th2 and is accompanied by the aggravation of clinical symptoms. Therefore, clinically, the elevation of eosinophils can be used as one of the reference indicators for judging airway allergic diseases. At the same time, the treatment of airway allergic diseases also needs to consider inhibiting the activity of eosinophils and reducing their number to relieve airway inflammation and symptoms. Currently, for the development of broad-spectrum therapies for eosinophils, one is to conduct non-specific eosinophil treatment by inhibiting several upstream or downstream immune pathways, and the other is to conduct eosinophil-targeted treatment by using biological agents. Therefore, monitoring eosinophil levels is of great significance for the diagnosis and treatment of airway allergic diseases. Hypobromous acid is a marker of eosinophil peroxidase activity in eosinophils, and its level detection is of great significance for measuring the activity of eosinophil peroxidase and monitoring the eosinophil level in tissue inflammatory infiltration.

[0003] Currently, there are various probes for detecting hypobromous acid, which are used in diseases such as arthritis, atherosclerosis, bacterial infection, and depression, but they have problems such as long reaction time, high detection limit, fluorescence quenching, and poor selectivity. Summary of the Invention

[0004] An object of the present invention is to provide a single-molecule fluorescent probe based on BODIPY derivative BP-BNO2, its synthesis method and application, and provide at least the advantages described hereinafter.

[0005] The present invention provides a single-molecule fluorescent probe based on BODIPY derivative BP-BNO2, its synthesis method and application. The probe has rapidity, sensitivity, fluorescence enhancement and high selectivity, providing support for the personalized diagnosis and treatment of allergic rhinitis diseases.

[0006] The technical solution of the present invention is as follows: A single-molecule fluorescent probe based on BODIPY derivative BP-BNO2, its structural formula is: 。

[0007] The Chinese name is 5,5-difluoro-1,3,7,9-tetramethyl-10-(4-nitrophenyl)-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborane, English name is 5,5-difluoro-1,3,7,9-tetramethyl-10-(4-nitrophenyl)-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine.

[0008] The present invention provides a method for synthesizing a single-molecule fluorescent probe based on the BODIPY derivative BP-BNO2, which comprises the following steps: p-Nitrobenzaldehyde (0.604 g, 4.00 mmol) and 2,4-dimethylpyrrole (0.902 mL, 8.76 mmol) were added to a 1% ethanol solution. Dissolve in 200 mL of dichloromethane, add trifluoroacetic acid (0.050 mL, 1.35 mmol) dropwise, and stir in the dark under a nitrogen atmosphere for 12 hours; Add 2,3-dichloro-5,6-dicyano-p-benzoquinone (0.908 g, 4.00 mmol) and stir for 40 minutes; Add triethylamine (9.000 mL, 65.00 mmol) and boron trifluoride etherate (9.000 mL, 72.00 mmol) in an ice bath and stir at room temperature for 2 hours; After the reaction, the solvent was removed under reduced pressure, the product was dried in vacuo, and the product was purified by silica gel column chromatography (eluent: petroleum ether: dichloromethane = 2:1, v:v) to obtain BP-BNO2 (yield 24%).

[0009] Note: The final structure of this probe uses the BODIPY fluorophore as the parent, with a p-nitrobenzene ring attached to its meso site to extend the conjugation and red-shift its emission wavelength, resulting in better tissue penetration. Because the synthesis process is only one step without intermediates, BODIPY is not reflected in the above steps.

[0010] The application of the single-molecule fluorescent probe based on the BODIPY derivative BP-BNO2 is used for the detection of hypobromous acid, and is applied to in vitro specific fluorescence detection, intracellular and extracellular visualization detection, or nasal detection in mice with allergic rhinitis.

[0011] The present invention has the following beneficial effects: The synthesis method of the BODIPY derivative BP-BNO2 is simple and can be completed in only one step, with convenient operation; Monitoring is carried out using a fluorescence detector, and the operation is simple. Moreover, this probe is a ratio-type fluorescence probe, with a more obvious detection signal, an accelerated reaction rate, and a visible color change to the naked eye; The small molecule probe has the ability to recognize hypobromous acid, providing a new option for detection in this field; The probe can distinguish between allergic rhinitis mice and normal mice in vivo and has potential value for the diagnosis of allergic rhinitis. At the same time, its applicability in cell and mouse experiments provides strong support for the subsequent research on allergic rhinitis.

[0012] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Description of the Drawings

[0013] Figure 1 is the 1H NMR spectrum of BP-BNO2; Figure 2 is the 13C NMR spectrum of BP-BNO2; Figure 3 is the spectral test chart of the reaction of BP-BNO2 with hypobromous acid at different concentrations; Figure 4 is the working curve of the fluorescence intensity and concentration of the reaction of BP-BNO2 with hypobromous acid at different concentrations; Figure 5 is the fluorescence bar chart of BP-BNO2 in different pH systems; Figure 6 is the fluorescence bar chart of BP-BNO2 with various analytes; Figure 7 is the toxicity test chart of BP-BNO2 on Hela cells; Figure 8 is the cell imaging chart of BP-BNO2 monitoring the hypobromous acid level in Hela cells; Figure 9 is the in vivo imaging chart of BP-BNO2 in mice. Detailed Embodiments

[0014] The following further elaborates on the present invention in conjunction with embodiments, enabling those skilled in the art to implement it with reference to the text of the specification.

[0015] It should be understood that terms such as "having", "comprising", and "including" as used herein do not preclude the presence or addition of one or more other elements or their combinations.

[0016] The present invention provides a single-molecule fluorescent probe based on the BODIPY derivative BP-BNO2, and its structural formula is: 。

[0017] The Chinese name is 5,5-difluoro-1,3,7,9-tetramethyl-10-(4-nitrophenyl)-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine, and the English name is 5,5-difluoro-1,3,7,9-tetramethyl-10-(4-nitrophenyl)-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine.

[0018] The present invention provides a synthesis method of the above-mentioned single-molecule fluorescent probe based on the BODIPY derivative BP-BNO2, which comprises the following steps: Dissolve p-nitrobenzaldehyde (0.604 g, 4.00 mmol) and 2,4-dimethylpyrrole (0.902 mL, 8.76 mmol) in 200 mL of dichloromethane, add trifluoroacetic acid (0.050 mL, 1.35 mmol), and stir in the dark for 12 hours under a nitrogen atmosphere; Add 2,3-dichloro-5,6-dicyano-p-benzoquinone (0.908 g, 4.00 mmol) and stir for 40 minutes; Add triethylamine (9.000 mL, 65.00 mmol) and boron trifluoride diethyl etherate (9.000 mL, 72.00 mmol) in an ice bath, and stir at room temperature for 2 hours; After the reaction is completed, remove the solvent under reduced pressure, dry in vacuo, and purify the product by silica gel column chromatography (eluent: petroleum ether:dichloromethane = 2:1, v:v) to obtain BP-BNO2 (yield 24%). 1 H NMR (500 MHz, DMSO- d 6) δ 8.40 (d, J =8.6 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 6.22 (s, 2H), 2.47 (s, 6H), 1.35 (s,6H), as Figure 1 shown. 13 C NMR (126 MHz, DMSO- d6) δ 155.68, 148.04, 142.57, 140.75, 139.26, 130.02, 129.96, 124.38, 121.81, 14.29, as Figure 2 shown: .

[0019] Application of the single-molecule fluorescence probe based on BODIPY derivative BP-BNO2, which is used for the detection of hypobromous acid, and is applied to in vitro specific fluorescence detection, intracellular and extracellular visualization detection or detection in the nasal cavity of allergic rhinitis mice.

[0020] Specifically as follows: Prepare a mixed buffer solution of acetonitrile and PBS (phosphate buffer solution with pH = 7.4 prepared with sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride) with a volume ratio of 3:7 (referred to as the test system), prepare a dimethyl sulfoxide solution of 2 mmol / L BP-BNO2 (referred to as the probe mother liquor), and prepare a 1.2 mmol / L HOBr solution. Example 1

[0021] Take 2.0 mL of the test system in a cuvette, add 10 μL of the probe mother liquor to it, gradually increase the HOBr concentration (0 - 50 μM), and measure the change in fluorescence intensity at the maximum emission wavelength (545 nm) under excitation at 525 nm. As the HOBr concentration increases, the fluorescence intensity at 545 nm gradually increases, as Figure 3 shown.

[0022] Plot a graph with the HOBr concentration as the abscissa and the fluorescence intensity as the ordinate to obtain the working curve of fluorescence intensity versus concentration; the linear regression equation is: F = 12.2302c + 34.7667, where the unit of c is 10 -6 mol / L, as Figure 4 shown. Example 2

[0023] Prepare PBS buffer solutions with pH = 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0. Add 10 μL of the probe mother liquor and 35 μL of the HOBr stock solution (4.2 μM) to cuvettes with different pH values respectively. After mixing evenly, record the fluorescence intensity of the probe at 545 nm in each pH environment.

[0024] As Figure 5 shown, the fluorescence intensity of the probe is the strongest at pH = 7.0. Example 3

[0025] Take 2.0 mL of the test system in a cuvette, add 10 μL of the probe mother liquor to it, and add 100 equivalents of other analytes to the cuvette respectively: 1. H2O2, 2. ·OH, 3. ONOO - 、4. ClO - 、5. 1 O2, 6. Thr, 7. Cys, 8. Ser, 9. Phe 10. Asp, 11. Val, 12. Glu, 13. Met, 14. GSH, 15. Hcy, 16. Ca 2+ 、17. Mg 2+ 、18. Na + 、19. Zn 2+ 、20. Cu 2 + 、21. Fe 2+ 、22. K + 、23. probe mother liquor, 24. HOBr. After mixing evenly, record the fluorescence intensities at 545 nm after adding different analytes respectively.

[0026] As Figure 6 shown, various analytes basically did not cause changes in the fluorescence intensity in the detection system. Example 4

[0027] Seed Hela cells into a 96-well plate and place it in an incubator for 24 hours. Then add probe solutions with different concentrations (0, 4, 8, 10, 15, 20 μM) and culture for 24 h. After that, add 90 μL of the diluted CCK-8 cell viability detection dye (take 6 μL of CCK-8 and add it to 6 mL of the culture medium containing DMEM) to each well, incubate for 40 min, and measure the cell viability under an enzyme-linked immunosorbent assay (ELISA) reader.

[0028] As Figure 7 shown, when the probe concentration is in the range of 0 - 20 μM, the cell survival rate is as high as over 85%. It indicates that the probe has good biocompatibility and can be applied to cell imaging. Example 5

[0029] Take 10 μL of the probe mother liquor and add it to 2 mL of PBS solution (the final concentration of the probe is 10 μM) for incubating Hela cells.

[0030] Hela cells were divided into 4 groups. The first group was incubated with the probe preparation solution (10 μM) for 15 min; the second group was incubated with the probe preparation solution (10 μM) for 15 min, then the probe preparation solution was aspirated, washed twice with PBS, and then HOBr (4.2 μM) was added and incubated for 15 min; the third group was incubated with Br− (100 μM) for 15 min, washed twice with PBS, and then the probe preparation solution (10 μM) was added and incubated for 15 min; the fourth group was first incubated with PBS containing NAC (a Br− scavenger) (100 μM) and Br− (100 μM) for 15 min, washed twice with PBS, and then the probe (10 μM) was added and incubated for 15 min.

[0031] The above four groups of cells were placed in a confocal laser microscope for confocal imaging. Fluorescence emission images in the range of 490 - 500 nm (green channel) were collected under excitation at 488 nm; fluorescence emission images in the range of 540 - 590 nm (red channel) were collected under excitation at 514 nm.

[0032] As Figure 8 shown, this probe can visualize the change in the content of HOBr in cells. Example 6

[0033] First, an allergic rhinitis mouse model was established by subcutaneous injection and intranasal administration of ovalbumin. The probe was used for intranasal administration to allergic rhinitis mice and healthy control mice. The mother liquor of the probe was added to physiological saline to prepare a test solution of 300 μM, and the mice were treated by intranasal administration (20 μL / nostril).

[0034] The treated mice were placed in a live fluorescence imager for imaging. The excitation channel was 535 nm and the emission channel was DsRed.

[0035] As Figure 9 shown, this probe can monitor the HOBr produced in the nasal cavity of allergic rhinitis mice, indicating that this probe has great potential for in vivo monitoring of EOS and HOBr levels in nasal secretions of allergic rhinitis.

[0036] The above experimental results show that BP - BNO2 is a good tool for detecting changes in HOBr in vitro, in cells, and in vivo.

[0037] The present invention provides a BODIPY derivative BP - BNO2, its synthesis method and application. The specific Chinese name of the derivative is 5,5 - difluoro - 1,3,7,9 - tetramethyl - 10 - (4 - nitrophenyl) - 5H - 4λ 4 ,5λ 4-Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine. The present invention also provides a synthesis method of the derivative, and applications of the derivative in detecting hypobromous acid in vitro, in cells and in the nasal cavity of allergic rhinitis mice. The method is based on the BODIPY derivative BP-BNO2, and the change in the concentration of hypobromous acid is detected by a fluorescence spectrophotometer in a mixed solution of phosphate buffer and acetonitrile (volume ratio 7:3). The method also realizes non-invasive in-situ imaging at the cell and mouse levels, can be used to in-situ monitor the level of hypobromous acid in the nasal cavity of allergic rhinitis mice, and assist in the diagnosis of allergic rhinitis.

[0038] Although the embodiments of the present invention have been disclosed as above, they are not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

Claims

1. A single-molecule fluorescent probe based on the BODIPY derivative BP-BNO2, characterized in that: The structural formula is as follows: 。 2. The synthesis method of the single-molecule fluorescence probe based on the BODIPY derivative BP-BNO2 according to claim 1, characterized in that, It includes the following steps: Dissolve 0.604 g of p-nitrobenzaldehyde and 0.902 mL of 2,4-dimethylpyrrole in 200 mL of dichloromethane, add dropwise 0.050 mL of trifluoroacetic acid, and stir in the dark for 12 hours under a nitrogen atmosphere; Add 0.908 g of 2,3-dichloro-5,6-dicyano-p-benzoquinone and stir for 40 minutes; Add 9.000 mL of triethylamine and 9.000 mL of boron trifluoride diethyl ether in an ice bath, and stir at room temperature for 2 hours; After the reaction is completed, remove the solvent under reduced pressure, dry in vacuo, and purify the product by silica gel column chromatography to obtain BP-BNO2, wherein the eluent is petroleum ether and dichloromethane with a volume ratio of 2:

1.

3. Use of the single-molecule fluorescence probe based on BODIPY derivative BP-BNO2 according to claim 1, characterized in that, It is used for the detection of hypobromous acid, and is applied to in vitro specific fluorescence detection, intracellular and extracellular visualization detection or detection in the nasal cavity of allergic rhinitis mice.

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