Preparation method and application of a fluorescent probe for screening monoamine oxidase B inhibitors

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a method for preparing and applying a fluorescent probe for screening monoamine oxidase B inhibitors. The method for preparing the fluorescent probe described in this invention is simple, convenient, and low in synthesis cost. The fluorescent probe prepared using this method exhibits good specificity and high sensitivity. The probe has a large fluorescence emission wavelength, which can effectively avoid fluorescence interference from chemical components in traditional Chinese medicine, thereby improving detection sensitivity. It shows promising application prospects in the screening of monoamine oxidase B inhibitors from traditional Chinese medicine.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for preparing and applying a fluorescent probe for screening monoamine oxidase B inhibitors. Background Technology

[0002] Monoamine oxidase (MAO, EC1.4.3.4) is a membrane-bound mitochondrial enzyme found in almost all vertebrate tissues, playing a crucial role in neurotransmitter metabolism. There are two subtypes of MAO: MAO-A and MAO-B. MAO-B is an important enzyme that degrades monoamine neurotransmitters, primarily phenylethylamine, dopamine, and benzylamine. MAO-B dysfunction can cause various neurological and psychiatric diseases, including Parkinson's disease (PD). Therefore, finding effective methods for detecting MAO-B is of great significance for a deeper understanding of the function of MAO in biological systems and for the clinical diagnosis and treatment of MAO-related diseases.

[0003] Currently, numerous methods for detecting monoamine oxidase B have been reported both domestically and internationally, including spectrophotometry, ultraviolet light methods, enzyme-linked immunosorbent assay (ELISA), and radiolabeling methods. Most of these methods suffer from low sensitivity and low selectivity. While ELISA offers fast detection speed and high sensitivity, its procedures are relatively cumbersome and its specificity is low. Radiolabeling methods also pose certain operational risks.

[0004] Fluorescent probes are biosignal molecular sensors that, upon specific binding to an analyte, selectively convert chemical signals into optical signals, thereby achieving detection by analyzing changes in fluorescence intensity. Compared to methods described above, this approach offers advantages such as simplicity, low synthesis cost, high selectivity, strong specificity, and good biocompatibility, making it widely applicable in medicine, environmental science, and life sciences. In recent years, numerous fluorescent probes for monoamine oxidase detection have been developed. However, many fluorescent probes suffer from poor water solubility, low sensitivity, and short emission wavelengths. Therefore, developing a simple and effective fluorescent probe for screening monoamine oxidase B inhibitors is essential. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for preparing and applying a fluorescent probe for screening monoamine oxidase B inhibitors. This probe is easy to synthesize, has good specificity, high sensitivity, and a suitable emission wavelength.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of this invention is to provide a fluorescent probe for screening monoamine oxidase B inhibitors, the fluorescent probe having the following structural formula:

[0008]

[0009] A second aspect of this invention is to provide a method for preparing a fluorescent probe for screening monoamine oxidase B inhibitors as described above, the specific steps of which are as follows:

[0010] (1) Dissolve isophorone in ethanol, add malononitrile and catalyst in sequence, reflux under nitrogen protection, cool to room temperature, stand overnight, filter, and dry to obtain compound 1;

[0011] (2) Compound 1, p-hydroxybenzaldehyde and piperidine were dissolved in ethanol and refluxed under nitrogen protection. After the reaction was completed, the mixture was purified by silica gel column chromatography to obtain compound 2.

[0012] (3) Dissolve 4-bromopyridine in dichloromethane, then add iodomethane dropwise, stir at room temperature, wash, and obtain compound 3;

[0013] (4) Dissolve compound 2 and sodium ethoxide in dichloromethane, stir and then add compound 3 dropwise. Stir at room temperature. After the reaction is complete, perform silica gel column chromatography purification to obtain crude fluorescent probe.

[0014] (5) Dissolve the crude fluorescent probe in dichloromethane, then add sodium borohydride, stir at room temperature, and after the reaction is complete, perform silica gel column chromatography purification and dry to obtain the fluorescent probe.

[0015] Preferably, in step (1), the catalyst is a mixed solvent of pyridine and glacial acetic acid, with a molar ratio of pyridine to glacial acetic acid of 1:1; glacial acetic acid can improve the reaction efficiency and accelerate the reaction rate.

[0016] Preferably, in step (2), the molar ratio of compound 1 to p-hydroxybenzaldehyde is 1:1.

[0017] Preferably, in step (3), the molar ratio of 4-bromopyridine to iodomethane is 1:2.

[0018] Preferably, in step (4), the molar ratio of compound 2: sodium ethoxide: compound 3 is 5:6:6.

[0019] Preferably, in step (5), the ratio of crude fluorescent probe to sodium borohydride is 1:4 by volume.

[0020] Preferably, in steps (2), (4), and (5), a mixed solvent of ethanol and dichloromethane is used as the eluent; wherein, in steps (2) and (4), the volume ratio of ethanol to dichloromethane is 1:100; and in step (5), the volume ratio of ethanol to dichloromethane is 1:50.

[0021] A third aspect of the present invention is to provide the use of the fluorescent probe described above for screening monoamine oxidase B inhibitors in screening monoamine oxidase B inhibitors.

[0022] Preferably, a third aspect of the present invention is to provide the application of the fluorescent probe described above for screening monoamine oxidase B inhibitors in screening monoamine oxidase B inhibitors in traditional Chinese medicine.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) The fluorescent probe of the present invention has good specificity, good chemical stability and photostability, high sensitivity, good cell and tissue penetration, and less side effects on cells;

[0025] (2) The fluorescent probe of this invention has a large fluorescence emission spectrum wavelength, which can effectively avoid fluorescence interference in the chemical components of traditional Chinese medicine and improve the sensitivity of detection. It has unique advantages in screening effective substances of traditional Chinese medicine.

[0026] (3) The fluorescent probe provided by the present invention has readily available raw materials, simple operation process and practical application significance. Attached Figure Description

[0027] Figure 1 This is a chemical structure diagram of the fluorescent probe of the present invention;

[0028] Figure 2 This is the 1H NMR spectrum of the fluorescent probe of this invention;

[0029] Figure 3 This is the mass spectrum of the fluorescent probe of the present invention;

[0030] Figure 4 (A) and (B) are fluorescence emission diagrams of the interaction between the fluorescent probe of the present invention and MAO-B;

[0031] Figure 4(C) is a fluorescence bar graph showing the interaction between the fluorescent probe of the present invention and MAO-B and other active substances, wherein: 1. Glutathione; 2. Cysteine; 3. Glutamic acid; 4. Sodium ion; 5. Manganese ion; 6. Copper ion; 7. Potassium ion; 8. Iron ion; 9. Aluminum ion; 10. Alanine; 11. Aspartic acid; 12. Hydroxyproline; 13. Methionine; 14. Phenylalanine; 15. Proline; 16. Threonine; 17. Kynureanine; 18. Chloride ion; 19. Bromide ion; 20. Nitrate ion; 21. Sulfate ion; 22. Peroxynitrite; 23. Superoxide ion; 24. Hypochlorous acid; 25. Hydrogen peroxide; 26. Butyrylcholinesterase; 27. Acetylcholinesterase; 28. Monoamine oxidase A; 29. ​​Monoamine oxidase B;

[0032] Figure 4 (D) shows the fluorescence response of the fluorescent probe to MAO-B in buffer solutions with different pH values;

[0033] Figure 5 (A) is a fluorescence bar graph of the fluorescent probes of this invention used to screen the monoamine oxidase B inhibitory activity of different monomers in the traditional Chinese medicine Cistanche deserticola. M (the first M from left to right in the figure), model group; Y, positive drug group (rasagiline); 2', 2'-acetylverascoside; A, tubuloside A; B, tubuloside B; F, tubuloside F; S, echinacoside; M (the second M from left to right in the figure), verbascoside; T, rhodioloside; Y, isoverascoside; DE, Decaffeoyl acteosid;

[0034] Figure 5 (B) represents the enzyme inhibition rate of echinacoside monomer against monoamine oxidase B;

[0035] Figure 6 (A) shows the cytotoxicity of the fluorescent probes of different concentrations in this invention;

[0036] Figure 6 (B) is a cell confocal imaging image. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] Example 1 Synthesis of Fluorescent Probe

[0039] (1) Isophorone (6.22 g, 45 mM) was dissolved in 90 mL of ethanol, and malononitrile (3.30 g, 50 mM), piperidine (0.4 mL, 4 mM) and glacial acetic acid (0.24 g, 4 mM) were added in sequence. The mixture was refluxed under nitrogen protection for 6 h, cooled to room temperature, and allowed to stand overnight. After filtration, it was recrystallized from ethanol to obtain white precipitate compound 1.

[0040] (2) Compound 1 (188 mg, 1 mM), p-hydroxybenzaldehyde (122 mg, 1 mM), and piperidine 200 μL were dissolved in 20 mL of ethanol and refluxed under nitrogen protection for 4 h. After the reaction was completed, the solution was passed through a silica gel column (ethanol: dichloromethane = 1:100, V / V) to obtain orange-red solid compound 2.

[0041] (3) Dissolve 4-bromopyridine (1.12 g, 7.09 mM) in 5 mL of dichloromethane, add iodomethane (2.01 g, 14.18 mM) while stirring at 0 °C, stir at room temperature for 24 h, wash with petroleum ether to obtain brown solid compound 3;

[0042] (4) Dissolve the fluorophore (145 mg, 0.5 mM) and sodium ethoxide (40.83 mg, 0.6 mM) in 20 mL of dichloromethane, stir the mixture for 10 min, add compound 3 (72.6 mg, 0.6 mM) dropwise, stir the mixture at room temperature for 24 h, and pass it through a silica gel column (ethanol: dichloromethane = 1:100, V / V) to obtain the crude fluorescent probe;

[0043] (5) Dissolve the crude fluorescent probe (192 mg, 0.5 mM) in 10 mL of dichloromethane, then add sodium borohydride (76 mg, 2 mM), stir overnight at room temperature, pass through a silica gel column (ethanol: dichloromethane = 1:50, V / V), and dry to obtain the fluorescent probe.

[0044] The chemical structure of the fluorescent probe is shown in [reference needed]. Figure 1 The proton NMR spectrum of the fluorescent probe is shown in [reference needed]. Figure 2 Mass spectrum can be found Figure 3 .

[0045] Example 2: Photophysical Detection Study of Monoamine Oxidase B Using Fluorescent Probes

[0046] (1) Prepare a 10 mM 20% DMSO / PBS buffer solution with pH 7.4, and prepare a 10 mM fluorescent probe solution using DMSO. Take 500 μL of the 20% DMSO / PBS buffer solution, first add the DMSO solution with a final concentration of 10 μM fluorescent probe, then add MAO-B at different gradients, and place the mixed solution in a 2 mL EP tube; shake at 37℃ for 5 h, and add the solution after the reaction to a fluorescent cuvette. Detect the fluorescence on a fluorescence spectrometer. As the MAO-B content increases, the fluorescence intensity gradually increases, see [see figure]. Figure 4 (A)(B).

[0047] (2) Prepare a 10 mM 20% DMSO / PBS buffer solution with pH 7.4, and prepare a 10 mM fluorescent probe solution using DMSO. Add the DMSO solution to the final diluted fluorescent probe solution to a concentration of 10 μM, and add the following analytes to a final concentration of 50 μM: GSH, Cys, Glu, Ala, Asp, Hyd, Met, Thr, Phe, Pro, Kyn, Na + Mn 2+ Fe 2+ Cu 2+ K + Al 3+ and Cl - , Br - NO 3- SO4 2- ONOO - H2O2, HClO, O2 - AChE, BChE, MAO-A, and MAO-B were placed in separate EP tubes and shaken at 37°C for 5 hours. The resulting solutions were then added to fluorescent cuvettes and detected on a fluorescence spectrometer. MAO-B significantly increased the fluorescence intensity at 560 nm, while MAO-A slightly increased it. Other analytes did not cause significant changes in fluorescence intensity. (See attached table). Figure 4 (C).

[0048] (3) Fluorescent probe solution with a final concentration of 10 μM and MAO-B with a final concentration of 50 μg / mL were added to 20% DMSO / PBS buffer solutions at different pH values. After thorough mixing, the solutions were incubated in a constant-temperature mixer for 5 h. After the reaction was completed, fluorescence spectroscopy was performed. The excitation wavelength λ of the fluorescence spectrometer was used. ex =486nm, wavelength range λ em =500-700nm, see Figure 4 (D)

[0049] Example 3: Screening Experiment for Monoamine Oxidase B Inhibitors Using Fluorescent Probes

[0050] (1) This embodiment uses the traditional Chinese medicine Cistanche deserticola as an example to study the screening of monoamine oxidase B inhibitors.

[0051] Accurately weigh each monomer from Cistanche deserticola (2'-acetylanoside; tubuloside A; tubuloside B; tubuloside F; echinacoside; verbascoside; rhodioloside; isoverascoside; Decaffeoyl acteosid), dissolve in 50% ethanol and water, and prepare 10 mM test solutions. Prepare a 10 mM 20% DMSO / PBS buffer solution at pH 7.4, and prepare a 10 mM fluorescent probe solution using DMSO.

[0052] (A) Add fluorescent probe (final concentration 10 μM, the same below) to EP tube, then add 490 μL of 20% DMSO / PBS buffer solution (pH = 7.4, concentration 10 mM, the same below) as blank control, the reaction system is 0.5 mL (the same below);

[0053] (B) Add fluorescent probe and MAO-B (final concentration of 50 μg / mL, the same below) to the EP tube, and then add 480 μL of 20% DMSO / PBS buffer solution as a model control group;

[0054] (C) Add the fluorescent probe to the EP tube, then add 10 μL of rasagiline buffer solution (final concentration is 100 μM), and finally add 475 μL of 20% DMSO / PBS buffer solution to make a positive control group;

[0055] (D) Take 10 μL of different monomer test solutions into EP tubes, add fluorescent probe and MAO-B respectively, and then add 475 μL of 20% DMSO / PBS buffer solution.

[0056] (E) Place the prepared samples at 37°C and shake for 5 hours, and use a fluorescence spectrophotometer to measure the fluorescence intensity of each solution.

[0057] Compared to other monomers, echinacoside exhibited the best inhibitory activity. (See...) Figure 5 (A)(Blanks have been removed).

[0058] (2) In this embodiment, the level of echinacoside inhibiting MAO-B activity was also investigated.

[0059] The total volume of the reaction system solution was 500 μL. Echinacea glycoside solutions with final concentrations of 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, and 700 μM were added. MAO-B with a final concentration of 50 μg / mL was added, and the mixture was thoroughly mixed. The mixture was incubated at 37 °C for 30 min. Then, a fluorescent probe solution with a final concentration of 10 μM was added, and the mixture was thoroughly mixed. The mixture was then transferred to a constant-temperature mixer and incubated for 5 h. After the reaction was complete, fluorescence spectroscopy was performed. ex =486nm, λ em =500-700nm. Experimental results show that as the concentration of echinacoside increases, the activity of MAO-B decreases, with an IC50 value of 0.15mM. See... Figure 5 (B)

[0060] Example 4: Probe toxicity to cells and cell confocal imaging

[0061] (1) CCK-8 assay for the cytotoxicity of fluorescent probes

[0062] After cell digestion, centrifugation, and resuspending, cell counting was performed, and the cell density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates, and incubated overnight at 37°C with 5% CO2. The next day, cells showed good growth. Different concentrations of fluorescent probes were then added and incubated for another 24 hours. After incubation, 10% CCK-8 solution was prepared, the original intervention medium was removed, and 100 μL of CCK-8 solution was added to each well. Cells were incubated at 37°C for 2 hours, and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability. Figure 6 As shown in (A), the cell survival rate was generally over 75% at different concentrations, proving that the probe has low toxicity.

[0063] (2) Cell confocal imaging

[0064] PC12 cells were seeded into confocal culture dishes, and the laser confocal microscope was used with an excitation wavelength of λ. ex =488nm, emission wavelength collection range λ em =500-700nm.

[0065] (A) Blank control group: The culture medium contained only 10 μM fluorescent probe and other compounds, and the cells were not treated in any way;

[0066] (B) Model group: PC12 cells containing 500 μM MPP + Pre-incubate in the culture medium for 12 h, wash three times with PBS, add the culture medium containing 10 μM fluorescent probe to the culture dish, incubate at 37℃ for 4 h, then wash three times with PBS, and add phenol red-free culture medium; Figure 6 As shown in (B), after successful modeling, the probe is added and red fluorescence is displayed during confocal imaging, proving that the probe has good water solubility.

[0067] Comparative Example 1

[0068] The difference between this comparative example and Example 1 is that DMF is used instead of dichloromethane in step (3) of the preparation method, while other conditions remain unchanged.

[0069] Experimental results show that using DMF as the solvent in step (3) of the preparation method will generate black impurities that are difficult to remove and cannot yield the final product.

[0070] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A fluorescent probe for screening monoamine oxidase B inhibitors, characterized in that, The structural formula of the fluorescent probe is: 。 2. The method for preparing the fluorescent probe for screening monoamine oxidase B inhibitors according to claim 1, characterized in that, The specific steps are as follows: (1) Dissolve isophorone in ethanol, add malononitrile and catalyst in sequence, reflux under nitrogen protection, cool to room temperature, stand overnight, filter, and dry to obtain compound 1; (2) Compound 1, p-hydroxybenzaldehyde and piperidine were dissolved in ethanol and refluxed under nitrogen protection. After the reaction was completed, the mixture was purified by silica gel column chromatography to obtain compound 2. (3) Dissolve 4-bromopyridine in dichloromethane, then add iodomethane dropwise, stir at room temperature, wash, and obtain compound 3; (4) Dissolve compound 2 and sodium ethoxide in dichloromethane, stir and add compound 3 dropwise. Stir at room temperature. After the reaction is complete, perform silica gel column chromatography to purify the crude fluorescent probe. (5) Dissolve the crude fluorescent probe in dichloromethane, then add sodium borohydride, stir at room temperature, and after the reaction is complete, perform silica gel column chromatography purification and dry to obtain the fluorescent probe.

3. The method for preparing a fluorescent probe for screening monoamine oxidase B inhibitors according to claim 2, characterized in that, In step (1), the catalyst is a mixed solvent of piperidine and glacial acetic acid, with a molar ratio of piperidine to glacial acetic acid of 1:

1.

4. The method for preparing a fluorescent probe for screening monoamine oxidase B inhibitors according to claim 2, characterized in that, In step (2), the molar ratio of compound 1 to p-hydroxybenzaldehyde is 1:

1.

5. The method for preparing a fluorescent probe for screening monoamine oxidase B inhibitors according to claim 2, characterized in that, In step (3), the molar ratio of 4-bromopyridine to iodomethane is 1:

2.

6. The method for preparing a fluorescent probe for screening monoamine oxidase B inhibitors according to claim 2, characterized in that, In step (4), the molar ratio of compound 2: sodium ethoxide: compound 3 is 5:6:

6.

7. The method for preparing a fluorescent probe for screening monoamine oxidase B inhibitors according to claim 2, characterized in that, In step (5), the ratio of crude fluorescent probe to sodium borohydride is 1:4 in molar ratio.

8. The method for preparing a fluorescent probe for screening monoamine oxidase B inhibitors according to claim 2, characterized in that, In steps (2), (4), and (5), a mixed solvent of ethanol and dichloromethane is used as the eluent. In steps (2) and (4), the volume ratio of ethanol to dichloromethane is 1:

100. In step (5), the volume ratio of ethanol to dichloromethane is 1:

50.

9. The use of the fluorescent probe for screening monoamine oxidase B inhibitors as described in claim 1 in the screening of monoamine oxidase B inhibitors.

10. The application according to claim 9, characterized in that, The application of the fluorescent probe described above for screening monoamine oxidase B inhibitors in screening monoamine oxidase B inhibitors in traditional Chinese medicine.

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