Probe molecules capable of selectively combining / marking amyloid protein, preparation method and application

Abstract

The invention discloses a probe molecule capable of selectively binding / marking amyloid protein, a preparation method and application of the probe molecule in brain slices of mice suffering from neurodegenerative diseases, and the probe molecule has a structure as shown in a formula I. In the formula I, the value range of n is 0 or 1; r is selected from at least one of p-aminobenzene, p-dimethylaminobenzene, julolidine and 2-dimethylaminonaphthalene. The invention designs a series of photosensitive labeling probes with amyloid variant protein binding capacity. The probe can generate a large amount of active oxygen under the condition of illumination. A marking substrate with nucleophilicity is added into a reaction system, and the probe can realize selective marking of amyloid protein. The probe can realize component analysis of amyloidosis protein in brain slices of mice suffering from Alzheimer's disease and component comparison of amyloidosis protein of mice with different neurodegenerative disease models.

Description

Technical Field

[0001] This application relates to a class of probe molecules that selectively bind to / label amyloid proteins, their preparation method, and their application in brain slices from mice with neurodegenerative diseases, belonging to the field of proteomics. Background Technology

[0002] Amyloid protein probes are a class of small molecules that can selectively bind to the cross-beta-sheet structure of proteins. When not bound to a protein, the probe itself is non-fluorescent. However, when the molecule binds to amyloid protein, the rotation of single bonds in the molecular structure is restricted, resulting in fluorescence and ultimately achieving specific recognition of amyloid protein. Currently, based on this principle, numerous amyloid protein recognition probes based on molecular backbones such as thiosulfinates, curcumin, and bodily compounds have been reported.

[0003] The aforementioned fluorescent probes for amyloid proteins have enabled the localization and tracing of amyloid proteins in cells, tissue sections, and in vivo. This provides a research foundation for the development and progression of amyloid protein-related diseases, such as neurodegenerative diseases.

[0004] However, the probes reported above can only be used for imaging amyloid proteins, not for identifying their components. Currently, analytical techniques for amyloid protein components mainly focus on proximity labeling techniques based on antibodies or enzymes. However, these proximity labeling techniques still suffer from high false-positive rates and complex operation.

[0005] Therefore, based on the traditional probe structure that can target amyloid proteins, molecular photosensitization can be achieved for the analysis of amyloid protein components deposited in tissue sections, which has important scientific significance and clinical value for studying the occurrence and development of neurodegenerative diseases. Summary of the Invention

[0006] Existing amyloid protein probes are mainly used for intracellular, brain slice, and in vivo amyloid protein localization imaging in mice. Currently, no small molecule probes have been reported for the analysis of amyloid protein components in in vivo models. This application synthesizes a series of photosensitive labeled probes with amyloid protein binding ability. These probes generate a large amount of reactive oxygen species under light irradiation. By adding nucleophilic labeled substrates to the reaction system, these probes can achieve selective labeling of amyloid proteins. These probes can enable the component analysis of amyloid proteins in brain slices from Alzheimer's disease mice, and the comparison of amyloid protein components in different neurodegenerative disease mouse models.

[0007] According to a first aspect of this application, a class of probe molecules that selectively bind to / label amyloid proteins is provided, said probe molecules having the structure shown in Formula I:

[0008]

[0009] In Equation I, the value of n is either 0 or 1;

[0010] R is selected from at least one of p-aminobenzene, p-dimethylaminobenzene, julonidine, and 2-dimethylaminonaphthalene.

[0011] According to a second aspect of this application, a method for preparing a class of probe molecules that selectively bind to / label amyloid proteins is provided.

[0012] When n is 0, the following steps are included:

[0013] Step I: Mix benzothiazole compounds with iodomethane and an organic solvent, react I, and obtain the intermediate product;

[0014] Step II: Disperse the intermediate product and salt compound described in Step I in a solvent to form a mixture, add an aldehyde compound, and react in step II to obtain the probe molecule;

[0015] When n is 1, the following steps are included:

[0016] Step I: Mix the benzothiazole compound with iodomethane and an organic solvent, and react in step I to obtain the intermediate product;

[0017] Step II: Disperse the intermediate product from Step I in a solvent to form a mixture, add an aldehyde compound, and react in step II to obtain the probe molecule.

[0018] Optionally, in step I, the benzothiazole compound is selected from at least one of 6-bromo-2-methyl-1,3-benzothiazole and 2-amino-6-bromobenzothiazole;

[0019] The organic solvent is selected from at least one of N,N-dimethylformamide, acetonitrile, tetrahydrofuran, and ethanol.

[0020] Optionally, in step I, the molar mass ratio of the benzothiazole compound to iodomethane and the organic solvent is 2471:478:1 to 7003:6366:1.

[0021] Optionally, in step I, the structure of the intermediate product is as shown in formula II:

[0022]

[0023] In Formula II, R is selected from at least one of methyl and amino groups.

[0024] Optionally, in step I, the reaction conditions for reaction I are as follows:

[0025] The temperature of reaction I is 80–85°C;

[0026] The reaction time for reaction I is 15 to 24 hours.

[0027] Optionally, in step II, the aldehyde compound is selected from at least one of p-aminobenzaldehyde, p-dimethylaminobenzaldehyde, julonidine formaldehyde, and 6-dimethylamino-2-naphthaldehyde.

[0028] The solvent is selected from at least one of ethanol and methanol;

[0029] The catalyst is selected from at least one of tris(2-carboxyethyl)phosphine hydrochloride and piperidine;

[0030] In the mixture, the concentration of the intermediate product is 6.0–8.0 mol / L.

[0031] Optionally, the mass ratio of the intermediate product to the aldehyde compound is 1:1 to 1:1.2.

[0032] Optionally, in step II, the conditions for reaction II are as follows:

[0033] The temperature of reaction II is 70–82°C;

[0034] The reaction time for reaction II is 12 to 16 hours.

[0035] According to a third aspect of this application, the application of a class of selectively binding / labeling amyloid protein probe molecules in brain slices of mice with neurodegenerative diseases is provided, characterized in that the probe molecules are mixed with the protein, added to a buffer solution, incubated, and the fluorescence intensity is monitored.

[0036] Optionally, the protein is at least one of A-beta protein, tau-K18 protein, and TTR protein;

[0037] Optionally, when the protein is tau-K18, dithiothreitol and heparin sodium need to be added.

[0038] Optionally, the mass ratio of the probe molecule to the protein is 1:4 to 1:10.

[0039] Optionally, the concentration of the buffer solution is 0.01 mol / L.

[0040] Optionally, the incubation conditions are as follows:

[0041] The incubation time is 0.5 to 1 hour.

[0042] The incubation temperature is 25–37°C.

[0043] The beneficial effects that this application can produce include:

[0044] 1) The probe provided in this application can selectively bind to amyloid protein.

[0045] 2) The probe provided in this application can selectively label amyloid proteins.

[0046] 3) The amyloid protein components deposited in amyloid-related diseases such as models of neurodegenerative diseases provided in this application can be used for identification, but are not limited to.

[0047] The probe of this invention, while possessing the ability to target amyloid proteins, can achieve selective labeling of amyloid proteins under conditions of light and the presence of a labeled substrate. This probe enables targeted labeling and component analysis of amyloid proteins in brain slices from animal models of neurodegenerative diseases. Attached Figure Description

[0048] Figure 1 This is a diagram illustrating the binding assay of the molecule to amyloid protein prepared in Example 1 of this application.

[0049] Figure 2 An experimental diagram of amyloid protein labeling in brain slices of a neurodegenerative disease model prepared in Example 1 of this application;

[0050] Figure 3 , Figure 4 The figure shows the results of molecular analysis of the amyloid protein components and functions in the brains of different neurodegenerative disease models prepared in Example 1 of this application;

[0051] Figure 5 The above is the hydrogen NMR spectrum of the molecule prepared in Example 1 of this application. Detailed Implementation

[0052] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0053] Unless otherwise specified, the raw materials and reagents used in the embodiments of this application were all purchased commercially.

[0054] The NMR data in this embodiment were obtained using a Bruker AVANCE III 700MHz NMR spectrometer.

[0055] The confocal fluorescence microscope used in this embodiment is an Olympus FV1000 FluoView. TM Confocal microscope.

[0056] The mass spectrometer used in this embodiment is an Orbitrap Exploris 480 mass spectrometer.

[0057] According to one embodiment of this application, a compound I is provided, which is a molecule capable of selectively binding amyloid protein. Under light irradiation and a labeled substrate, this molecule can selectively label amyloid protein. The above-mentioned properties of this molecule can be used for labeling and component analysis of amyloid protein deposited in brain slices of a mouse model of neurodegenerative diseases. The compound I is selected from at least one compound having the chemical formula shown in Formula I.

[0058]

[0059] In Formula I, n represents the number of carbon atoms, which can be selected from either 0 or 1;

[0060] R is selected from: p-aminobenzene, p-dimethylaminobenzene, julonidine, 2-dimethylaminonaphthalene.

[0061] According to one embodiment of this application, a method for preparing the above-mentioned compound I is provided, comprising at least the following steps:

[0062] (1) For compounds with n=0:

[0063] Iodomethane and N,N-dimethylformamide were added to compound II, and the mixture was filtered after reaction to obtain a precipitate. This precipitate was mixed with ethylene glycol and potassium hydroxide and refluxed. The reaction product was neutralized with hydrochloric acid and extracted with chloroform to obtain an intermediate product.

[0064]

[0065] The intermediate product was dissolved in ethanol with tris(2-carboxyethyl)phosphine hydrochloride. After reacting at room temperature for 30 minutes, compound III was added, and the mixture was refluxed overnight. The final product I was obtained by filtration.

[0066] Compound III is selected from: p-aminobenzaldehyde, p-dimethylaminobenzaldehyde, and julonidine formaldehyde.

[0067] (2) For compounds with n=1:

[0068] Iodomethane and acetonitrile were added to compound IV, and the intermediate product was obtained by filtration after the reaction.

[0069]

[0070] The intermediate product and compound V were dissolved in methanol, refluxed overnight, and filtered to give the final product I.

[0071] Compound V is selected from: p-aminobenzaldehyde, p-dimethylaminobenzaldehyde, julonidine formaldehyde, and 6-dimethylamino-2-naphthaldehyde.

[0072] Example 1

[0073] 6.7 mmol of 6-bromo-2-methyl-1,3-benzothiazole and 7.37 mmol of iodomethane were dissolved in 60 mL of acetonitrile and reacted at 82 °C for 24 hours to give 0.98 g of intermediate product.

[0074] The intermediate (1.2 mmol, 0.433 g) and 9-aldehyde julonidine (1.0 mmol, 0.201 g) were dissolved in 30 mL of methanol. 400 μL of piperidine was added to the reaction mixture, and the mixture was refluxed at 70 °C for 12 h. The resulting precipitate was hot-extracted and washed three times with cold methanol to obtain probe I (0.364 g, 66% yield). The specific reaction process is shown in Figure a.

[0075]

[0076] Probe I was tested using nuclear magnetic resonance spectroscopy, and the results are as follows: δ 8.51 (s, 1H), 7.95-7.87 (m, 3H), 7.51 (s, 2H), 7.39 (d, J = 14.9 Hz, 1H), 4.11 (s, 3H), 3.39 (t, J = 5.9 Hz, 4H), 2.73 (t, J = 6.3 Hz, 4H), 1.90 (t, J = 6.1 Hz, 4H); 13 C10 NMR (176MHz, DMSO-d6) δ 170.8, 151.1, 148.7, 141.8, 131.9, 128.8, 126.4, 121.8, 121.2, 119.6, 117.4, 104.2, 50.2, 49.7, 49.1, 40.3, 39.6, 35.7, 27.4, 21.0. The molecular weight of product I was monitored using high-resolution mass spectrometry, and the results are as follows: HRMS (ESI+) m / z: [M]+ calcd 425.0682, found 425.0669. The specific 1H NMR spectrum is shown below. Figure 5 As shown.

[0077] Test Example 1

[0078] The binding of probe I to amyloid protein

[0079] Take 1.0 mmol of probe I prepared in Example 1 and mix it with 2 mg / mL (146 μM) of tau-K18 protein. Add 3 μL of 100 mmol / L dithiothreitol and 4 mL of 200 μmol / L heparin sodium to make the concentration of probe I 20 μmol / L, the concentration of tau-K18 protein 100 μmol / L, the concentration of dithiothreitol 1 mmol / L, and the concentration of heparin sodium 2.5 μmol / L. Incubate at 37°C for 72 hours and detect the fluorescence intensity of probe I. The results are as follows. Figure 1 As shown in the left figure, probe I's ability to monitor tau-K18 protein aggregation is similar to that of the classic ThT molecule of amyloid proteins.

[0080] The docking of probe I with amyloid proteins Aβ and tau was simulated using AutoDock software, and the results are as follows: Figure 1 As shown in the right figure, this probe can bind well to the cross-β-sheet structure of amyloid protein.

[0081] Test Example 2

[0082] Probe I was used to label amyloid protein deposits in brain slices from a mouse model of neurodegenerative diseases.

[0083] Frozen sections (6 μm thick) were subjected to gradient warming and washed three times with PBS buffer (0.01 M NaCl, 0.01 M KCl, 0.01 M disodium hydrogen phosphate, 0.01 M sodium dihydrogen phosphate). The target area was then circled with a PAP pen (immunohistochemistry pen). The sections were then stained with 200 μL of 20 μmol / L probe I for 30 min. Afterward, they were washed three times with PBS. Finally, the sections were incubated with 200 μL of 10 mmol / L propargyl-PEG-1-amine for 10 min. The solution was applied at a power of 25 mW·cm⁻¹. -2 After 10 minutes of exposure to white light, the slide was allowed to stand for 10 minutes. It was then washed once with PBS, followed by a click chemistry reaction (FITC-N3), washed three times with PBS, and mounted using an anti-fluorescence quenching mounting medium. The confocal imaging results are shown below. Figure 2 As shown. Red represents the fluorescence color of probe I, and green represents the fluorescence color of the click-labeled FITC, i.e., amyloid protein labeled with probe I.

[0084] Test Example 3

[0085] Probe I was used to detect amyloid protein components deposited in brain slices of a mouse model of neurodegenerative diseases.

[0086] Frozen sections (6 μm thick) were rewarmed and washed three times with PBS buffer (0.01 M NaCl, 0.01 M KCl, 0.01 M disodium hydrogen phosphate, 0.01 M sodium dihydrogen phosphate). The target area was circled with a PAP pen (immunohistochemistry pen). The sections were then stained with 200 μL of probe I at a concentration of 20 μmol / L for 30 min. They were then washed three times with PBS. Finally, the sections were incubated with 200 μL of biotin-PEG 1-amine at a concentration of 10 mmol / L for 10 min. The solution was then applied at a power of 25 mW·cm⁻¹. -2After irradiation under white light for 10 min, the tissue was allowed to stand for 10 min. The tissue was washed once with PBS, dissolved in 200 μL of 8 mol / L urea, and diluted to 1 mol / L with 50 mmol / L ammonium bicarbonate solution. Streptavidin-containing magnetic beads were added, and the mixture was incubated in a shaker at 50 rpm / min at room temperature for 1 min, repeated three times. The magnetic beads were then washed twice each with 8 mol / L urea and 2 mol / L sodium chloride. The tissue was denatured at 95 °C, cooled, and then digested with 10 μg of trypsin at 37 °C for 16 h. The supernatant was collected and desalted. The samples were lyophilized and then loaded onto mass spectrometers for data analysis. The experimental results are as follows: Figure 3 and Figure 4 As shown, compared with control mice without neurodegenerative diseases, this method identified a large number of upregulated proteins in mice with neurodegenerative diseases. Data analysis revealed that more proteins were associated with metabolic pathways, with the mitochondrial and proline metabolic pathways being the most prominent.

[0087] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A class of probe molecules that selectively bind to / label amyloid proteins, characterized in that, The probe molecule has the structure shown in Formula I: In Equation I, the value of n is either 0 or 1; R is selected from at least one of p-aminobenzene, p-dimethylaminobenzene, julonidine, and 2-dimethylaminonaphthalene.

2. A method for preparing a class of selectively binding / labeling amyloid protein probe molecules, characterized in that... The synthesis steps are simple and do not require separation and purification by chromatography. When n is 0, the following steps are included: Step I: Mix benzothiazole compounds with iodomethane and an organic solvent, react I, and obtain the intermediate product; Step II: Disperse the intermediate product and salt compound described in Step I in a solvent to form a mixture, add an aldehyde compound, and react in step II to obtain the probe molecule; When n is 1, the following steps are included: Step I: Mix the benzothiazole compound with iodomethane and an organic solvent, and react in step I to obtain the intermediate product; Step II: Disperse the intermediate product from Step I in a solvent to form a mixture, add an aldehyde compound, and react in step II to obtain the probe molecule.

3. The preparation method according to claim 2, characterized in that, In step I, the benzothiazole compound is selected from at least one of 6-bromo-2-methyl-1,3-benzothiazole and 2-amino-6-bromobenzothiazole; The organic solvent is selected from at least one of N,N-dimethylformamide, acetonitrile, tetrahydrofuran, and ethanol.

4. The preparation method according to claim 2, characterized in that, In step I, the molar mass ratio of the benzothiazole compound to iodomethane and the organic solvent is 2471:478:1 to 7003:6366:

1.

5. The preparation method according to claim 2, characterized in that, In step I, the structure of the intermediate product is shown in formula II: In Formula II, R is selected from at least one of methyl and amino groups.

6. The preparation method according to claim 2, characterized in that, In step I, the reaction conditions for reaction I are as follows: The temperature of reaction I is 80–85°C; The reaction time for reaction I is 15 to 24 hours.

7. The preparation method according to claim 2, characterized in that, In step II, the aldehyde compound is selected from at least one of p-aminobenzaldehyde, p-dimethylaminobenzaldehyde, julonidine formaldehyde, and 6-dimethylamino-2-naphthaldehyde. The solvent is selected from at least one of ethanol and methanol; The catalyst is selected from at least one of tris(2-carboxyethyl)phosphine hydrochloride and piperidine; In the mixture, the concentration of the intermediate product is 6.0–8.0 mol / L; Preferably, the molar mass ratio of the intermediate product to the aldehyde compound is 1:1 to 1:1.

2. Preferably, the conditions for reaction II are as follows: The temperature of reaction II is 70–82°C; The reaction time for reaction II is 12 to 16 hours.

8. The application of a class of selectively binding / labeling amyloid protein probe molecules in brain slices of mice with neurodegenerative diseases, characterized in that, The probe molecules were mixed with the protein, a buffer solution was added, and the mixture was incubated. The fluorescence intensity was then monitored. The probe molecule is the probe molecule of claim 1 or the probe molecule prepared by any one of the preparation methods of claims 1 to 7.

9. The application according to claim 8, characterized in that, The protein is at least one of A-beta protein, tau-K18 protein, and TTR protein; The buffer solution is a phosphate buffer solution; Preferably, when the protein is tau-K18, dithiothreitol and heparin sodium need to be added additionally; Preferably, the molar mass ratio of the probe molecule to the protein is 1:4 to 1:10; Preferably, the concentration of the buffer solution is 0.01 mol / L.

10. The application according to claim 8, characterized in that, The incubation conditions are as follows: The incubation time is 0.5 to 1 hour. The incubation temperature is 25–37°C.

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