Chemical probe as well as preparation method and application thereof in detection of alpha-synuclein mutant aggregate

Abstract

The invention belongs to the technical field of protein detection, and particularly relates to a chemical probe, a preparation method of the chemical probe and application of the chemical probe to detection of alpha-synuclein mutant aggregates. The chemical probe can specifically recognize the alpha-synuclein E46K mutant aggregate, and after the chemical probe is combined with the alpha-synuclein E46K mutant aggregate, a fluorescence signal can be remarkably enhanced, so that the alpha-synuclein E46K mutant aggregate is detected; and the method is not interfered by other amyloid protein aggregates (such as A beta protein aggregates, Tau protein aggregates or TDP-43 protein aggregates and the like).

Description

Technical Field

[0001] This invention belongs to the field of protein detection technology, specifically relating to chemical probes and their preparation methods, and their application in detecting α-synuclein mutant aggregates. Background Technology

[0002] Neurodegenerative diseases are a group of neurological disorders characterized by the progressive loss of neuronal structure and function, including Parkinson's disease, Alzheimer's disease, and Lewy body dementia. Their incidence is increasing annually with population aging, placing a heavy burden on patients' families and the social healthcare system. Abnormal aggregation of α-synuclein is a core pathological feature of synuclein diseases such as Parkinson's disease and Lewy body dementia, and its aggregates (such as Lewy bodies and Lewy neurites) are considered important biomarkers for disease diagnosis and monitoring disease progression. Therefore, achieving highly specific and sensitive detection of α-synuclein aggregates is of great significance for the early diagnosis, drug development, and pathological mechanism research of neurodegenerative diseases.

[0003] Currently, methods for detecting α-synuclein aggregates mainly include immunohistochemistry, Western blot, enzyme-linked immunosorbent assay (ELISA), and fluorescent probe labeling. Immunohistochemistry relies on specific antibodies, which, while enabling targeted recognition, suffers from limitations such as complex operation, the need for sample fixation or fragmentation, and difficulty in real-time in vivo detection. Fluorescent probe methods have gained widespread attention due to their ease of operation and real-time imaging capabilities. Commonly used probes, such as thioflavin (T / S) and Congo red derivatives, generate fluorescent signals by binding to the β-sheet structure of amyloid protein. However, existing fluorescent probes generally suffer from insufficient targeting specificity, often exhibiting cross-reactivity with other types of amyloid aggregates (such as Aβ and Tau protein aggregates in Alzheimer's disease, or TDP-43 protein aggregates in frontotemporal dementia), leading to false positives and failing to meet the need for accurate identification of α-synuclein aggregates in complex pathological samples.

[0004] Furthermore, α-synuclein mutants exacerbate the abnormal aggregation and neurotoxicity of α-synuclein. Studies have shown that the E46K mutation is the most toxic to primary neurons in rats compared to wild-type and other mutants. The E46K mutation promotes the pathogenicity of α-synuclein mutant fibers. Therefore, achieving specific detection of wild-type or E46K mutant α-synuclein aggregates is crucial for understanding the pathological mechanisms of sporadic and familial Parkinson's disease.

[0005] Addressing the specificity challenge in detecting α-synuclein mutant aggregates, the development of molecular probes capable of distinguishing α-synuclein E46K mutants from other amyloid aggregates has become a research hotspot in the field. An ideal detection probe must possess high affinity, high specificity, good biocompatibility, and optical signal response to achieve accurate identification and quantitative analysis of α-synuclein E46K mutant aggregates in pathological samples. However, current technologies lack mature probes that can completely eliminate interference from other amyloid aggregates. Therefore, developing a detection tool for α-synuclein E46K mutant aggregates with both high specificity and high sensitivity has significant clinical value and scientific importance for advancing the diagnosis and research of neurodegenerative diseases. Summary of the Invention

[0006] Based on this, the present invention provides a chemical probe that can specifically recognize α-synuclein (hereinafter also referred to as α-synuclein) E46K mutant aggregates and is not interfered with by other amyloid protein aggregates (such as Aβ protein aggregates, Tau protein aggregates or TDP-43 protein aggregates, etc.).

[0007] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0008] This invention provides a chemical probe, the structural formula of which is shown below:

[0009]

[0010] Wherein, R is selected from alkyl groups.

[0011] Preferably, in the above chemical probe, the number of carbon atoms in the alkyl group is 1 to 5.

[0012] More preferably, in the above chemical probe, the alkyl group is methyl.

[0013] Another aspect of the present invention provides a method for preparing the above-mentioned chemical probe, the method comprising: mixing and reacting intermediate 4 and intermediate 5 to obtain the chemical probe;

[0014] The structural formulas of intermediate 4 and intermediate 5 are as follows:

[0015] Intermediate 4:

[0016] Intermediate 5:

[0017] Preferably, in the above preparation method, the preparation method of intermediate 4 includes:

[0018] (1) Intermediate 1 and boron trifluoride diethyl ether are mixed and reacted to obtain intermediate 2;

[0019] (2) Intermediate 2, intermediate 3 and tetrahydroquinoline react to obtain intermediate 4.

[0020] The structural formulas of intermediate 1, intermediate 2, and intermediate 3 are as follows:

[0021] Intermediate 1:

[0022] Intermediate 2:

[0023] Intermediate 3:

[0024] In another aspect, the present invention provides a detection reagent for detecting α-synuclein mutant aggregates, comprising the aforementioned chemical probe.

[0025] Preferably, the above-mentioned detection reagents also include PBS solution and DMSO.

[0026] More preferably, the volume ratio of the above PBS solution to DMSO is (8-10):1.

[0027] Preferably, the concentration of the chemical probe in the above-mentioned detection reagent is 0.5 μM to 1.5 μM.

[0028] In another aspect, the present invention provides an application of the above-mentioned chemical probe or the above-mentioned detection product, the application including:

[0029] (i) The use of chemical probes or detection products in the in vitro detection of α-synuclein mutant aggregates, wherein the α-synuclein mutant is the E46K mutant;

[0030] (ii) Application of chemical probes or detection products in the preparation of disease diagnostic reagents, wherein the disease is a disease with α-synuclein mutant aggregates as biomarkers, and the α-synuclein mutant is the E46K mutant.

[0031] The beneficial effects of this invention include: the chemical probe provided by this invention can specifically recognize α-synuclein E46K mutant aggregates, and can significantly enhance the fluorescence signal after binding to α-synuclein E46K mutant aggregates, thereby realizing the detection of α-synuclein E46K mutant aggregates; and is not affected by other amyloid protein aggregates (such as Aβ protein aggregates, Tau protein aggregates, or TDP-43 protein aggregates). Attached Figure Description

[0032] Figure 1 The UV absorption spectra of probe CLead1 in different solvents;

[0033] Figure 2The fluorescence emission spectra of probe CLead1 in different solvents;

[0034] Figure 3 The UV absorption spectrum of probe CLead1 co-incubated with α-synuclein E46K mutant aggregates;

[0035] Figure 4 The fluorescence emission spectra of probe CLead1 and monomers and aggregates of α-synuclein E46K mutant are shown.

[0036] Figure 5 The fluorescence response of probe CLead1 to protein aggregates of different concentrations (0-5 μM) is shown.

[0037] Figure 6 The fluorescence response of probe CLead1 to different protein aggregates;

[0038] Figure 7 The affinity test results for probe CLead1 with α-synuclein E46K mutant aggregates;

[0039] Figure 8 For comparison, the chemical structure of probe Clead1A is required;

[0040] Figure 9 To compare the fluorescence response of probe CLead1A with aggregates of Aβ or α-synuclein E46K mutants;

[0041] Figure 10 For comparison, the chemical structure of probe CRANAD15 is required;

[0042] Figure 11 The fluorescence response of different probes to α-synuclein E46K mutant aggregates is shown. Detailed Implementation

[0043] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0044] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0045] In this invention, the terms "testing reagent" or "diagnostic reagent" are used in a broad sense, that is, including reagents, kits or other products used for testing or diagnosis.

[0046] In a first aspect, embodiments of the present invention provide a chemical probe, the structural formula of which is shown below:

[0047]

[0048] Wherein, R is selected from alkyl groups.

[0049] It should be noted that the chemical probe in this invention can specifically recognize α-synuclein E46K mutant aggregates. After binding to α-synuclein E46K mutant aggregates, it can significantly enhance the fluorescence signal, thereby realizing the detection of α-synuclein E46K mutant aggregates. Moreover, it is not affected by other amyloid protein aggregates (such as Aβ protein aggregates, Tau protein aggregates, or TDP-43 protein aggregates).

[0050] In some specific examples, the number of carbon atoms in the alkyl group in the above chemical probes is 1 to 5.

[0051] It should be noted that the number of carbon atoms in the alkyl group in this invention can be 1 to 5, that is, the alkyl group can be methyl, ethyl, propyl, butyl, or pentyl. Furthermore, it should be understood that propyl includes n-propyl or isopropyl; butyl includes n-butyl, isobutyl, sec-butyl, tert-butyl, and other isomers; and pentyl includes n-pentyl, sec-pentyl, isopentyl, tert-pentyl, 2-methylbutyl, 3-methylbutyl, or 1-methylbutyl, and other isomers.

[0052] In some specific examples, the alkyl group in the above chemical probe is methyl.

[0053] It should be noted that the alkyl group in this invention is preferably methyl. When the alkyl group is methyl, the chemical probe exhibits a greater fluorescence enhancement factor after binding to the α-synuclein E46K mutant aggregate, and the Kd value of binding to the α-synuclein E46K mutant aggregate is smaller.

[0054] Secondly, embodiments of the present invention provide a method for preparing the above-mentioned chemical probe, the method comprising: mixing and reacting intermediate 4 and intermediate 5 to obtain the chemical probe;

[0055] The structural formulas of intermediate 4 and intermediate 5 are as follows:

[0056] Intermediate 4:

[0057] Intermediate 5:

[0058] In some specific examples, the preparation method of intermediate 4 in the above preparation method includes:

[0059] (1) Intermediate 1 and boron trifluoride diethyl ether are mixed and reacted to obtain intermediate 2;

[0060] (2) Intermediate 2, intermediate 3 and tetrahydroquinoline react to obtain intermediate 4.

[0061] The structural formulas of intermediate 1, intermediate 2, and intermediate 3 are as follows:

[0062] Intermediate 1:

[0063] Intermediate 2:

[0064] Intermediate 3:

[0065] It should be noted that the chemical probes in this invention can be prepared using the methods listed above. Of course, it should be understood that the chemical probes in this invention can also be prepared using other methods.

[0066] Thirdly, embodiments of the present invention provide a detection reagent for detecting α-synuclein E46K mutant aggregates, which includes the aforementioned chemical probe.

[0067] It should be noted that, as described above, the chemical probe of this invention can efficiently recognize α-synuclein E46K mutant aggregates. Therefore, α-synuclein E46K mutant aggregates can be prepared into detection reagents by adding other auxiliary reagents. It should be understood that other auxiliary reagents, either individually or together with the chemical probe, can be packaged according to the specific detection requirements.

[0068] In some specific examples, the above-mentioned test reagents also include PBS solution and DMSO.

[0069] It should be noted that, as described above, the α-synuclein E46K mutant aggregates are prepared into a detection reagent for the detection of α-synuclein E46K mutant aggregates by adding other auxiliary reagents. The auxiliary reagents are those known in the art, such as PBS solution and DMSO. Specifically, the PBS solution refers to all PBS solutions known in the art. It should be understood that the main functions of the PBS solution and DMSO are to dissolve the chemical probe and to establish a suitable protein detection environment.

[0070] In some specific examples, the volume ratio of the above PBS solution to DMSO is (8-10):1.

[0071] It should be noted that the volume ratio of PBS solution to DMSO in this invention can be (8-10):1, for example 8.5:1, 9:1 or 9.5:1, etc.

[0072] In some specific examples, the concentration of the chemical probe in the above-mentioned detection reagent is 0.5 μM to 1.5 μM, such as 0.7 μM, 1 μM or 1.3 μM.

[0073] Fourthly, embodiments of the present invention provide an application of the above-described chemical probe or the above-described detection product, the application including:

[0074] (i) The use of chemical probes or detection products in the in vitro detection of α-synuclein E46K mutant aggregates;

[0075] (ii) Application of chemical probes or detection products in the preparation of disease diagnostic reagents, wherein the disease is a disease for which α-synuclein E46K mutant aggregates are used as biomarkers.

[0076] It should be noted that the diseases using α-synuclein E46K mutant aggregates as biomarkers in this invention are well-known in the art, such as Parkinson's disease, Alzheimer's disease, or Lewy body dementia. During diagnosis, the presence of α-synuclein E46K mutant aggregates in the blood can be detected using the chemical probes described in this invention, thereby achieving the diagnostic purpose.

[0077] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0078] In the following examples, the PBS solution (10 mM, pH 7.4) was purchased from SPERIKON (Cat.SP00104-0500).

[0079] Probe preparation

[0080] Preparation Example 1

[0081] In this embodiment of the invention, probe CLead1 is synthesized according to the following synthetic route;

[0082]

[0083] The specific preparation steps are as follows:

[0084] (1) Compound 1 (3g, 29.97mmol, 3.08mL, 1eq) was dissolved in dichloromethane, and K2CO3 (4.14g, 29.97mmol, 1eq) and BF3·Et2O (8.86g, 29.97mmol, 7.68mL, 48% purity, 1eq) were added; nitrogen was used for purging, and the mixture was stirred at 20℃ for 2h. The reaction of compound 1 was monitored by TLC until it was complete; the reaction was quenched with 50mL H2O in an ice bath and stirred at 20℃ for 0.5h. The mixture was extracted with ethyl acetate (30mL×3), the organic layers were combined and washed with saturated NaCl aqueous solution (20mL×1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50:1-0:1) to give 4.0g of yellow intermediate 2 solid, with a yield of 90.25%.

[0085] (2) Intermediate 2 (200 mg, 1.35 mmol, 1 eq) and compound 3 (216.59 mg, 1.08 mmol, 0.8 eq) were dissolved in 5 mL of acetonitrile, and acetic acid (284.19 mg, 4.73 mmol, 270.92 μL, 3.5 eq) and 1,2,3,4-tetrahydroisoquinoline (54.03 mg, 405.64 μmol, 50.78 μL, 0.3 eq) were added dropwise. The reaction solution was stirred at 60 °C for 12 h. After the reaction of intermediate 2 was completed by LCMS analysis, 30 mL of H2O was added for dilution and extraction with ethyl acetate (15 mL × 3). The organic layers were combined and washed with saturated NaCl aqueous solution (25 mL × 2). The mixture was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1-1:1) to give 100.0 mg of red intermediate 4 solid, with a yield of 22.4%.

[0086] (3) Intermediate 4 (50 mg, 151.45 μmol, 1 eq) and compound 5 (35.48 mg, 227.18 μmol, 30.99 μL, 1.5 eq) were dissolved in 5 mL of ethyl acetate, and tributyl borate (69.71 mg, 302.91 μmol, 81.73 μL, 2 eq) and n-butylamine (3.32 mg, 45.44 μmol, 4.49 μL, 0.3 eq) were added dropwise. The reaction was stirred at 60 °C for 8 h, and LCMS analysis showed that intermediate 4 had completed the reaction. The solvent was evaporated under reduced pressure, and the compound was separated using preparative liquid chromatography (column: WePure Biotech XPtC18).

[0087] 150×40×7μm; mobile phase A: H2O (10mM NH4HCO3), mobile phase B: MeCN; elution gradient: 75%-95% B0-8.0min), yielded 10.0mg of red CLead1 solid, yield 12.83%.

[0088] Probe characterization

[0089] The probe CLead1 (hereinafter referred to as probe CLead1) prepared in Preparation Example 1 was subjected to proton nuclear magnetic resonance spectroscopy, carbon-13 nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry, respectively. The results are shown below:

[0090] 1 H NMR (400MHz, DMSO-d6): δppm 8.70-8.85(m,2H),8.41(d,J=8.5Hz,1H),8.28(d,J=8.8Hz,1H),8.12-8.22(m,3H),8.02-8.09(m,1H),7.94-8.01(m,1H) ,7.57-7.74(m,5H),7.45-7.54(m,2H),7.33(d,J=15.6Hz,1H),7.02(s,1H),4.44(q,J=6.9Hz,2H),1.52(t,J=6.9Hz,3H).

[0091] 13 C NMR (100MHz, DMSO-d6): δ179.50,159.30,142.21,139.85,133.92,133.01,132.72,129.09,12 8.20,127.11,126.32,125.15,124.86,124.48,115.31,114.80,102.70,65.58,53.71,15.20.

[0092] HR-MS(m / z): calc'd for [M+Na] + C 29 H 23 BF2NaO3491.1606, found 491.1613.

[0093] In addition, the optical properties of probe CLead1 in different solvents were tested, as follows:

[0094] The probe CLead1 was prepared as a DMSO stock solution (concentration 5 mM). At room temperature, the DMSO stock solution was diluted in quartz cuvettes with PBS (10 mM, pH 7.4), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), dichloromethane (DCM), ethyl acetate (EA), ethanol (EtOH), methanol (MeOH), and glycerin (glycerin) to a final concentration of 10 μM. The absorption spectra were then measured using a UV spectrophotometer, and the maximum absorption wavelength was recorded. The emission spectra were also measured using a fluorescence spectrophotometer, and the maximum emission wavelength was recorded. The UV absorption spectra of probe CLead1 in different solvents are shown below. Figure 1 As shown, the fluorescence emission spectra in different solvents are as follows: Figure 2 As shown in the figure, the results indicate that the maximum absorption wavelength and maximum emission wavelength of the probe are affected by the solvent, and the wavelength redshifts as the polarity of the solvent increases.

[0095] Specifically, CLead1 possesses a DAD conjugated system, with two naphthyl groups acting as electron donors (D) and difluoroborate esters acting as electron acceptors (A). Like most difluoroborate derivatives, CLead1 exhibits intramolecular charge transfer (ICT) photophysical properties. CLead1 displays extremely weak fluorescence in PBS, but exhibits strong fluorescence signals in nonpolar solvents such as dichloromethane, ethyl acetate, or glycerol due to its ICT properties. This indicates that the fluorescence intensity of CLead1 depends on the polarity of the solvent, suggesting its sensitivity to changes in the hydrophobicity of the surrounding environment caused by protein aggregation. PBS solution, as a commonly used buffer solution, maintains pH stability and has minimal interference with biomolecules (such as proteins), reducing protein denaturation or activity loss during detection. Although its UV absorption intensity is not the highest, in biological detection scenarios, compatibility and low interference are more critical than signal intensity alone; therefore, PBS solution is preferred as the dilution solvent in this invention.

[0096] Probe applications

[0097] In the following examples, the preparation method of α-synuclein E46K mutant aggregates is as follows: 1 mg of α-synuclein E46K mutant monomer (Qiangyo Biotechnology, Shanghai, the same below) was dissolved in 1 mL of PBS (10 mM, pH 7.4) (final concentration 1 mg / mL), placed in a magnetic stirrer (37℃ water bath, 600 rpm, IKA), and incubated for 7 days; the formation of α-synuclein E46K mutant aggregates was verified by ThT.

[0098] Application Example 1

[0099] In this embodiment of the invention, the UV absorption spectrum of probe CLead1 in a PBS solution containing 10% DMSO (i.e., the volume ratio of DMSO to PBS solution is 1:9, the same applies below) was tested; the details are as follows:

[0100] At room temperature, probe CLead1 was dissolved in 10 mM PBS solution containing 10% DMSO into a quartz microcuvette (10 μM). Then, 28.2 μL of 10 μM α-synuclein E46K mutant aggregate (labeled Probe+α-synaggregates) and 28.2 μL of PBS solution (labeled Probe) were added, resulting in a final volume of 200 μL and a final probe concentration of 10 μM. The mixture was incubated at room temperature for 1 h, and the UV absorption spectrum was recorded using a UV spectrophotometer. The results are as follows: Figure 3 As shown, after co-incubation of the probe CLead1 with the α-synuclein E46K mutant aggregate, the maximum absorption wavelength exhibits a blue shift. The results indicate that CLead1 binds to the hydrophobic region of the protein, and the probe's maximum absorption wavelength shifts to blue in response to changes in the surrounding environment.

[0101] Application Example 2

[0102] In this embodiment of the invention, the fluorescence emission spectrum of probe CLead1 in PBS solution containing 10% DMSO was tested and compared with that of the α-synuclein E46K mutant monomer; details are as follows:

[0103] At room temperature, probe CLead1 was dissolved in PBS solution (10 mM, pH 7.4) containing 10% DMSO into a quartz microcuvette (1 μM). Then, 2 μM of α-synuclein E46K mutant monomer (labeled Probe+α-syn Monomer), 5.6 μL of α-synuclein E46K mutant aggregate (labeled Probe+α-syn aggregates), and 5.6 μL of PBS solution (labeled Probe only) were added, resulting in a final volume of 200 μL and a final probe concentration of 1 μM. After incubation at room temperature for 1 h, the fluorescence emission spectrum was recorded using a fluorescence spectrophotometer. The results are shown below. Figure 4 As shown.

[0104] In addition, the fluorescence changes after the probe and the α-synuclein E46K mutant monomer and the α-synuclein E46K mutant aggregate were calculated, and the results are shown in Table 1. The fluorescence growth factor was calculated as: fluorescence of probe and protein binding / fluorescence of probe added to PBS solution (the same below).

[0105] Table 1. Fluorescence growth of α-synuclein E46K mutant monomers / α-synuclein E46K mutant aggregates.

[0106] Analyte (2 μM) Fluorescence growth factor α-synuclein E46K mutant monomer 1.0 α-synuclein E46K mutant aggregate 3.5

[0107] From Table 1 and Figure 4 It can be seen that, compared with the probe group and the protein monomer co-incubation group, the fluorescence was significantly enhanced after the probe and protein aggregate were co-incubated for 1 hour.

[0108] Application Example 3

[0109] This invention tested the fluorescence response of probe CLead1 to aggregates of α-synuclein E46K mutants at different concentrations; details are as follows:

[0110] (1) At room temperature, the probe CLead1 was dissolved in PBS solution containing 10% DMSO, and then different concentrations of α-synuclein E46K mutant aggregates were added to make the final concentrations of α-synuclein E46K mutant aggregates 0, 0.1 μM, 0.5 μM, 1 μM, 2 μM and 5 μM. The mixture was incubated at room temperature for 1 h to obtain the reaction solution.

[0111] (2) Transfer the reaction solution to 384-well plates (final volume 50 μL, n=3) and use a microplate reader (PerkinElmerVICTOR Nivo) to read the solution. TM ) Test the fluorescence signal of each well;

[0112] The results are as follows Figure 5 As shown, after the probe was incubated with aggregates of different concentrations of α-synuclein E46K mutant for 1 h, the fluorescence signal showed a linear increase, indicating that the fluorescence change depended on the concentration of α-synuclein E46K mutant aggregates.

[0113] Application Example 4

[0114] In this embodiment of the invention, the Kd value of the probe CLead1 and the α-synuclein E46K mutant aggregate was tested and calculated; the details are as follows:

[0115] (1) At room temperature, the α-synuclein E46K mutant aggregate was prepared into a solution with a final concentration of 1 μM using PBS solution containing 10% DMSO.

[0116] (2) Dispense the solutions into 96-well plates with black background and black frame, and then add different volumes of probe CLead1 (CLead1 is diluted with PBS solution containing 10% DMSO to 1mM) stock solution to make the final concentration of probe CLead1 0-1000nM, and incubate at room temperature for 1h.

[0117] (3) After incubation, use an ELISA reader to collect the fluorescence intensity at the maximum emission wavelength, plot the obtained data and calculate the Kd value;

[0118] The results are as follows Figure 7 As shown in the figure, the results indicate that the Kd value of the probe CLead1 aggregate with the α-synuclein E46K mutant is 218.8 nM.

[0119] Application Comparative Example 1

[0120] This invention comparatively tested the fluorescence response of probe CLead1 with other different protein aggregates, and compared it with the α-synuclein E46K mutant aggregate; the details are as follows:

[0121] (1) At room temperature, probe CLead1 (final concentration 1 μM) was dissolved in PBS solution (10 mM, pH 7.4) containing 10% DMSO. Then, α-synuclein E46K mutant aggregates (1.4 μL), Aβ protein aggregates (1.8 μL), Tau protein aggregates (1.8 μL), TDP-43 protein aggregates (5 μL), BSA protein (BSA), and PBS solution (5 μL, blank control, probe only) were added to a final volume of 50 μL. The final concentration of the probe was 1 μM, and the final concentration of the protein aggregates was 2 μM. The mixture was incubated at room temperature for 1 h to obtain the reaction solution. The amount of different protein aggregates added was specified, and the final volume of the reaction solution was 50 μL.

[0122] (2) Transfer each reaction solution to a 384-well plate (final volume 50 μL, n = 3), and test the fluorescence signal of each well using a microplate reader (e.g., ...). Figure 6 (as shown in the figure), and the fluorescence growth fold of the remaining groups was calculated. The results are shown in Table 2.

[0123] Table 2. Fluorescence growth of probe CLead1 on different protein aggregates.

[0124] Analyte (2 μM) Fluorescence growth factor α-synuclein E46K mutant aggregate 2.7 Aβ protein aggregates 0.9 Tau protein aggregates 1.1 TDP-43 protein aggregates 1.1 BSA protein 1.0

[0125] As shown in Table 2 above, the fluorescence growth fold of the probe CLead1 of the present invention after binding to the α-synuclein E46K mutant aggregate is much higher than that of other protein aggregates, which indicates that the probe CLead1 of the present invention has strong selectivity for the α-synuclein E46K mutant aggregate.

[0126] Application Comparative Example 2

[0127] This invention comparatively tested the probe CLead1A (structural formula see...). Figure 8 The fluorescence response of the α-synuclein E46K mutant aggregate and the Aβ protein aggregate are as follows:

[0128] (1) At room temperature, probe CLead1A (final concentration 1 μM) was dissolved in PBS solution containing 10% DMSO (10 mM, pH 7.4). Then, 2 μM α-synuclein E46K mutant aggregates (5.6 μL, α-syn aggregates), 2 μM Aβ protein aggregates (7.2 μL, Aβ aggregates), and PBS solution (7.2 μL, blank control, probe only) were added to a final volume of 200 μL, and the final probe concentration was 1 μM. Different reaction solutions were obtained by incubation at room temperature for 1 h. The fluorescence emission spectra were recorded using a fluorescence spectrophotometer.

[0129] (2) The results are as follows Figure 9 As shown in the results, there was no change in fluorescence when CLead1A was co-incubated with the α-synuclein E46K mutant aggregate.

[0130] Application Comparative Example 3

[0131] This invention comparatively tested the fluorescence response of other probes to α-synuclein E46K mutant aggregates and Aβ protein aggregates, respectively; details are as follows:

[0132] (1) At room temperature, probe CLead1 (final concentration 1 μM) and probe CRANAD15 (structural formula shown) were dissolved in PBS solution containing 10% DMSO (10 mM, pH 7.4). Figure 10 The final concentration was 1 μM. Then, α-synuclein E46K mutant aggregate (1.4 μL, probe + α-syn aggregates), Aβ protein aggregate (1.8 μL, probe + Aβ aggregate), and PBS solution (1.8 μL, blank control, probe only) were added to a final volume of 50 μL. The final concentration of the probe was 1 μM and the final concentration of the protein aggregate was 2 μM. The solutions were incubated at room temperature for 1 h to obtain different reaction solutions.

[0133] (2) Transfer each reaction solution to a 384-well plate (final volume 50 μL, n = 3), and test the fluorescence signal of each well using a microplate reader (e.g., ...). Figure 11 (As shown).

[0134] In addition, the Kd value of the binding of probe CRANAD15 to the α-synuclein E46K mutant aggregate was tested and calculated according to the method of application example 4 above, and compared with probe CLead1. The results are shown in Table 3 below.

[0135] Table 3. Kd values ​​of different probes binding to α-synuclein E46K mutant aggregates

[0136] probe Kd(α-synaggregates) CLead1 218.8nM CRANAD15 ND

[0137] Based on Table 3 above Figure 11 It can be seen that CRANAD15 did not show significant fluorescence enhancement after binding to the α-synuclein E46K mutant aggregate, and the Kd value was not significant; indicating that even if the probes CLead1 and CRANAD15 have some structural similarities... The ability to detect α-synuclein E46K mutant aggregates varies significantly depending on the other functional groups.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A chemical probe, the structural formula of which is shown below: in, R is selected from alkyl groups.

2. The chemical probe according to claim 1, characterized in that, Alkyl groups have 1 to 5 carbon atoms.

3. The chemical probe according to claim 1, characterized in that, The alkyl group is methyl.

4. The method for preparing the chemical probe according to any one of claims 1 to 3, characterized in that, The preparation method includes: reacting intermediate 4 and intermediate 5 together to obtain a chemical probe; The structural formulas of intermediate 4 and intermediate 5 are as follows: Intermediate 4: Intermediate 5:

5. The preparation method according to claim 4, characterized in that, The preparation method of intermediate 4 includes: (1) Intermediate 1 and boron trifluoride diethyl ether are mixed and reacted to obtain intermediate 2; (2) Intermediate 2, intermediate 3 and tetrahydroquinoline react to obtain intermediate 4. The structural formulas of intermediate 1, intermediate 2, and intermediate 3 are as follows: Intermediate 1: Intermediate 2: Intermediate 3:

6. A detection reagent for detecting α-synuclein mutant aggregates, characterized in that, Includes the chemical probe described in any one of claims 1 to 3.

7. The detection reagent according to claim 6, characterized in that, The test reagents also include PBS solution and DMSO.

8. The detection reagent according to claim 7, characterized in that, The volume ratio of PBS solution to DMSO is (8-10):

1.

9. The detection reagent according to any one of claims 6 to 8, characterized in that, The concentration of the chemical probe is 0.5 μM to 1.5 μM.

10. The application of the chemical probe according to any one of claims 1 to 3 or the detection product according to any one of claims 6 to 8, wherein the application includes: (i) The use of chemical probes or detection products in the in vitro detection of α-synuclein mutant aggregates, wherein the α-synuclein mutant is the E46K mutant; (ii) Application of chemical probes or detection products in the preparation of disease diagnostic reagents, wherein the disease is a disease with α-synuclein mutant aggregates as biomarkers, and the α-synuclein mutant is the E46K mutant.

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