Fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a fluorescent probe and a preparation method and application thereof.The structural formula of the fluorescent probe is shown in the formula I. The preparation method comprises the steps that a raw material A and a raw material B serve as reaction raw materials and are added into an organic solvent, then a catalyst tetrabutylammonium bromide is added, a condensation reaction is carried out under the sealing condition at the temperature of 60-80 DEG C, cyclization is carried out, and the fluorescent probe is obtained. A series of fluorescent probes are synthesized, high sensitivity and rapid response detection of A beta are realized by using a twisted intramolecular charge transfer mechanism, and meanwhile, the probe is ensured to have good biocompatibility so as to meet the requirements of early diagnosis and treatment research of Alzheimer's disease. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of probes, and particularly to a fluorescent probe, a preparation method thereof, and an application thereof. Background Art

[0002] Alzheimer's Disease (AD) is a progressive neurodegenerative disease characterized by memory loss, cognitive impairment, and behavioral abnormalities, which seriously affects the quality of life of millions of elderly people globally. Its pathological features mainly include the deposition of extracellular β-amyloid (Aβ) plaques, the formation of intracellular neurofibrillary tangles (NFTs), as well as neuronal loss and synaptic dysfunction. Among them, the abnormal aggregation of Aβ is considered to be the core link in the pathogenesis of AD.

[0003] Currently, the detection methods for Alzheimer's disease include magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), optical imaging, cerebrospinal fluid detection, and blood detection, etc., but all have some deficiencies. The sensitivity of MRI for detecting Aβ is low, the process is complex and the cost is high; PET and SPECT are expensive, the equipment accessibility is poor, the operation is complex, and there are also radiation exposure problems; although optical imaging has high sensitivity, it shows deficiencies in imaging depth, resolution, and signal-to-noise ratio; cerebrospinal fluid detection is invasive due to lumbar puncture, the patient compliance is low and the results are easily affected by errors; blood detection is simple and inexpensive, but the biomarker concentration is low and it is easily interfered, and the results of different laboratories lack consistency and standardization. Although the fluorescent probe technology has advantages, some probes have poor stability, insufficient specificity, limited imaging depth and resolution, and lack of efficient near-infrared fluorescent probes, making it difficult to achieve multi-channel dynamic real-time imaging. Therefore, developing a highly sensitive and specific Aβ protein detection tool with a simple preparation method is still a research hotspot in this field. Summary of the Invention

[0004] Objectives of the Invention: The first objective of the present invention is to provide a fluorescent probe with high sensitivity and rapid response to Aβ, and good biocompatibility at the same time; the second objective of the present invention is to provide a preparation method of the probe; the third objective of the present invention is to provide an application of the fluorescent probe in detecting Aβ protein; the fourth objective of the present invention is to provide an intermediate; the fifth objective of the present invention is to provide a preparation method of the intermediate.

[0005] Technical Solution: The fluorescent probe of the present invention has the following structural formula:

[0006]

[0007] Among them, R1 is -OR or -N-(R)2; R2 is -OR, -N-(R)2 or -COO-R, where R is a C1-C4 alkyl group.

[0008] Preferably, R1 is -N-(R)2 and R2 is -COO-R.

[0009] Preferably, the structural formula of the fluorescent probe is as follows:

[0010]

[0011] Preferably, the fluorescent probe is BTAN-MF, which has the longest fluorescence wavelength. Long-wavelength fluorescence not only has stronger tissue penetration ability but also can effectively reduce background interference, making it suitable for biological tissue imaging; the BTAN-MF probe is responsive to different aggregation forms of Aβ (such as monomers, oligomers, and fibrils), and has the strongest response to Aβ aggregates with high selectivity; the BTAN-MF probe can be used to detect Aβ plaques in brain slices of AD mice, indicating that the probe is not only suitable for in vitro experiments but also has the potential to detect Aβ in biological tissues, providing a powerful tool for the pathological study of AD.

[0012] The preparation method of the fluorescent probe described in the present invention includes the following steps:

[0013] Using raw material A and raw material B as reaction raw materials, adding them to an organic solvent, then adding the catalyst tetrabutylammonium bromide, and carrying out a condensation reaction and ring formation at 60-80°C under sealed conditions to obtain the fluorescent probe; the synthesis route is as follows:

[0014]

[0015] The application of the fluorescent probe described in the present invention in the detection of β-amyloid (Aβ). Further, the application of the fluorescent probe in the responsive detection of Aβ protein in Alzheimer's disease.

[0016] The intermediate described in the present invention has the following structural formula:

[0017]

[0018] Among them, R is a C1-C4 alkyl group.

[0019] The preparation method of the intermediate described in the present invention includes the following steps:

[0020] (1) Dissolve 6-aminobenzothiazole in an organic solvent, and then sequentially add dry triethylamine and alkyl acid to the reaction mixture. Under the protection of an inert gas, carry out a Mannich reaction to obtain the product 6-dialkylaminobenzothiazole.

[0021] (2) Under the protection of inert gas, dissolve the mixture of 6-dialkylaminobenzothiazole and base in a solvent, and carry out an alkaline ring-opening reaction. After the reaction is completed, cool the reaction solution, acidify the mixture to obtain 2-amino-5-dialkylaminobenzenethiol;

[0022] (3) Add 2-amino-5-dimethylaminobenzenethiol to an organic solvent, then add glacial acetic acid (AcOH) and malononitrile, and carry out a condensation reaction to obtain intermediate I;

[0023] The synthesis route is as follows:

[0024]

[0025] Preferably, the reaction temperature in step (1) is 80-100 °C. Preferably, the organic solvent is dry dimethyl sulfoxide (DMSO).

[0026] Preferably, in step (1), after the reaction is completed, purification is also included. The purification is by extraction and column chromatography separation. The extraction solvent is dichloromethane, the stationary phase of column chromatography is silica gel, and the mobile phase is a mixed solvent of petroleum ether-ethyl acetate.

[0027] Preferably, the reaction temperature in step (2) is 60-100 °C. Preferably, the solvent is water and the base is potassium hydroxide (KOH).

[0028] Preferably, in step (2), after the reaction is completed, extraction is also included. The extraction solvent is dichloromethane.

[0029] Preferably, the reaction temperature in step (3) is 60-100 °C. Preferably, the organic solvent is ethanol.

[0030] Preferably, in step (3), after the reaction is completed, purification is also included. The purification is by extraction and column chromatography separation. The extraction solvent is dichloromethane, the stationary phase of column chromatography is silica gel, and the mobile phase is a mixed solvent of dichloromethane - petroleum ether-ethyl acetate.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: (1) A series of fluorescent probes are synthesized in the present invention, and high-sensitivity and rapid-response detection of Aβ are achieved by using the twisted intramolecular charge transfer (TICT) mechanism, while ensuring that the probes have good biocompatibility to meet the needs of early diagnosis and treatment research of Alzheimer's disease; (2) Good biocompatibility: Preliminary studies show that the probes have good biocompatibility and are suitable for detecting biological samples, providing a safe and reliable tool for in vivo experiments; (3) Simple preparation and low cost: The preparation method is relatively simple and the cost is low, which is suitable for large-scale production and application, providing an economical and efficient solution for the clinical diagnosis and research of AD; (4) Viscosity response mechanism: The probes achieve specific detection of Aβ by responding to viscosity changes; (5) Blood-brain barrier penetration potential: The LogP value of the BTAN-MF probe is 1.56, and the molecular weight is small, having the potential to cross the blood-brain barrier and can be used in in vivo experiments (such as mouse models), providing a potential auxiliary tool for the diagnosis and treatment research of Alzheimer's disease (AD). Description of the Drawings

[0032] Figure 1 Are the ultraviolet absorption spectra of the probes BTAN-MF, BTAN-MO, BTAO-MF, BTAO-MO, BTAO-NE and BTAO-NM in DMSO;

[0033] Figure 2 Are the fluorescence spectrum of the probe BTAN-MF itself and the fluorescence response spectrum of Aβ;

[0034] Figure 3 Are the fluorescence spectrum of the probe BTAN-MO itself and the fluorescence response spectrum of Aβ;

[0035] Figure 4 Are the fluorescence spectrum of the probe BTAO-MF itself and the fluorescence response spectrum of Aβ;

[0036] Figure 5 Are the fluorescence spectrum of the probe BTAO-MO itself and the fluorescence response spectrum of Aβ;

[0037] Figure 6 Are the fluorescence spectrum of the probe BTAO-NE itself and the fluorescence response spectrum of Aβ;

[0038] Figure 7 Are the fluorescence spectrum of the probe BTAO-NM itself and the fluorescence response spectrum of Aβ;

[0039] Figure 8 Is the response curve of the BTAN-MF probe to Aβ;

[0040] Figure 9Viscosity response of the BTAN-MF probe in solution systems with different viscosities prepared from glycerol and ethylene glycol, and the corresponding fitted straight lines;

[0041] Figure 10 Selectivity of the BTAN-MF probe towards different ions and biomolecules;

[0042] Figure 11 Cell viability after incubation of the probe BTAN-MF at different concentrations with PC12 cells;

[0043] Figure 12 Fluorescence images of brain tissue sections of APP mice stained with BTAN-MF. Detailed implementation mode

[0044] The technical solution of the present invention will be further described below in conjunction with the embodiments.

[0045] Example 1

[0046] The intermediate of the present invention has the following structural formula:

[0047]

[0048] The synthesis route is as follows:

[0049]

[0050] The preparation method includes the following steps:

[0051] (1) Synthesis of compound 6-dimethylaminobenzothiazole

[0052] First, dissolve 6-aminobenzothiazole (1 g, 6.7 mmol) in dry dimethyl sulfoxide (DMSO). Subsequently, sequentially add dry triethylamine (3.5 mL, 25 mmol) and formic acid (1.2 mL, 30 mmol). Under nitrogen protection, heat the reaction mixture to 125 °C (oil bath temperature) and continuously stir for 72 hours until the reaction is complete. After the reaction is completed, cool the mixture to room temperature, adjust it to pH = 3 with 10% aqueous sodium hydroxide solution, then extract the reaction mixture with dichloromethane, combine the organic layers and wash them with brine. Dry the combined organic layers with anhydrous sodium sulfate (Na2SO4), and finally evaporate the solvent under reduced pressure to obtain the product 6-dimethylaminobenzothiazole.

[0053] (2) Synthesis of compound 2-amino-5-dimethylaminobenzenethiol

[0054] Under the protection of nitrogen gas (N2), a mixture of 6-dimethylaminobenzothiazole (5 g, 28 mmol) and potassium hydroxide (KOH, 25 g, 500 mmol) was dissolved in 75 ml of water, stirred and heated to reflux at 100 °C for 24 hours. After the reaction was completed, the mixture was cooled and adjusted to pH = 7 with dilute hydrochloric acid. Subsequently, the resulting suspension was extracted with dichloromethane, the organic layers were combined and washed with brine. Finally, the combined organic layers were dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain 2-amino-5-dimethylaminobenzenethiol.

[0055] (3) Synthesis of compound intermediate (I) 6-dimethylaminobenzothiazole-2-acetonitrile

[0056] At 25 °C, glacial acetic acid (0.38 mL, 6.4 mmol) and malononitrile (0.85 g, 12.9 mmol) were added to a solution of 2-amino-5-dimethylaminobenzenethiol (1.1 g, 6.463 mmol) in ethanol (60 mL). Subsequently, the mixture was stirred at 80 °C for 18 hours. After the reaction was completed, the resulting solid was obtained by filtration, which was the product intermediate (I) 6-dimethylaminobenzothiazole-2-acetonitrile, showing a pale yellow color. 1 H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 9.1 Hz, 1H), 7.09 (d, J = 2.5 Hz, 1H), 7.00–6.90 (m, 1H), 4.16 (s, 2H), 3.04 (s, 6H).

[0057] Example 2

[0058] The fluorescent probe BTAN-MO of the present invention is prepared by the following steps:

[0059] Weighed 6-dimethylaminobenzothiazole-2-acetonitrile (65.2 mg, 0.3 mmol) prepared in Example 1 and 4-methoxysalicylaldehyde (54.72 mg, 0.36 mmol) were added to 2 mL of ethanol, and then tetrabutylammonium bromide (12.89 mg, 0.04 mmol) was added. The mixture was refluxed at 80 °C (oil bath) for 10 h under sealed conditions. After the reaction was complete, it was allowed to stand to room temperature, and a solid precipitated. It was filtered by suction and washed three times with absolute ethanol to obtain 21.3 mg of a yellow solid product. 1 H NMR (300 MHz, CDCl3) δ 7.87 (d, J = 9.0 Hz, 2H), 7.37 (d, J = 2.5 Hz, 1H), 7.28 (s, 1H), 7.11 (tt, J = 5.7, 2.5 Hz, 2H), 7.02 (s, 1H), 6.98 (d, J = 2.6 Hz, 1H), 4.18 (s, 3H), 3.06 (s, 6H).

[0060] Example 3

[0061] The fluorescent probe BTAN-MF of the present invention has a preparation method including the following steps:

[0062] Weigh 6-dimethylaminobenzothiazole-2-acetonitrile (65.2 mg, 0.3 mmol) prepared in Example 1 and methyl 3-formyl-5-hydroxybenzoate (69.49 mg, 0.36 mmol), add them to 2 mL of ethanol, then add tetrabutylammonium bromide (12.89 mg, 0.04 mmol), and reflux and react for 10 h under sealed conditions at 80 °C (oil bath). After the reaction is complete, let it stand to room temperature, and a solid will precipitate. Filter by suction and wash three times with absolute ethanol to obtain 28.1 mg of a brick-red solid product. 1 H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.36 (d, J = 2.5 Hz, 1H), 7.20–7.06 (m, 2H), 6.92 (dd, J = 9.0, 2.9 Hz, 1H), 6.75 (d, J = 3.0 Hz, 1H), 3.90 (s, 3H), 2.97 (s, 6H).

[0063] Example 4

[0064] The fluorescent probe BTAN-NM of the present invention has a preparation method including the following steps:

[0065] Weigh 6-dimethylaminobenzothiazole-2-acetonitrile (65.2 mg, 0.3 mmol) prepared in Example 1 and 4-(dimethylamino)salicylaldehyde (59.46 mg, 0.36 mmol), add them to 2 mL of ethanol, then add tetrabutylammonium bromide (12.89 mg, 0.04 mmol), and reflux and react for 10 h under sealed conditions at 80 °C (oil bath). After the reaction is complete, let it stand to room temperature, and a solid will precipitate. Filter by suction and wash three times with absolute ethanol to obtain 23.6 mg of a yellow solid product. 1 H NMR (300 MHz, Chloroform-d) δ 8.78 (s, 1H), 8.30 (s, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 2.4 Hz, 1H), 6.98–6.90 (m, 2H), 2.93 (d, J = 3.5 Hz, 12H).

[0066] Example 5

[0067] The fluorescent probe BTAN-NE of the present invention has a preparation method including the following steps:

[0068] 6-(Dimethylamino)benzothiazole-2-acetonitrile (65.2 mg, 0.3 mmol) and 4-(diethylamino)salicylaldehyde (69.49 mg, 0.36 mmol) prepared in Example 1 were added to 2 mL of ethanol, and then tetrabutylammonium bromide (12.89 mg, 0.04 mmol) was added. The mixture was refluxed under sealed conditions at 80 °C (oil bath) for 10 h. After the reaction was complete, the mixture was allowed to stand to room temperature, and a solid precipitated. The solid was filtered by suction and washed three times with absolute ethanol to obtain 12.3 mg of a brown solid product. 1 H NMR (300 MHz, CDCl3) δ 8.78 (s, 1H), 8.30 (s, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 2.3 Hz, 1H), 7.17 (s, 1H), 6.95 (dd, J = 7.1, 2.4 Hz, 1H), 6.83 (dd, J = 8.3, 2.3 Hz, 1H), 3.46 (q, J = 7.0 Hz, 4H), 2.94 (s, 6H), 1.12 (t, J = 7.0 Hz, 6H).

[0069] Example 6

[0070] The fluorescent probe BTAO-MO of the present invention has a preparation method comprising the following steps:

[0071] (1) Synthesis of 6-methoxybenzothiazole-2-acetonitrile, and the synthesis route is as follows:

[0072]

[0073] The preparation method comprises the following steps:

[0074] (a) Under the protection of nitrogen (N2), a mixture of 2-amino-6-methoxybenzothiazole (4.6 g, 28 mmol) and potassium hydroxide (KOH, 25 g, 500 mmol) was dissolved in 75 ml of water, stirred and heated to reflux at 100 °C for 24 h. After the reaction was complete, the mixture was cooled and adjusted to pH = 7 with dilute hydrochloric acid. Subsequently, the resulting suspension was extracted with dichloromethane, and the organic layers were combined and washed with brine. Finally, the combined organic layers were dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain 2-amino-5-methoxythiophenol.

[0075] (b) At 25 °C, glacial acetic acid (0.38 mL, 6.4 mmol) and malononitrile (0.85 g, 12.9 mmol) were added to a solution of 2-amino-5-methoxythiophenol (1 g, 6.1 mmol) in ethanol (60 mL). Subsequently, the mixture was stirred at 80 °C for 18 h. After the reaction was completed, the solid obtained after filtration was 6-methoxybenzothiazole-2-acetonitrile, which was bright yellow.1 1H NMR (300 MHz, CDCl3) δ 7.94 (d, J = 9.0 Hz, 1H), 7.35 (d, J = 2.5 Hz, 1H), 7.14 (dd, J = 9.0, 2.5 Hz, 1H), 4.23 (s, 2H), 3.91 (s, 3H).

[0076] (2) Synthesis of BTAO-MO

[0077] Weigh 6-methoxybenzothiazole-2-acetonitrile (61.2 mg, 0.3 mmol) prepared in step (1) and 4-methoxysalicylaldehyde (54.72 mg, 0.36 mmol), add them to 2 mL of ethanol, then add tetrabutylammonium bromide (12.89 mg, 0.04 mmol), and reflux the reaction for 10 h under sealed conditions at 80 °C (oil bath). After the reaction is complete, let it stand to room temperature, solid precipitates out, filter by suction, wash three times with absolute ethanol to obtain 28.2 mg of a brown solid product. 1 1H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H), 7.93 (d, J = 9.0 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 2.5 Hz, 1H), 7.11 (dd, J = 8.9, 2.6 Hz, 1H), 6.81 (d, J = 9.4 Hz, 2H), 3.90 (s, 3H), 3.89 (s, 3H).

[0078] Example 7

[0079] The fluorescent probe BTAO-MF of the present invention has a preparation method including the following steps:

[0080] Weigh 6-methoxybenzothiazole-2-acetonitrile (61.2 mg, 0.3 mmol) prepared in Example 5 and methyl 3-formyl-5-hydroxybenzoate (64.8 mg, 0.36 mmol), add them to 2 mL of ethanol, then add tetrabutylammonium bromide (12.89 mg, 0.04 mmol), and reflux the reaction for 10 h under sealed conditions at 80 °C (oil bath). After the reaction is complete, let it stand to room temperature, solid precipitates out, filter by suction, wash three times with absolute ethanol to obtain 25.6 mg of a yellow solid product. 1 1H NMR (300 MHz, CDCl3) δ 8.44 (s, 1H), 8.08 (s, 1H), 7.98 (d, J = 9.1 Hz, 2H), 7.73 (s, 1H), 7.37 (s, 1H), 7.17 (d, J = 9.0 Hz, 1H), 3.98 (s, 3H), 3.93 (s, 3H).

[0081] Example 8

[0082] The fluorescent probe BTAO-NM of the present invention, the preparation method comprises the following steps:

[0083] Weigh 6-methoxybenzothiazole-2-acetonitrile (61.2 mg, 0.3 mmol) prepared in Example 5 and 4-(dimethylamino)salicylaldehyde (59.46 mg, 0.36 mmol), add them into 2 mL of ethanol, then add tetrabutylammonium bromide (12.89 mg, 0.04 mmol), and reflux and react for 10 h under the conditions of sealing and 80 °C (oil bath). After the reaction is complete, let it stand to room temperature, solid will precipitate out, filter by suction, wash three times with absolute ethanol, and obtain 23.6 mg of yellow solid product. 1 H NMR (300 MHz, DMSO) δ 8.48 (s, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.65 (d, J = 2.7 Hz, 1H), 7.55 (s, 1H), 7.11 (dd, J = 8.9, 2.6 Hz, 1H), 6.66 (dd, J = 8.8, 2.5 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 3.85 (s, 3H), 3.05 (s, 6H).

[0084] Example 9

[0085] The fluorescent probe BTAO-NE of the present invention, the preparation method comprises the following steps:

[0086] Weigh 6-methoxybenzothiazole-2-acetonitrile (61.2 mg, 0.3 mmol) prepared in Example 5 and 4-(diethylamino)salicylaldehyde (69.49 mg, 0.36 mmol), add them into 2 mL of ethanol, then add tetrabutylammonium bromide (12.89 mg, 0.04 mmol), and reflux and react for 10 h under the conditions of sealing and 80 °C (oil bath). After the reaction is complete, let it stand to room temperature, solid will precipitate out, filter by suction, wash three times with absolute ethanol, and obtain 18.3 mg of red solid product. 1 H NMR (300 MHz, CDCl3) δ 8.84 (s, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.9 Hz, 1H), 7.40 (d, J = 2.6 Hz, 1H), 7.28 (s, 1H), 7.11 (dd, J = 8.9, 2.6 Hz, 1H), 6.68 (dd, J = 9.0, 2.4 Hz, 1H), 6.61–6.54 (m, 1H), 3.91 (d, J = 3.2 Hz, 3H), 3.48 (q, J = 7.1 Hz, 4H), 1.28 (d, J = 7.0 Hz, 6H).

[0087] Performance test

[0088] 1. Absorption spectrum test

[0089] Test method: The probe was prepared into a mother liquor of the probe solution with a concentration of 5 mM using 1 mL of dimethyl sulfoxide (DMSO). The probe mother liquor was diluted with DMSO to a standard solution of 1 μM. Prepare two 1.0 cm quartz cuvettes, and use a pipette to aspirate 1 mL of DMSO buffer solution into each of the two cuvettes, and place them in a UV-spectrophotometer to measure the baseline. Fix the blank cell cuvette, add 1 mL of the probe standard solution to the other cuvette, put it into the UV-spectrophotometer, set the spectral scanning range of the UV-spectrophotometer to be 300 nm - 900 nm, generate the absorption spectrum corresponding to the probe, and the test results are as Figure 1 shown.

[0090] It can be Figure 1 seen that the UV absorption wavelengths of the six probes, namely BTAN-MF, BTAN-MO, BTAO-MF, BTAO-MO, BTAO-NE, and BTAO-NM, were measured to be 467 nm, 516 nm, 408 nm, 404 nm, 462 nm, and 460 nm respectively, providing important basic data for subsequent research.

[0091] 2. Fluorescence response test of the probe with Aβ1-42 aggregates

[0092] Test method: The prepared probe mother liquor was diluted to a standard solution of 500 nM with PBS (10 mM, pH = 7.4) buffer solution, and the emission wavelength of the probe was measured using an F-7000 fluorescence spectrophotometer (slit 10 / 10, response time 0.5 s, PMT voltage 700 V). When preparing Aβ aggregates, 7.8 mg of sodium dihydrogen phosphate dihydrate (10 mM) and 1.86 mg of EDTA (1 mM) were dissolved in 5 mL of ultrapure water, and after filtration, 0.55 mg of Aβ1-42 protein powder (prepared with TFA) was added. 2.5 mL of buffer solution was added in batches. After the solution became turbid, ammonia gas was injected into it with a syringe multiple times until it became clear, and it was incubated at 37 °C and 120 r / min for 48 hours to obtain 48.74 μM of Aβ1-42 aggregates, which were stored at -20 °C. The Aβ1-42 aggregates and the probe mother liquor were respectively diluted to 1 μM with PBS, and 500 μL of each was taken into a four-sided light-transmitting cuvette, and the fluorescence response of the probe with Aβ1-42 aggregates was measured using an F-7000 fluorescence spectrophotometer (parameters as above), and the test results are as Figures 2 - 7 shown.

[0093] It can be Figures 2 - 7It can be seen that the fluorescence emission wavelengths of the probes BTAN-MF, BTAN-MO, BTAO-MF, BTAO-MO, BTAO-NE, and BTAO-NM after binding to Aβ1-42 aggregates are 594 nm, 386 nm, 529 nm, 531 nm, 529 nm, and 523 nm respectively. The diversity of this wavelength distribution provides a key spectral basis for subsequent selective detection and structure-activity relationship studies.

[0094] 3. Dissociation Constant Test

[0095] Test method: Dilute the prepared Aβ1-42 aggregate solution (50 μM) to a standard solution of 500 nM with PBS buffer solution (10 mM, pH = 7.4); at the same time, dilute the standard mother solution of probe BTAN-MF (5 mM) to a standard solution of 20 μM with PBS buffer solution. When measuring the dissociation constant, add 950 μL of Aβ1-42 aggregate standard solution (500 nM) to the cuvette, and then sequentially add different volumes of the probe standard solution (20 μM), set the probe concentration gradient (such as 0, 5, 10, 15, 20... 1000 nM) to ensure the effectiveness of each gradient point. Use an F-7000 fluorescence spectrophotometer (slit 10 / 10, response time 0.5 s, PMT voltage 700 V) to measure the fluorescence response of the probe to Aβ1-42 aggregates and the fluorescence intensity of the probe itself at different concentrations until the fluorescence response tends to be stable, ensuring that there are no less than 8 effective gradient points. Subtract the two sets of spectra to obtain the final dissociation constant spectrum, and finally process the data through Origin software to calculate the dissociation constant (Kd). The results are as Figure 8 shown.

[0096] It can be Figure 8 seen that the measured Kd value is 32.83 nM, indicating that BTAN-MF has high sensitivity to Aβ1-42 protein.

[0097] 4. Viscosity Response Test

[0098] Test method: Prepare viscosity solutions with different ratios using glycerol and ethylene glycol (the proportion of glycerol is 0%, 10%, 20%... 100%). Dilute the mother solution of probe BTAN-MF (5 mM) to a probe standard solution of 50 μM with PBS buffer solution (10 mM, pH = 7.4). Use a pipette to accurately measure 950 μL of the viscosity solution and add it to the cuvette, then add 50 μL of the probe standard solution (50 μM), gently pipette and mix well and shake. Use an F-7000 fluorescence spectrophotometer (slit 10 / 10, response time 0.5 s, PMT voltage 700 V) to record the fluorescence curve. Repeat the above steps to sequentially measure the viscosity solutions with all glycerol ratios, ensure that the measurement conditions are the same each time, record and analyze the trend of the fluorescence curve with the change of viscosity. The test results are asFigure 9 as shown

[0099] From Figure 9 it can be seen that as the viscosity increases, the fluorescence response is significantly enhanced, indicating that the response of the BTAN-MF probe to Aβ protein partly stems from the fact that Aβ protein restricts the distortion of the probe structure, thereby enhancing the fluorescence signal.

[0100] 5. Selectivity of the Probe to Different Analytes

[0101] Testing method: First, prepare selective solutions containing 10 mM different metal ions and amino acids with PBS buffer (10 mM, pH = 7.4), and dilute the 5 mM BTAN-MF probe mother solution to a 50 μM standard solution. During the experiment, mix 50 μL of the probe standard solution with 950 μL of each selective solution in a cuvette, vortex and shake, and then use an F-7000 fluorescence spectrophotometer (slit width 10 / 10 nm, response time 0.5 s, PMT at 700 V) to record the fluorescence spectrum. By cyclically replacing the selective solutions and repeating the above operations, the response characteristics of the probe to different analytes are systematically measured, and the test results are as Figure 10 shown

[0102] From Figure 10 it can be seen that the probe BTAN-MF shows high specificity for Aβ protein.

[0103] 6. Biosafety Evaluation of the Probe BTAN-MF

[0104] Testing method: The CCK-8 method is used to evaluate the cytotoxicity of the probe: After subculturing PC12 cells and discarding the supernatant after centrifugation, resuspend with 1 mL of medium, take 10 μL of the suspension and mix it with 90 μL of medium for dilution, adjust the density after counting, seed 5000 cells / well into a 96-well plate, and culture for 48 hours. Replace it with a gradient concentration of BTAN-MF probe culture medium (0, 0.5, 1, 2, 5, 10, 20, 40 μM) under light-proof conditions, continue to culture for 24 hours, then remove the drug solution, add fresh medium containing 10% CCK-8, incubate for 2 hours under light-proof conditions, and finally calculate the cell survival rate by measuring the absorbance at 450 nm with an enzyme-labeled instrument to quantify the toxic effect. The test results are as Figure 11 shown

[0105] From Figure 11 it can be seen that the toxicity of the probe to PC12 cells is extremely low, showing good biocompatibility.

[0106] 7. Fluorescence Imaging Test of Brain Slices

[0107] Test method: Take the brain tissue of euthanized mice, and rinse it with physiological saline or PBS buffer solution. Freeze the tissue in liquid nitrogen for 10 seconds, and then store it in a -80 °C refrigerator. Then place the tissue in an embedding cassette, inject OCT (Optimal Cutting Temperature compound) freezing embedding medium, remove the outer shell, and cut it into 20-μm-thick sections with a cryostat. Place the sections on a room-temperature glass slide and dry for half an hour. Next, prepare a dilution buffer solution (0.2 g BSA / 10 mL, 1% goat serum, 99% TBS solution, 10% Triton) and a blocking buffer solution (2 mL goat serum, 10 mL TBS). Wash the brain sections three times with the dilution buffer solution on a shaker for 10 minutes each time, and then block them with the blocking buffer solution at room temperature for 30 minutes. Circle the range of the brain sections with a transparent marker pen. Dilute the mother solution of the BTAN-MF probe to 25 μM with 50% ethanol, drop it on the brain sections and stain for 20 minutes, then wash with ethanol for 1 minute, repeat twice, and finally wash with water twice. After sucking off the excess moisture, use a confocal microscope with green and red excitation channels to obtain fluorescence images. The test results are as Figure 12 shown.

[0108] As Figure 12 shown, the BTAN-MF probe can effectively recognize Aβ in brain sections, showing good specificity and imaging effects.

Claims

1. A fluorescent probe, characterized in that, The structural formula is as follows: Wherein, R1 is -OR or -N-(R)2; R2 is -OR, -N-(R)2 or -COO-R, where R is a C1-C4 alkyl group.

2. The fluorescent probe according to claim 1, wherein Said R1 is -N-(R)2 and R2 is -COO-R.

3. The fluorescent probe according to claim 1, wherein The structural formula of the fluorescent probe is as follows:

4. A method for preparing the fluorescent probe according to any one of claims 1 to 3, characterized in that, It includes the following steps: Using raw material A and raw material B as reaction raw materials, adding them into an organic solvent, then adding the catalyst tetrabutylammonium bromide, and under sealed conditions, carrying out a condensation reaction and ring formation at 60-80 °C to obtain the fluorescent probe; the synthesis route is as follows:

5. Use of the fluorescent probe according to any one of claims 1 to 3 in the detection of β-amyloid protein.

6. An intermediate, characterized in that, The structural formula is as follows: Wherein, R is a C1-C4 alkyl group.

7. A method for preparing the intermediate according to claim 6, characterized in that, It includes the following steps: (1) Dissolve 6-aminobenzothiazole in an organic solvent, and then sequentially add dry triethylamine and an alkyl acid to the reaction mixture. Under the protection of an inert gas, carry out a Mannich reaction to obtain the product 6-dialkylaminobenzothiazole. (2) Under the protection of an inert gas, dissolve the mixture of 6-dialkylaminobenzothiazole and a base in water, carry out an alkaline ring-opening reaction. After the reaction is completed, cool the reaction solution, acidify the mixture to obtain 2-amino-5-dialkylaminobenzenethiol; (3) Add 2-amino-5-dimethylaminobenzenethiol to an organic solvent, then add glacial acetic acid and malononitrile, and carry out a condensation reaction to obtain intermediate I; The synthesis route is as follows:

8. A method for preparing the intermediate according to claim 7, characterized in that, In step (1), the reaction temperature is 80-100 °C.

9. A method for preparing the intermediate according to claim 7, characterized in that, In step (2), the reaction temperature is 60-80 °C.

10. A method for preparing the intermediate according to claim 7, characterized in that, In step (3), the reaction temperature is 60-80 °C.

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