A difluoroborahydrazine pyrrole fluorescent probe, a preparation method thereof and a beta-amyloid detection application

Abstract

The application belongs to a kind of difluoroborane hydrazine pyrrole fluorescent probe and its preparation method and beta amyloid detection application, belongs to the field of fluorescence detection technology. The probe is composed of BOPHY fluorescent mother body and recognition group, and its preparation method includes three steps of BOPHY precursor preparation, fluorescent mother body synthesis and target probe molecule synthesis. The prepared series of probes have large Stokes shift, can effectively reduce imaging interference, and show high sensitivity and high selectivity of fluorescence response to A beta aggregate, wherein the performance of N-BOPHY is optimal. Experiments show that the binding affinity of the probe to A beta aggregate is high, the staining imaging result of A beta plaque in brain slice has high colocalization with commercial probe thioflavin-T, and the cell toxicity is low and the biocompatibility is good.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection technology, and specifically relates to a difluoroborosilicate pyrrole fluorescent probe, its preparation method, and its application in the detection of β-amyloid protein. Background Technology

[0002] Alzheimer's disease (AD) is a slowly progressing, insidious, chronic neurodegenerative disease with a complex pathogenesis. Early diagnosis of AD can intervene in disease progression as early as possible, which is of great significance for improving the clinical symptoms of AD patients. Currently, the pathogenesis of AD is not fully understood, and many possible mechanisms have been proposed. Among them, the amyloid cascade hypothesis is one of the mainstream and accepted hypotheses. This hypothesis suggests that the abnormal deposition of extracellular β-amyloid protein (Aβ) forming senile plaques is one of the key factors in the development of AD. Aβ is produced by the cleavage of amyloid precursor protein by β- and γ-secretases. The C-terminal cleavage of APP yields two types of Aβ peptides, with the most predominant isoform being Aβ. 1-40 Secondly, Aβ 1-42 With Aβ 1-40 In comparison, Aβ 1-42 More hydrophobic, Aβ peptides are more prone to aggregation and thus more toxic. The β-sheet structure of Aβ peptides tends to aggregate, resulting in various forms such as Aβ monomers, Aβ oligomers, Aβ fibrils, and Aβ aggregates. Generally, in the healthy human brain, the production and clearance of Aβ are in dynamic equilibrium. When this equilibrium is disrupted, Aβ is overproduced and accumulates in the brain, causing neurotoxicity. Studies have shown that excessive Aβ production can induce oxidative stress in the brain, triggering neuroinflammation, accelerating neuronal apoptosis, and exacerbating the development of Alzheimer's disease (AD). Therefore, highly sensitive detection of Aβ is of great significance for the early diagnosis of AD, delaying the onset of the disease, and alleviating clinical symptoms in patients.

[0003] Currently, fluorescent probes used for Aβ detection mainly rely on benzothiazole, BODIPY, curcumin, and electron donor-acceptor (DA) conjugated polyene structures as fluorescent parent structures, and the recognition groups mainly depend on... N , N -Dimethylamino, N , N Aβ fluorescent probes can be constructed by adjusting the number of conjugated double bonds or introducing other conjugated groups such as thiophene, benzene rings, and furan, using diethylamino, piperidinyl, tetrahydropyrrolyl, and cyclohexylimino groups. While these traditional probes can recognize and detect Aβ aggregates to some extent, their sensitivity and selectivity remain limited. Therefore, developing new fluorescent parent structures and recognition groups to improve the detection sensitivity and selectivity of Aβ aggregates has significant universal implications. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a difluoroborate pyrrole fluorescent probe, its preparation method, and its application in β-amyloid protein detection.

[0005] This invention is achieved through the following technical solution: A bis(fluoroborohydrazine) pyrrole-based fluorescent probe includes a fluorescent parent structure of bis(fluoroborohydrazine) pyrrole (BOPHY) and a recognition group structure. The structure of the fluorescent probe is as follows: .

[0006] Furthermore, the difluoroborate pyrrole fluorescent probe is one of the following compounds: .

[0007] The present invention also provides a method for preparing the bis(fluoroborate)hydrazine pyrrole fluorescent probe, comprising the following steps: S1, Preparation of BOPHY precursor: 3,5-dimethyl-2-pyrrolecarboxaldehyde was reacted with hydrazine hydrate in ethanol, and condensation was catalyzed by adding an amount of glacial acetic acid. The solid was precipitated at room temperature, filtered, and washed with glacial methanol to obtain the BOPHY precursor. S2, Synthesis of BOPHY fluorescent precursor: Under nitrogen protection, the precursor was dissolved in toluene, triethylamine was added dropwise at 0°C, boron trifluoride diethyl ether was added dropwise at room temperature, and the mixture was heated to reflux at 110°C; after the reaction, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain the BOPHY fluorescent precursor; S3. Synthesis of target probe molecule: 4-azacyclohexylbenzaldehyde derivative and BOPHY were dissolved in toluene, and piperidine and p-toluenesulfonic acid were added as catalysts. The mixture was heated to reflux at 130 °C. After the reaction was completed, the mixture was extracted with ethyl acetate / water, the organic phase was concentrated, and purified by column chromatography to obtain the target probe molecule. .

[0008] Furthermore, the 4-azacyclohexylbenzaldehyde derivative is one of the following compounds:

[0009] Furthermore, the molar ratio of 3,5-dimethyl-2-pyrrolecarboxaldehyde to hydrazine hydrate in S1 is 9:5.

[0010] Furthermore, the volume ratio of triethylamine to boron trifluoride diethyl ether in S2 is 2:1.7.

[0011] Furthermore, the molar ratio of 4-azacyclohexylbenzaldehyde derivative to BOPHY in S3 is 3.6:3.1; the addition ratio of toluene, piperidine, and p-toluenesulfonic acid is 10 mL:1 mL:30 mg.

[0012] The present invention also provides the application of the aforementioned difluoroborosilicate pyrrole fluorescent probe in the detection of β-amyloid protein.

[0013] The beneficial technical effects of the present invention are as follows: (1) The fluorescent probe prepared by this invention has a large Stokes shift, which can effectively reduce the interference of the excitation source on the imaging effect during imaging. It has good fluorescence response and concentration dependence on Aβ aggregates in solution.

[0014] (2) This invention proposes a novel fluorescent parent structure, BOPHY, which can be used to construct Aβ fluorescent probes and has strong universality. Compared with BODIPY, the BOPHY fluorescent parent structure proposed in this invention significantly reduces the specific binding of BSA protein, ensuring accurate imaging capability of Aβ in complex brain slices.

[0015] (3) Among the Aβ fluorescent probes prepared in this invention, C-BOPHY, N-BOPHY, O-BOPHY and S-BOPHY can all provide sensitive fluorescence response to Aβ aggregates. The N-BOPHY probe has the highest signal-to-noise ratio, the best recognition sensitivity, good selectivity and high binding affinity for the fluorescence response of Aβ aggregates.

[0016] (4) Among the Aβ fluorescent probes prepared in this invention, C-BOPHY, N-BOPHY, O-BOPHY and S-BOPHY can all stain and image Aβ plaques in brain slices of AD mice, and their staining results have a high co-localization effect with the commercial probe thiosulfate-T (ThT). Attached Figure Description

[0017] Figure 1 This is the C-BOPHY normalized ultraviolet absorption spectrum.

[0018] Figure 2 This is the normalized UV absorption spectrum of N-BOPHY.

[0019] Figure 3 This is the normalized UV absorption spectrum of O-BOPHY.

[0020] Figure 4 This is the S-BOPHY normalized ultraviolet absorption spectrum.

[0021] Figure 5 The normalized fluorescence spectrum is for C-BOPHY.

[0022] Figure 6 The normalized fluorescence spectrum is N-BOPHY.

[0023] Figure 7 This is the normalized fluorescence spectrum of O-BOPHY.

[0024] Figure 8 The normalized fluorescence spectrum is S-BOPHY.

[0025] Figure 9 The fluorescence signal enhancement curve of C-BOPHY on viscosity.

[0026] Figure 10 The fluorescence signal enhancement curve of N-BOPHY on viscosity.

[0027] Figure 11 The fluorescence signal enhancement curve of O-BOPHY on viscosity.

[0028] Figure 12 The fluorescence signal enhancement curve of S-BOPHY on viscosity.

[0029] Figure 13 The curve shows the fluorescence signal enhancement of Aβ aggregates by C-BOPHY.

[0030] Figure 14 The curve shows the fluorescence signal enhancement of Aβ aggregates by N-BOPHY.

[0031] Figure 15 The curve shows the fluorescence signal enhancement of Aβ aggregates by O-BOPHY.

[0032] Figure 16 The curve shows the fluorescence signal enhancement of Aβ aggregates by S-BOPHY.

[0033] Figure 17 The fluorescence signal enhancement curves of the control probe N-BODIPY with Aβ aggregates and BSA are shown.

[0034] Figure 18 The signal relationship between the fluorescence intensity of C-BOPHY and the concentration of Aβ addition is shown.

[0035] Figure 19 The signal relationship between the fluorescence intensity of N-BOPHY and the concentration of Aβ addition is shown.

[0036] Figure 20 The signal relationship between the fluorescence intensity of O-BOPHY and the concentration of Aβ addition is shown.

[0037] Figure 21 The signal relationship between the fluorescence intensity of S-BOPHY and the concentration of Aβ addition is shown.

[0038] Figure 22 Normalized fluorescence intensity competition curves for the C-BOPHY probe to displace ThT from Aβ aggregates / commercial Aβ dye ThT complex.

[0039] Figure 23Normalized fluorescence intensity competition curves for N-BOPHY probe to displace ThT from Aβ aggregates / commercial Aβ dye ThT complex.

[0040] Figure 24 Normalized fluorescence intensity competition curves for the O-BOPHY probe to displace ThT from Aβ aggregates / commercial Aβ dye ThT complex.

[0041] Figure 25 Normalized fluorescence intensity competition curves for the S-BOPHY probe to displace ThT from Aβ aggregates / commercial Aβ dye ThT complex.

[0042] Figure 26 The binding affinity (Kd) of C-BOPHY to Aβ aggregates.

[0043] Figure 27 The binding affinity (Kd) of N-BOPHY to Aβ aggregates.

[0044] Figure 28 The binding affinity (Kd) of O-BOPHY to Aβ aggregates.

[0045] Figure 29 The binding affinity (Kd) of S-BOPHY to Aβ aggregates.

[0046] Figure 30 This represents the selectivity of C-BOPHY for Aβ aggregates.

[0047] Figure 31 This represents the selectivity of N-BOPHY for Aβ aggregates.

[0048] Figure 32 This represents the selectivity of O-BOPHY for Aβ aggregates.

[0049] Figure 33 This represents the selectivity of S-BOPHY for Aβ aggregates.

[0050] Figure 34 The results show the fluorescence staining imaging of Aβ aggregates in solution by C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY, as well as their co-localization with the commercial Aβ dye ThT.

[0051] Figure 35 Fluorescent staining imaging of Aβ in the cortical and hippocampal regions of brain slices from AD mice using C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY, and co-localization with the commercially available Aβ dye ThT.

[0052] Figure 36 This is the cytotoxicity of C-BOPHY.

[0053] Figure 37 This is due to the cytotoxicity of N-BOPHY.

[0054] Figure 38 This is the cytotoxicity of O-BOPHY.

[0055] Figure 39 This is the cytotoxicity of S-BOPHY. Detailed Implementation

[0056] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Unless otherwise specified, all experimental methods used are conventional methods.

[0057] Example 1: Synthesis of probe C-BOPHY (1) Synthesis of the BOPHY precursor: 3,5-dimethyl-2-pyrrolecarboxaldehyde (1.00 g, 8.1 mmol) and hydrazine hydrate (0.45 g, 4.5 mmol) were added to 30 mL of ethanol. A catalytic amount of glacial acetic acid was slowly added to the reaction system. When a solid was formed, the reaction was carried out at room temperature until the reaction was complete. The solid was obtained by filtration and washed with glacial methanol to obtain the solid BOPHY precursor. 1 H NMR(CDCl3) 8.36 (s, 2H), 5.80 (s, 2H), 2.26 (s, 6H), 2.17 (s, 6H). HRMS (ESI)cacld for C 14 H 19 N4 [M+H] + : 243.161, found 243.1597.

[0058] (2) Synthesis of the fluorescent precursor of compound BOPHY: Under nitrogen protection, the BOPHY precursor was dissolved in 30 mL of toluene, and 2 mL of triethylamine was added dropwise at 0 °C. After 15 minutes, 1.7 mL of boron trifluoride diethyl ether was added dropwise at room temperature. The mixture was heated to reflux at 110 °C. After the reaction was complete, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. Solid BOPHY was obtained by column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v). 1 H NMR (CDCl3) 7.93 (s, 2H), 6.18 (s, 2H), 2.49 (s, 6H), 2.32 (s, 6H). HRMS (ESI) cacld for C 14 N4H 16 B2F4Na [M+Na] + :361.1389, found361.1389.

[0059] (3) Synthesis of compound C-BOPHY: 4-piperidin-1-ylbenzaldehyde (0.136 g, 0.72 mmol) and BOPHY (0.224 g, 0.62 mmol) were dissolved in 10 mL of toluene. 1 mL of piperidine and 30 mg of p-toluenesulfonic acid were slowly added dropwise as a catalyst, and the mixture was heated to reflux at 130 °C. After the reaction was complete, the reaction was cooled to room temperature, and the crude product was extracted with water and ethyl acetate. After removing the solvent under reduced pressure, C-BOPHY was purified by column chromatography (petroleum ether / dichloromethane = 1 / 1, volume ratio) to obtain C-BOPHY. 1 H NMR (CDCl3) δ9.11 (s, 1H), 9.00 (s, 1H), 7.42–7.36 (m, 2H), 7.33 (d, J=14.2 Hz, 1H), 7.18 (d, J=14.1 Hz, 1H), 6.69–6.61 (m, 3H), 6.36–6.32 (m, 1H), 3.38 (m, 4H), 2.42 (s, 3H), 2.22 (s, 3H), 2.16 (s, 3H), 1.73–1.57 (m, 6H). HRMS (ESI) cacld forC 26 H 30 B2F4N5 [M+H] + : 510.2618, found 510.260.

[0060] Example 2 Synthesis of probe N-BOPHY (1) Synthesis of the BOPHY precursor: 3,5-dimethyl-2-pyrrolecarboxaldehyde (1.00 g, 8.1 mmol) and hydrazine hydrate (0.45 g, 4.5 mmol) were added to 30 mL of ethanol. A catalytic amount of glacial acetic acid was slowly added to the reaction system. When a solid was formed, the reaction was carried out at room temperature until the reaction was complete. The solid was obtained by filtration and washed with glacial methanol to obtain the solid BOPHY precursor. 1 H NMR(CDCl3) 8.36 (s, 2H), 5.80 (s, 2H), 2.26 (s, 6H), 2.17 (s, 6H). HRMS (ESI)cacld for C 14 H 19 N4 [M+H] + : 243.161, found 243.1597.

[0061] (2) Synthesis of the fluorescent precursor of compound BOPHY: Under nitrogen protection, the BOPHY precursor was dissolved in 30 mL of toluene, and 2 mL of triethylamine was added dropwise at 0 °C. After 15 minutes, 1.7 mL of boron trifluoride diethyl ether was added dropwise at room temperature. The mixture was heated to reflux at 110 °C. After the reaction was complete, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. Solid BOPHY was obtained by column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v). 1 H NMR (CDCl3) 7.93 (s, 2H), 6.18 (s, 2H), 2.49 (s, 6H), 2.32 (s, 6H). HRMS (ESI) cacld for C 14 N4H 16 B2F4Na [M+Na] + :361.1389, found361.1389.

[0062] (3) Synthesis of compound N-BOPHY: 4-(4-methylpiperazinyl)benzaldehyde (0.148 g, 0.72 mmol) and BOPHY (0.224 g, 0.62 mmol) were dissolved in 10 mL of toluene. 1 mL of piperidine and 30 mg of p-toluenesulfonic acid were slowly added dropwise as a catalyst, and the mixture was heated to reflux at 130 °C. After the reaction was complete, the reaction was cooled to room temperature, and the crude product was extracted with water and ethyl acetate. After removing the solvent under reduced pressure, N-BOPHY was purified by column chromatography (dichloromethane / methanol = 40 / 1, v / v) to obtain N-BOPHY. 1 H NMR (CDCl3) δ9.11 (s, 1H), 9.00 (s, 1H), 7.42-7.36 (m, 2H), 7.33 (d, J=14.2 Hz, 1H), 7.18 (dd, J=14.1, 0.9 Hz, 1H), 6.77-6.71 (m, 2H), 6.67 (s, 1H), 6.34 (d, J=1.2 Hz, 1H), 3.28-3.17 (m, 4H), 2.80 (m, 2H), 2.57 (m, 2H), 2.42 (m, 3H), 2.29 (m, 3H), 2.16 (m, 6H). HRMS (ESI) cacld for C 26 H 31 B2F4N6 [M+H] + :525.2727, found525.27711.

[0063] Example 3 Synthesis of probe O-BOPHY (1) Synthesis of the BOPHY precursor: 3,5-dimethyl-2-pyrrolecarboxaldehyde (1.00 g, 8.1 mmol) and hydrazine hydrate (0.45 g, 4.5 mmol) were added to 30 mL of ethanol. A catalytic amount of glacial acetic acid was slowly added to the reaction system. When a solid was formed, the reaction was carried out at room temperature until the reaction was complete. The solid was obtained by filtration and washed with glacial methanol to obtain the solid BOPHY precursor. 1 H NMR(CDCl3) 8.36 (s, 2H), 5.80 (s, 2H), 2.26 (s, 6H), 2.17 (s, 6H). HRMS (ESI)cacld for C 14 H 19 N4 [M+H] + : 243.161, found 243.1597.

[0064] (2) Synthesis of the fluorescent precursor of compound BOPHY: Under nitrogen protection, the BOPHY precursor was dissolved in 30 mL of toluene, and 2 mL of triethylamine was added dropwise at 0 °C. After 15 minutes, 1.7 mL of boron trifluoride diethyl ether was added dropwise at room temperature. The mixture was heated to reflux at 110 °C. After the reaction was complete, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. Solid BOPHY was obtained by column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v). 1 H NMR (CDCl3) 7.93 (s, 2H), 6.18 (s, 2H), 2.49 (s, 6H), 2.32 (s, 6H). HRMS (ESI) cacld for C 14 N4H 16 B2F4Na [M+Na] + :361.1389, found361.1389.

[0065] (3) Synthesis of compound O-BOPHY: 4-(4-morpholine)benzaldehyde (0.138 g, 0.72 mmol) and BOPHY (0.224 g, 0.62 mmol) were dissolved in 10 mL of toluene, and 1 mL of piperidine and 30 mg of p-toluenesulfonic acid were slowly added dropwise. The mixture was heated to reflux at 130 °C. After the reaction was completed, the reaction was cooled to room temperature, and the crude product was extracted with water and ethyl acetate. After removing the solvent under reduced pressure, O-BOPHY was purified by column chromatography (petroleum ether / dichloromethane = 1 / 1, volume ratio) to obtain O-BOPHY. 1H NMR (CDCl3) δ 9.11 (s, 1H), 9.00 (s, 1H), 7.42-7.36 (m, 2H), 7.33 (d, J=14.2 Hz, 1H), 7.18 (d, J=14.1 Hz, 1H), 6.73-6.66 (m, 3H), 6.36-6.32 (m, 1H), 3.80 (m, 4H), 3.21 (m, 4H), 2.42 (s, 3H), 2.16 (d, 6H). HRMS (ESI) cacld for C 25 H 28 B2F4N5O [M+H] + :512.2411, found 510.2404.

[0066] Example 4 Synthesis of probe S-BOPHY (1) Synthesis of the BOPHY precursor: 3,5-dimethyl-2-pyrrolecarboxaldehyde (1.00 g, 8.1 mmol) and hydrazine hydrate (0.45 g, 4.5 mmol) were added to 30 mL of ethanol. A catalytic amount of glacial acetic acid was slowly added to the reaction system. When a solid was formed, the reaction was carried out at room temperature until the reaction was complete. The solid was obtained by filtration and washed with glacial methanol to obtain the solid BOPHY precursor. 1 H NMR(CDCl3) 8.36 (s, 2H), 5.80 (s, 2H), 2.26 (s, 6H), 2.17 (s, 6H). HRMS (ESI)cacld for C 14 H 19 N4 [M+H] + : 243.161, found 243.1597.

[0067] (2) Synthesis of the fluorescent precursor of compound BOPHY: Under nitrogen protection, the BOPHY precursor was dissolved in 30 mL of toluene, and 2 mL of triethylamine was added dropwise at 0 °C. After 15 minutes, 1.7 mL of boron trifluoride diethyl ether was added dropwise at room temperature. The mixture was heated to reflux at 110 °C. After the reaction was complete, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. Solid BOPHY was obtained by column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v). 1 H NMR (CDCl3) 7.93 (s, 2H), 6.18 (s, 2H), 2.49 (s, 6H), 2.32 (s, 6H). HRMS (ESI) cacld for C 14 N4H 16 B2F4Na [M+Na]+ :361.1389, found361.1389.

[0068] (3) Synthesis of Compound 1: Thiomorpholine (2.98 g, 24 mmol) and 4-fluorobenzaldehyde (2.06 g, 20 mmol) were dissolved in 30 mL of dimethyl sulfoxide, and 10 g of potassium carbonate was added. The mixture was heated to reflux at 120 °C. After the reaction was completed, the reaction was cooled to room temperature, and the crude product was extracted with a large amount of water and ethyl acetate. After removing the solvent under reduced pressure, Compound 1 was purified by column chromatography (petroleum ether / dichloromethane = 1 / 1, volume ratio) to obtain Compound 1. 1 H NMR (CDCl3) 9.78(s, 1H), 7.74-7.77(d, 2H), 6.85-6.89(d, 2H), 3.83(d, 4H), 2.70-2.72(t, 4H).

[0069]

[0070] (4) Synthesis of compound S-BOPHY: Compound 1 (0.149 g, 0.72 mmol) and BOPHY (0.224 g, 0.62 mmol) were dissolved in 10 mL of toluene. 1 mL of piperidine and 30 mg of p-toluenesulfonic acid were slowly added dropwise as a catalyst, and the mixture was heated to reflux at 130 °C. After the reaction was complete, the reaction was cooled to room temperature, and the crude product was extracted with water and ethyl acetate. After removing the solvent under reduced pressure, S-BOPHY was purified by column chromatography (petroleum ether / dichloromethane = 1 / 1, volume ratio) to obtain S-BOPHY. 1 H NMR (CDCl3) δ 9.11 (s, 1H), 9.00 (s, 1H), 7.42-7.36 (m, 2H), 7.33 (d, J=14.2 Hz, 1H), 7.18 (dd, J=14.1, 0.9 Hz, 1H), 6.76-6.70 (m, 2H), 6.67 (s, 1H), 6.36-6.32 (m, 1H), 3.64 (m, J=6.1, 3.6, 2.5 Hz, 4H), 2.78 (m, J=6.4, 5.5, 3.6 Hz, 4H), 2.42 (s, 3H), 2.16 (s, 6H). HRMS (ESI) cacld for C 25 H 28 B2F4N5S [M+H] + :528.2182, found528.2153.

[0071] Comparative Example 1 (1) Synthesis of the BODIPY fluorescent precursor: 3,5-dimethyl-1H-pyrrole-2-methyl (246 mg, 2 mmol) was dissolved in 10 mL of CH2Cl2, and POCl3 (0.22 mL, 2.4 mmol) was added dropwise over 1 minute at 0°C. The solution was slowly brought to room temperature and stirred for 12 hours. The mixture was cooled to 0°C, and Et3N (1.4 mL, 10 mmol) was added dropwise over 5 minutes. After stirring for 15 minutes, BF3·OEt2 (2.0 mL, 16 mmol) was added dropwise over 5 minutes. The reaction mixture was brought to room temperature and stirred for 12 hours. The mixture was filtered through a short silica gel sieve and washed with dichloromethane to remove polar impurities. The solvent was removed under reduced pressure. The residue was dissolved in dichloromethane and water was added, and the mixture was stirred overnight at room temperature (to decompose excess BF3·OEt2 and other impurities). The organic layer was washed with water and brine and dried with Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by rapid chromatography (5% EtOAc / hexane) to obtain BODIPY as a red solid. 1 H NMR (400MHz, CDCl3) δ 6.96 (s, 1H), δ 5.97 (s, 2H), δ 2.46 (s, 6H), δ 2.17 (s, 6H). EI-MS m / z cacld forC 13 H 15 N2BF2, 248.08; found, 248.

[0072] (2) Synthesis of compound N-BODIPY: 4-(4-methylpiperazinyl)benzaldehyde (0.149 g, 0.72 mmol) and BODIPY (0.224 g, 0.62 mmol) were dissolved in 10 mL of toluene. 1 mL of piperidine and 200 μL of acetic acid were slowly added dropwise as a catalyst, and the mixture was heated to reflux at 130 °C. After the reaction was complete, the reaction mixture was cooled to room temperature, and the crude product was extracted with water and dichloromethane. After removing the solvent under reduced pressure, the product was purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v) to obtain N-BODIPY. 1H NMR (CDCl3)δ 7.42–7.36 (m, 2H), 7.38–7.31 (m, 2H), 7.18 (d, J=14.1 Hz, 1H), 7.03 (s, 1H), 6.88 (s, 1H), 6.77–6.71 (m, 2H), 3.28–3.17 (m, 4H), 2.80 (m, 4H), 2.62 (d, J=1.1 Hz, 3H), 2.31–2.24 (m, 9H). HRMS (ESI) cacld for C 25 H 30 BF2N4 [M+H] + :435.2526, found 435.2508.

[0073] Performance testing (1) Spectral properties of probes C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY Probes C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY were prepared into 1 mM stock solutions using dimethyl sulfoxide (DMSO). Working concentrations of 5 μM were then prepared using different solvents (toluene, dichloromethane, ethyl acetate, DMSO, and methanol). The UV-Vis absorption and fluorescence emission spectra were measured using both a UV-Vis spectrophotometer and a fluorescence spectrophotometer. The normalized UV absorption spectra of probes C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY are shown below. Figure 1-4 As shown; normalized fluorescence spectrum as shown Figure 5-8 As shown, the UV absorption wavelengths of the probes in different solvents vary with the solvent. The maximum absorption wavelengths of C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY are 565 nm (in toluene), 551 nm (in toluene), 601 nm (in DMSO), and 544 nm (in DMSO), respectively. The emission wavelengths of the probes in different solvents also vary with the solvent. The maximum emission wavelengths of C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY are 647 nm (in DMSO), 655 nm (in DMSO), 664 nm (in DMSO), and 655 nm (in DMSO), respectively.

[0074] (2) Fluorescence response of probes C-BOPHY, N-BOPHY, O-BOPHY and S-BOPHY to viscosity Solvents of varying viscosities (water / glycerol ratio 0-100%) were prepared. Using these solvents, 1 μM probe solutions of C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY were prepared. The probe response to viscosity was then tested using a Hitachi FL-4600 fluorescence spectrophotometer. Figure 9-12 As shown, the fluorescence intensity of probes C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY gradually increases with increasing viscosity, indicating that viscosity restricts molecular motion and dihedral twisting, leading to enhanced fluorescence.

[0075] (3) The fluorescence enhancement of Aβ aggregates by probes C-BOPHY, N-BOPHY, O-BOPHY, S-BOPHY, and N-BODIPY. The following test solutions were prepared: PBS solution (pH=7.2-7.4), probe PBS solution (final probe concentration 1 μM), bovine serum albumin (BSA) PBS solution (10 mg / mL), probe + BSA solution (final probe concentration 1 μM, final BSA concentration 10 mg / mL), and probe + Aβ aggregate solution (final probe concentration 1 μM, final Aβ aggregate concentration 40 μM). Their fluorescence emission spectra were measured using a fluorescence spectrophotometer. Figure 13-16 As shown, the fluorescence intensity of probes C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY after binding to Aβ aggregates was increased by 48, 42, 37, and 26 times, respectively, compared to the probes themselves. Furthermore, the fluorescence intensity after binding to BSA showed almost no change, indicating specific fluorescence enhancement for Aβ aggregates. As a control, N-BODIPY, after incubation with Aβ aggregates for a period of time, also produced several-fold fluorescence enhancement, but it exhibited more significant non-specific fluorescence enhancement when interacting with bovine serum albumin (BSA). Figure 17 This result makes it difficult for N-BODIPY probes to effectively distinguish Aβ plaques from surrounding proteins in brain slices. In contrast, the BOPHY-type fluorescent probes proposed in this invention demonstrate a significant advantage in detecting Aβ.

[0076] (4) Concentration dependence of probes C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY on Aβ aggregates Solutions of probe + Aβ aggregate were prepared separately (probe final concentration 1 μM, Aβ aggregate final concentrations 0-10 μM), and fluorescence spectra were measured using a fluorescence spectrophotometer. Figure 18-21 As the concentration of Aβ aggregates increased, probes C-BOPHY, O-BOPHY, N-BOPHY, and S-BOPHY all exhibited concentration-dependent fluorescence enhancement.

[0077] (5) Competition curves of probes C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY displacing ThT from Aβ aggregates / ThT. Prepare an Aβ aggregate (40 μM) / ThT (1 μM) complex, add 0–1 μM of the above molecular probe to it, and measure the fluorescence spectrum using a fluorescence spectrophotometer to plot a competition curve. Figure 22-25 As shown, for C-BOPHY, the fluorescence intensity of ThT decreased significantly after the addition of 0.1 μM C-BOPHY. As the concentration of added C-BOPHY increased, the fluorescence intensity of C-BOPHY gradually increased and reached saturation, while the fluorescence intensity of ThT gradually increased, but the final fluorescence intensity was still lower than that of C-BOPHY. For N-BOPHY, a similar trend was observed to that of C-BOPHY. For O-BOPH, the fluorescence intensity of ThT decreased significantly after the addition of 0.1 μM O-BOPHY. As the concentration of added O-BOPHY increased to 0.3 μM, the fluorescence intensity of O-BOPHY gradually increased and reached saturation, while the fluorescence intensity of ThT gradually decreased. For S-BOPHY, the fluorescence intensity of ThT decreased significantly after the addition of 0.1 μM O-BOPHY. As the concentration of added O-BOPHY increased to 0.8 μM, the fluorescence intensity of O-BOPHY gradually increased and reached saturation, while the fluorescence intensity of ThT gradually decreased. The above results demonstrate that the probe of this invention can replace ThT, and the probe of this invention has a superior affinity for Aβ aggregates compared to ThT. The probe of this invention can detect signals more sensitively and accurately than the traditional ThT.

[0078] (6) Binding affinity (K) of probes C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY to Aβ aggregates d ) Prepare Aβ (3 μM) solutions containing molecular probes (10, 30, 50, 70, 90, 100, 120, 150, 200, 250, 300, 500, 1000 nM, final concentration), and measure the fluorescence spectra using a fluorescence spectrophotometer. Quantify the fluorescence intensity to determine the binding affinity. Figure 26-29 ).like Figure 26-29 As shown, the binding affinities of C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY to Aβ aggregates are 5.57, 1.87, 18.85, and 20.95 nM, respectively, indicating that they have high binding affinity to Aβ aggregates.

[0079] (7) Selectivity of probes (a) C-BOPHY, (b) N-BOPHY, (c) O-BOPHY, and (d) S-BOPHY for Aβ aggregates Interference solutions were prepared for probes (a) C-BOPHY, (b) N-BOPHY, (c) O-BOPHY, and (d) S-BOPHY, respectively, to achieve a final probe concentration of 1 μM in the interference solutions. The interferences were K... + Na + Mg 2+ Cu 2+ Ca 2+ Fe 2+ Fe 3+ Glutathione, L-lysine, L-arginine, and L-proline were selected as interfering substances. The fluorescence spectra of the probe in different interfering substances were measured using a fluorescence spectrophotometer and compared with the fluorescence spectrum of "probe + Aβ" to generate a selectivity bar chart. Figures 30-33 As shown, the probe did not show a significant change in fluorescence intensity for the interfering substance, but it showed a significant increase in fluorescence intensity for Aβ, indicating that the probe has good selectivity for Aβ.

[0080] (8) Solution staining imaging of Aβ aggregates using probes (a) C-BOPHY, (b) N-BOPHY, (c) O-BOPHY, and (d) S-BOPHY. Probes (a) C-BOPHY, (b) N-BOPHY, (c) O-BOPHY, and (d) S-BOPHY were added to the prepared Aβ aggregate solution to achieve a final probe concentration of 1 μM; ThT was added to achieve a final ThT concentration of 10 μM. After mixing, 20 μL of each probe was placed on a glass slide, and the Aβ aggregates were imaged and analyzed using a laser confocal fluorescence microscope (Leica SP5). Figure 34 As shown, Aβ aggregates were stained green using commercially available ThT, while C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY stained Aβ aggregates red. ImageJ was used to analyze the co-localization effect. The results showed that the Pearson correlation coefficient R for the staining co-localization of the probe and ThT was greater than 0.7, indicating a good co-localization effect and confirming the probe's specific imaging and analysis capability for Aβ aggregates.

[0081] In summary, the probe of this invention can accurately identify and bind to Aβ aggregates. A Pearson correlation coefficient R greater than 0.7 indicates a high degree of overlap between the probe and ThT markers under the microscope, proving that the probe accurately located the target. The probe in this embodiment emits red fluorescence, which contrasts sharply with the green fluorescence of ThT. This color distinction gives the probe a significant advantage during detection, allowing for clearer observation of the morphology and distribution of Aβ aggregates.

[0082] (9) Fluorescent staining imaging of Aβ in the cortical and hippocampal regions of brain slices from AD mice by probes (a) C-BOPHY, (b) N-BOPHY, (c) O-BOPHY, and (d) S-BOPHY, and colocalization with the commercial Aβ dye ThT. 5 μM brain sections from paraffin-embedded APPswe / PSEN1 or wild-type mice were subjected to in vitro fluorescent staining. Sections were dewaxed by immersion in xylene for 5 minutes, then washed with ethanol for 2 minutes, followed by 5 minutes of water. The sections were incubated with 40 μL of 1 mg / mL ThT for 10 minutes, then washed with 50% ethanol for 13 minutes. After incubation with 40 μL of 10 μM probe for 30 minutes, followed by washing with 50% ethanol for 15 minutes, the sections were treated with an anti-fluorescence quencher and sealed with neutral resin. Brain sections were imaged under a laser scanning confocal microscope (Leica SP5). Figure 35 As shown, Aβ aggregates were stained green using commercially available ThT staining, while C-BOPHY, N-BOPHY, O-BOPHY, and S-BOPHY staining were stained red. Fluorescence imaging was performed on the hippocampal and cortical regions of the brain slices, and ImageJ was used to analyze co-localization. The results showed that the Pearson correlation coefficients (R) for co-localization between the probe and ThT staining were all greater than 0.8, indicating good co-localization and confirming the probe's specific imaging and analysis capabilities for Aβ aggregates in a real brain slice environment. The high overlap between the probe's staining signal and the ThT staining signal indicates that the probe can specifically identify and label Aβ aggregates, rather than binding to non-specific tissue components. The probe effectively imaged these key brain regions, demonstrating its potential to comprehensively assess the pathological distribution of Aβ and cover the most important lesion areas in disease progression.

[0083] (10) Cytotoxicity of probes (a) C-BOPHY, (b) N-BOPHY, (c) O-BOPHY, and (d) S-BOPHY PC12 cells were cultured to the logarithmic growth phase at 37°C with 5% CO2, and then cultured at a rate of 1×10⁻⁶ cells / year. 4 Inoculate 1 / mL of probe into 86-well plates and incubate for 24 hours. Then, add a gradient concentration of probe and incubate for another 24 hours. Measure the absorbance at 490 nm according to the MTT instructions. Figures 36-39 As shown, all probes exhibited cytotoxicity exceeding 80%, indicating excellent biocompatibility. They can accurately label Aβ without disrupting normal cellular physiological activities, fully meeting the safety requirements of bioimaging.

Claims

1. A bis(fluoroborate)hydrazine pyrrole-based fluorescent probe, characterized in that: The fluorescent probe structure, including the fluorescent parent structure bis(fluoroborohydrazine)pyrrole (BOPHY) and the recognition group structure, is as follows: 。 2. The bis(fluoroborate)hydrazine pyrrole fluorescent probe according to claim 1, characterized in that: The difluoroborosilicate pyrrole fluorescent probe is one of the following compounds: 、 、 、 。 3. A method for preparing a difluoroborate pyrrole-based fluorescent probe as described in claim 1 or 2, characterized in that: Includes the following steps: S1, Preparation of BOPHY precursor: 3,5-dimethyl-2-pyrrolecarboxaldehyde was reacted with hydrazine hydrate in ethanol, and condensation was catalyzed by adding an amount of glacial acetic acid. The solid was precipitated at room temperature, filtered, and washed with glacial methanol to obtain the BOPHY precursor. S2, Synthesis of BOPHY fluorescent precursor: Under nitrogen protection, the precursor was dissolved in toluene, triethylamine was added dropwise at 0°C, boron trifluoride diethyl ether was added dropwise at room temperature, and the mixture was heated to reflux at 110°C; after the reaction, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain the BOPHY fluorescent precursor; S3. Synthesis of target probe molecule: 4-azacyclohexylbenzaldehyde derivative and BOPHY were dissolved in toluene, and piperidine and p-toluenesulfonic acid were added as catalysts. The mixture was heated to reflux at 130 °C. After the reaction was completed, the mixture was extracted with ethyl acetate / water, the organic phase was concentrated, and purified by column chromatography to obtain the target probe molecule.

4. The method for preparing the difluoroborate pyrrole fluorescent probe according to claim 3, characterized in that: In S1, the molar ratio of 3,5-dimethyl-2-pyrrolecarboxaldehyde to hydrazine hydrate is 9:

5.

5. The method for preparing the difluoroborate pyrrole fluorescent probe according to claim 3, characterized in that: The volume ratio of triethylamine to boron trifluoride diethyl ether in S2 is 2:1.

7.

6. The method for preparing the difluoroborate pyrrole fluorescent probe according to claim 3, characterized in that: The molar ratio of 4-azacyclohexylbenzaldehyde derivative and BOPHY in S3 is 3.6:3.

1.

7. The method for preparing the difluoroborate pyrrole fluorescent probe according to claim 3, characterized in that: The addition ratio of toluene, piperidine, and p-toluenesulfonic acid in S3 is 10 mL: 1 mL: 30 mg.

8. The application of a difluoroborosilicate pyrrole fluorescent probe as described in claim 1 or 2 in the detection of β-amyloid protein.

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