A responsive near-infrared fluorescent probe targeting butyrylcholinesterase and beta amyloid and a preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and beta amyloid protein as well as a preparation method and application thereof. The responsive near-infrared fluorescent probe is formed by connecting a positively charged hemicyanine as a strong electron-withdrawing group, a carbon-carbon double bond and thiophene as a bridging unit, and a phenylcyclopropane carboxylic acid ester with a BChE recognition group, so as to form an organic small-molecule fluorescent compound with a typical D-pi-A structure. The fluorescent compound not only prolongs the conjugated structure to reach the near-infrared region, but also shows good biocompatibility, no obvious toxic side effects, and binding with the main marker BChE of AD, and is converted from non-fluorescence to fluorescence mode. The fluorescent mode probe is further combined with A beta protein to solve the defect of single target response, to enhance the specific response, and to effectively solve the problem of single target false positive.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and beta amyloid as well as a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a progressive central nervous system degenerative disease. The disease is more insidious, and the incubation period can last for more than ten years. Since the early symptoms of AD are not obvious, the disease has developed to a very serious condition when it is discovered, which often delays treatment, so the early diagnosis of Alzheimer's disease is particularly important, which can help patients to be discovered and treated early, effectively control the disease, and delay the speed of disease deterioration, which has important significance for protecting people's health and improving the quality of life. Among the current clinical diagnosis methods, cerebrospinal fluid (CSF) detection has high accuracy, but the collection process of cerebrospinal fluid puncture is invasive, and therefore is not widely accepted.

[0003] Imaging techniques mainly include fluorescence imaging (FI), computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and single photon emission computed tomography (SPECT), etc. At present, the diagnosis and treatment effect of AD in the clinic needs to be evaluated through multiple reviews and long-term observation. These imaging techniques cannot achieve real-time monitoring and evaluation.

[0004] Due to the complexity of the cause of AD, although the research on it has been carried out for more than 100 years, so far the exact cause of AD has not been completely understood. This has led to the fact that in the past few decades of research, the AD drug development strategy based on a single target has not been successful. Obviously, a single target drug is not enough to improve the pathological condition of AD or even delay its progression. Therefore, the complex mechanism of the disease and the interaction between multiple factors make it necessary to develop a diagnosis and treatment method targeting multiple targets.

[0005] The currently developed molecular probes have the phenomenon of poor selectivity, and false positive results are easy to occur. For example, there are two hydrolytic enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the brain, and the structure and function of AChE and BChE are very similar, which leads to the fact that many probes have low selectivity for them. The probe targeting butyrylcholinesterase has been reported in AD, but the abnormal level of cholinesterase in the brain is also related to other diseases such as Parkinson's disease, frontotemporal dementia and Lewy body dementia, which is easy to confuse. Therefore, the accuracy of the double-target probe is higher. SUMMARY

[0006] The technical problem solved by the present application is to provide a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and beta amyloid protein, which is combined with the main marker BChE of AD, is converted from non-fluorescence to fluorescence mode, and further combines with Aβ protein in the fluorescence mode, thereby enhancing specific response and effectively solving the problem of single target false positive.

[0007] The technical scheme adopted by the present application to solve the above-mentioned problems is:

[0008] A responsive near-infrared fluorescent probe targeting butyrylcholinesterase and beta amyloid protein has the following general structure: In the formula, R is selected from methyl or ethyl.

[0009] According to the above scheme, when R is selected from methyl or ethyl, the responsive near-infrared fluorescent probe is abbreviated as HCy-Me or HCy-Et, respectively, and the specific structure is as shown below:

[0010]

[0011] In the present application, the responsive near-infrared fluorescent probe is composed of a positively charged half-cyanine moiety as an electron-withdrawing moiety and a phenylcyclopropyl carboxylate moiety as a BChE recognition group, which are connected through a carbon-carbon double bond and a bridging unit thiophene group. The maximum ultraviolet absorption wavelength of HCy-Me and HCy-Et in DMSO solution is about 475 nm and about 480 nm, respectively, and the emission wavelength is about 647 nm and about 622 nm, respectively. The maximum ultraviolet absorption wavelength of HMy-OH and HEy-OH in DMSO solvent is about 515 nm and about 520 nm, respectively, and the emission wavelength is about 652 nm and about 653 nm, respectively.

[0012] The synthesis method of the above-mentioned responsive near-infrared fluorescent probe comprises the following steps:

[0013] 1) Substitution reaction of 2,3,3-trimethyl-3H-indole and iodide to introduce a substituent group at N to synthesize an electron acceptor moiety, i.e. a positively charged half-cyanine, with the general structure being R is methyl or ethyl;

[0014] 2) Suzuki coupling reaction of 4-hydroxyphenylboronic acid and 5-bromothiophene-2-carboxaldehyde to obtain an electron donor moiety, i.e. 5-(4-hydroxyphenyl)thiophene-2-carboxaldehyde, with the general structure being

[0015] 3) Knovengel condensation reaction of the products of step 1) and step 2) to obtain an intermediate compound R is methyl or ethyl; wherein, when R is methyl, the intermediate compound is HMy-OH; and when R is ethyl, the intermediate compound is HEy-OH, with the structures as follows:

[0016]

[0017] 4) Compound HMy-OH and HEy-OH are subjected to nucleophilic substitution reaction with cyclopropylcarbonyl chloride respectively to obtain responsive near-infrared fluorescent probes with the structures of HCy-Me or HCy-Et respectively.

[0018] According to the above scheme, in step 1), the iodide is selected from one or both of methyl iodide or ethyl iodide; the molar ratio between 2,3,3-trimethyl-3H-indole and the iodide is in the range of (1-3):3, so that the substitution reaction of step 1) can occur; the time of the substitution reaction is 10-16 h, and the temperature is 55-85℃.

[0019] According to the above scheme, in step 1), acetonitrile is used as the reaction solvent, and the concentration of the iodide in acetonitrile is preferably 0.5-3 mol / L; after the substitution reaction is completed, cold ether (0℃) is added to precipitate the solid product, which is washed with ether and then filtered and dried.

[0020] According to the above scheme, in step 2), the molar ratio between 4-hydroxyphenylboronic acid and 5-bromothiophene-2-carboxaldehyde is in the range of (1-3):1, so that the Suzuki coupling reaction of step 2) can occur; the time of the Suzuki coupling reaction is 10-22 h, and the temperature is 50-90℃.

[0021] According to the above scheme, in step 2), the Suzuki coupling reaction uses tetrakis(triphenylphosphine)palladium as the catalyst, tetrahydrofuran as the reaction solvent, and nitrogen or inert gas or vacuum as the protective atmosphere; the addition amount of the catalyst in the reaction solvent is 3-8 mg / mL; 4-hydroxyphenylboronic acid and 5-bromothiophene-2-carboxaldehyde are each dissolved in tetrahydrofuran under a protective atmosphere to obtain a 4-hydroxyphenylboronic acid solution with a concentration of 0.5-1.5 mmol / mL and a 5-bromothiophene-2-carboxaldehyde solution with a concentration of 0.5-1.5 mmol / mL respectively; then the 5-bromothiophene-2-carboxaldehyde solution is injected into the 4-hydroxyphenylboronic acid solution (the catalyst is pre-added to the 4-hydroxyphenylboronic acid solution), an aqueous sodium carbonate solution is added to adjust the pH to 8.5-9, the temperature is raised to 70-90℃, and the reaction is carried out for 10-22 hours; after the Suzuki coupling reaction is completed, extraction is carried out with a water and dichloromethane system, and then column chromatography is carried out with an eluent prepared by mixing petroleum ether and ethyl acetate in a volume ratio of (4-6):1 to obtain the product of step 2).

[0022] According to the above scheme, in step 3), the product of step 1) and the product of step 2) are subjected to the Claisen condensation reaction in a molar ratio of 1:(1-3), the reaction time is 1.5-3h, and the temperature is 50-80℃.

[0023] According to the above scheme, in step 3), ethanol is used as the reaction solvent, and a small amount of piperidine is also added to the reaction solvent, the volume of piperidine is 2-3% of the volume of ethanol, and the concentration of the product of step 2) in ethanol is 0.02-0.05mmol / mL; a protective atmosphere of nitrogen or inert gas or vacuum is used during the reaction; after the reaction is completed, dichloromethane and methanol are mixed in a volume ratio of (15-25):1 to form an eluent for column chromatography.

[0024] According to the above scheme, in step 4), the molar ratio of the intermediate compound obtained in step 3) to cyclopropylcarbonyl chloride is (0.5-1.5):2, the reaction time is 1.5-3h, and the temperature is -5℃-0℃.

[0025] According to the above scheme, in step 4), dichloromethane is used as the reaction solvent, the addition concentration of cyclopropylcarbonyl chloride in the reaction solvent is 0.03-0.05mmol / mL, and a certain amount of triethylamine needs to be added, the concentration of triethylamine in the reaction solvent is 2-5mg / mL; a protective atmosphere of nitrogen or inert gas or vacuum is used during the reaction; after the reaction is completed, dichloromethane and methanol are mixed in a volume ratio of (15-25):1 to form an eluent for column chromatography.

[0026] The technical concept of the present application is as follows:

[0027] The responsive near-infrared fluorescent probe involved in the present application includes HCy-Me and HCy-Et, which have weak or no fluorescence in the initial state, and after contacting with butyrylcholinesterase (BChE), they rapidly hydrolyze the ester bond and return to HMy-OH and HEy-OH (at this time they can be called hydrolyzed fluorescent probes), and the fluorescence instantaneously enhances. During the binding process of the probe and the marker beta amyloid protein, the hydroxyl groups of HMy-OH and HEy-OH form two or one hydrogen bonds with the active site F20 on the beta amyloid protein, and the hydrogen bond force restricts the torsional motion of the probe molecule, thereby inhibiting the intramolecular charge transfer (ICT) process. The ICT process usually causes fluorescence quenching, so its inhibition will make the fluorescence significantly enhance. In the brain of AD patients, the probes HCy-Me and HCy-Et can sensitively detect the main marker BChE, and convert from no fluorescence to fluorescence mode, and then make the fluorescent mode probe further bind to the Aβ protein and specifically respond to enhance, which can help AD patients to be found and treated early, and effectively control the disease.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] 1. The present application provides a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and beta-amyloid protein, which is first reacted with iodide through a hemicyanine structure to introduce a substituent group on N, forming a stronger electron acceptor, and the middle of the molecular structure is composed of a vinyl group and a heterocycle to expand the conjugated system; the binding group of BChE mainly consists of a phenylcyclopropane carboxylate, the cyclopropane carboxylate is a leaving group, and finally a phenolic hydroxyl group is formed as an electron donor group. This kind of fluorescent compound can expand the conjugation degree of the original structure, making the wavelength red shift.

[0030] 2. The responsive near-infrared fluorescent probe provided by the present application is hydrolyzed and removed after being combined with BChE, and is converted from a non-fluorescent state to a fluorescent mode; at the same time, the hydroxyphenyl group can act as an electron donor group to form a "push-pull" structure with the indole group. This probe is very sensitive to the environment solvent and is prone to aggregation leading to luminescence quenching (ACQ), and has a very low fluorescence quantum yield in aqueous solution. However, after the probe is combined with the Aβ protein, the intramolecular rotation is limited, and the fluorescence signal of the probe molecule is significantly enhanced due to the intramolecular charge transfer (ICT) effect, solving the problem of single target false positive and the defect of single target response. The above-mentioned new responsive near-infrared fluorescent probe increases the accuracy of fluorescent probe diagnosis and provides a new idea for early and accurate diagnosis of Alzheimer's disease.

[0031] 3. The responsive near-infrared fluorescent probe has good liposolubility, can quickly pass through the blood-brain barrier, has good biocompatibility, and successfully performs diagnostic imaging on an Alzheimer's disease model mouse (C57BL / 6, APP / PS1) in vivo, which can realize real-time visualization and specific detection of early molecular changes of the disease.

[0032] 4. The responsive near-infrared fluorescent probe has high selectivity for BChE. The active site of BChE is different in structure from the active site of acetylcholinesterase (AChE), especially the active cavity of BChE is larger, and lacks the narrow aromatic amino acid residues (such as Tyr124 and Phe337) in AChE. According to this structural feature, a larger steric hindrance group-cyclopropyl is introduced as a recognition unit in the present application to adjust the size of the binding group of BChE to construct a highly selective probe for BChE. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The present application is a synthetic route map of the fluorescent probe.

[0034] Figure 2Response and selectivity study of the probes of the present application to BChE. a-b are the fluorescence emission spectra of HCy-Me and HCy-Et (5 μΜ, final concentration after mixing) after incubation with different concentrations of BChE in PBS (10 mM, pH 7.4), (HCy-Me: λex= 475 nm; HCy-Et: λex= 480 nm). c-d are the plots of fluorescence intensity as a function of BChE concentration. e-f are the selectivity studies of HCy-Me and HCy-Et with potential competing substances (metal ions, amino acids, AChE, etc.) and BChE.

[0035] Figure 3 Response and selectivity study of the probes of the present application to Αβ fibrils. a-b are the fluorescence emission spectra of hydrolytic fluorescent probes HMy-OH and HEy-OH after incubation with different concentrations of Αβ aggregates in PBS (10 mM, pH 7.4), (HMy-OH: λex= 515 nm; HEy-OH: λex= 520 nm). c-d are the plots of fluorescence intensity of HMy-OH and HEy-OH as a function of Αβ aggregates concentration. e is the selectivity study of HMy-OH and HEy-OH with potential competing substances and Αβ aggregates.

[0036] Figure 4 Fluorescence imaging study of the fluorescent probes HCy-Me and HCy-Et in brain sections of the present application,

[0037] Figure 5 Fluorescence imaging study of the hydrolytic fluorescent probes HMy-OH and HEy-OH in brain sections of the present application, (HMy-OH: λex= 515 nm, λem= 640-650 nm; HEy-OH: λex= 520 nm, λem= 640-650 nm); where a, b are the brain sections of age-matched Tg mice (APP / PS1, 12 months old) stained with HMy-OH and ThT in vitro, d, e are the brain sections of WT mice (12 months old) stained with HMy-OH and ThT in vitro, c, f are their merged images. g, h are the brain sections of age-matched Tg mice (APP / PS1, 12 months old) stained with HEy-OH and ThT in vitro, j, k are the brain sections of WT mice (12 months old) stained with HEy-OH and ThT in vitro, i, 1 are their merged images.

[0038] Figure 6 In vivo fluorescence imaging of BChE of the present application, (HCy-Me: λex= 475 nm, λem= 640-650 nm; HCy-Et: λex= 480 nm, λem= 620-640 nm); where a, b are the fluorescence images of wild type mice and APP / PS1 mice (12 months old) at different time points before and after tail vein injection of HCy-Me and HCy-Et, respectively. DETAILED DESCRIPTION

[0039] In order to better understand the present application, the following further illustrates the content of the present application in conjunction with examples, but the present application is not limited to the following examples only.

[0040] Example 1

[0041] Referring to Figure 1 The synthesis method of a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and beta amyloid (abbreviated as HCy-Me) is shown in the following specific steps:

[0042] (1) Synthesis of compound 1:

[0043] Methyl iodide (0.93 mL, 15 mM) and 2,3,3-trimethyl-3H-indole (1.6 mL, 10 mM) were added to acetonitrile (15 mL) as a reaction solvent. They were placed in an oil bath pot and refluxed at 80°C under vigorous stirring for 12 h to obtain a pink solution. The stirring was stopped and the pink solution was naturally cooled to room temperature. An excess of cold ether was added to precipitate the solid, which was filtered under reduced pressure and washed with ether 3 times to obtain a filter cake. The filter cake was placed in a vacuum drying oven to dry to obtain a clean product, i.e. compound 1, which did not need further purification. Compound 1 was a pink solid with a yield of 78.0% (1.7 g).

[0044] (2) Synthesis of compound 3:

[0045] Tetrakis(triphenylphosphine)palladium (0.1 g) was placed in a three-necked flask, 4-hydroxyphenylboronic acid (10 mmol) was dissolved in 10 mL of tetrahydrofuran and injected into the three-necked flask under nitrogen protection, and a syringe was used to inject 5-bromothiophene-2-carboxaldehyde (10 mmol) dissolved in 10 mL of tetrahydrofuran into the three-necked flask, and then sodium carbonate (2.76 g) was dissolved in water (6 mL) and injected, at which time the pH was 8.5-9, and the temperature was raised to 80°C and stirred for 16 h. After the reaction was completed, the temperature was lowered to room temperature, and then 100 mL of water was added, and extracted with dichloromethane (3 x 50 mL), and the obtained organic phase was dried with anhydrous sodium sulfate, filtered and evaporated to obtain a crude product. The crude product was subjected to column chromatography with petroleum ether and ethyl acetate (5 / 1 by volume) as eluent to obtain yellow solid compound 3 (136 mg, 67%).

[0046] (3) Synthesis of compound HMy-OH:

[0047] Compound 1 (162.5 mg, 0.93 mmol) and compound 3 (300 mg, 1.40 mmol) were mixed and dissolved in ethanol (30 mL) and piperidine (4 drops), and then heated to 60 °C and stirred for 2 h under nitrogen. After the reaction was completed, the mixture was filtered and evaporated to give the crude product. The crude product was eluted with DCM / MeOH (20 / 1, volume ratio of dichloromethane to methanol) and then subjected to column chromatography to give the dark red solid compound HMy-OH (134 mg, 40%).

[0048] (4) Synthesis of compound HCy-Me:

[0049] Compound HMy-OH (100 mg, 0.27 mmol) and cyclopropylformyl chloride (56 mg, 0.54 mmol) were dissolved in dichloromethane (15 mL), and triethylamine (54 mg) was slowly added dropwise. The mixture was then stirred at 0 °C for 2 h under a nitrogen atmosphere. After the reaction was complete, the mixture was filtered and evaporated to obtain the crude product. The crude product was eluted with DCM / MeOH (20 / 1, v / v) and then subjected to column chromatography to obtain a deep red solid compound HCy-Me (86 mg, 75%), which is a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid protein (abbreviated as HCy-Me).

[0050] Example 2

[0051] Reference Figure 1 As shown, a method for synthesizing a responsive near-infrared fluorescent probe (abbreviated as HCy-Et) targeting butyrylcholinesterase and β-amyloid protein specifically includes the following steps:

[0052] (1) Synthesis of compound 2:

[0053] Iodoethane (1.20 mL, 15 mM) and 2,3,3-trimethyl-3H-indole (1.6 mL, 10 mM), followed by toluene (15 mL) as the reaction solvent, were reacted in an oil bath and refluxed at 80 °C for 12 h with vigorous stirring to obtain a pink solution. Stirring was stopped, and the pink solution was allowed to cool naturally to room temperature. Excess cold diethyl ether was added to precipitate the solid. The precipitate was filtered under reduced pressure and washed three times with diethyl ether to obtain a filter cake. The filter cake was dried in a vacuum drying oven to obtain a clean product, compound 2, which required no further purification. Compound 2 was a pink solid with a yield of 75.0% (1.7 g).

[0054] (2) Synthesis of compound 3: Same as in Example 1.

[0055] (3) Synthesis of compound HEy-OH:

[0056] Compound 2 (100 mg, 0.53 mmol) and compound 3 (163 mg, 0.8 mmol) were mixed and dissolved in ethanol (30 mL) and piperidine (4 drops), then heated and stirred at 60 °C under nitrogen atmosphere for 2 h. After the reaction was completed, filtration and evaporation were performed to obtain a crude product. The crude product was subjected to column chromatography with DCM / MeOH (20 / 1, volume ratio) as the eluent to obtain compound HEy-OH (79 mg, 40%) as a dark red solid.

[0057] (4) Synthesis of compound HCy-Et:

[0058] Compound HEy-OH (180 mg, 0.48 mmol) and cyclopropylcarbonyl chloride (100 mg, 0.96 mmol) were dissolved in dichloromethane (20 mL), and triethylamine (97 mg) was slowly added dropwise, then the reaction was stirred at 0 °C under nitrogen atmosphere for 2 h. After the reaction was completed, filtration and evaporation were performed to obtain a crude product. The crude product was subjected to column chromatography with DCM / MeOH (20 / 1, volume ratio) as the eluent to obtain compound HCy-Et (160 mg, 75%) as a dark red solid, which is a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and beta amyloid (abbreviated as HCy-Et).

[0059] Application Example 1

[0060] The responsive near-infrared fluorescent probes HCy-Me and HCy-Et prepared in Examples 1-2 were tested for their response ability and specificity to BChE.

[0061] The response ability of the probes to BChE was tested as follows: BChE (0-1.6 U / mL, the concentration refers to the final concentration in PBS, the same below) was mixed with HCy-Me and HCy-Et (5 μM) in PBS (pH 7.4) respectively, vortexed for 10 seconds, and then placed in a constant temperature incubator at 37 °C without shaking for 60 minutes to test their fluorescence emission spectra. The specificity of the probes to BChE was tested as follows: the probes HCy-Me and HCy-Et (5 μM, the concentration refers to the final concentration after mixing, the same below) were mixed with 1: Na + (10 μM), 2: Mg 2+ (10 μM), 3: K + (10 μM), 4: Fe 2+ (10 mM), 5: Cu 2+ (10 μM), 6: Ca 2+ (10 μM), 7: Zn 2+(10 mM), 8: Ser. (10 mM), 9: Lys (10 mM), 10: Arg. (10 mM), 11: GSH (10 mM), 12: Gly. (10 mM), 13: Phe. (10 mM), 14: Cys (10 mM), 15: Ala. (10 mM), 16: Pepsin (10 mg / mL), 17: HSA (10 mg / mL), 18: BSA (10 mg / mL), 19: AChE (1 U / mL), 20: BChE (1 U / mL) in PBS (pH 7.4) were mixed and incubated in a 37 °C incubator for 30 min, and then their fluorescence emission spectra were tested. All samples were configured in triplicate as self-control groups.

[0062] From Figure 2 a, b, it can be seen that the fluorescence of probes HCy-Me and HCy-Et is enhanced with the increase of the concentration of BChE, which indicates that probes HCy-Me and HCy-Et can sensitively recognize BChE. Figure 2 c, d, the functional relationship between the fluorescence intensity of the probe and the concentration of BChE, can be known that there is a good linear relationship between the two. From Figure 2 e, f, it can be known that the fluorescence emission of the probe is not observed to be enhanced or quenched in the environment of part of metal ions, amino acids and AChE, while it shows a phenomenon of significantly enhanced fluorescence intensity after being combined with the marker BChE, which indicates that the probe indeed has a specific response to the marker BChE.

[0063] Application Example 2

[0064] The hydrolytic fluorescent probes HMy-OH and HEy-OH prepared in Examples 1-2 were tested for application, and the response ability and specificity of the probes to Aβ fibrils were tested.

[0065] The response ability of the probe to Aβ 42 was tested as follows: the incubated Aβ 42 aggregates (0-10 mM, the concentration refers to the final concentration after mixing, the same below) were mixed with HMy-OH and HEy-OH (10 mM) respectively, vortexed for 10 seconds, and then placed in a constant temperature incubator without shaking at 37 °C for 30 minutes, and the fluorescence emission spectra were tested. The specificity of the probe to Aβ fibrils was tested as follows: the fluorescent probes HMy-OH and HEy-OH (10 mM) were mixed with Aβ monomers, Aβ oligomers and Aβ aggregates in PBS (pH 7.4) respectively, and incubated in a 37 °C constant temperature incubator for 30 min, and then the fluorescence emission spectra were tested. All samples were configured in triplicate as self-control groups.

[0066] From Figure 3As shown in Fig. 1a and Fig. 1b, the fluorescence of probes HMy-OH and HEy-OH increased with the increase of the concentration of Aβ, which indicated that probes HMy-OH and HEy-OH could sensitively recognize Aβ. Figure 3 c and d are the functional relationship between the fluorescence intensity of the probe and the concentration of Aβ, and it can be seen from the figure that there is a good linear relationship between the two. From the data of e, it can be seen that the fluorescence emission of the probe did not increase or quench in the environment of Aβ monomer and Aβ oligomer, but showed a phenomenon of significantly increased fluorescence intensity after binding with the marker Aβ aggregate, which indicated that the probe indeed had specific response to the marker Aβ. Figure 3

[0067] Application Example 3

[0068] The in vitro section staining effect of the fluorescent probes HCy-Me and HCy-Et prepared in Examples 1-2 was tested. The specific process was as follows: the brain tissues of 8-month-old APP / PS1 mice and normal mice of the same age were taken out under perfusion, after the brain tissue section, the adjacent two layers of sections were respectively infiltrated with HCy-Me (10 μM, prepared by PBS with pH 7.4) and HCy-Et (10 μM, prepared by PBS with pH 7.4) to fully infiltrate the tissues, and incubated at room temperature for 15 minutes, and then the tissue sections were washed with normal saline. The results are shown in Fig. 2, which shows that no fluorescence signal is observed in normal mice; in the adjacent brain sections of AD model mice, the fluorescence intensity is obviously increased, thereby proving that the probes HCy-Me and HCy-Et both have the ability of specific recognition of BChE. Figure 4

[0069] Application Example 4

[0070] The in vitro section staining effect of the hydrolyzed fluorescent probes HMy-OH and HEy-OH prepared in Examples 1-2 was tested, and the specific process was as follows: the brain tissues of 8-month-old APP / PS1 mice and normal mice of the same age were taken out under perfusion, after the brain tissue section, the adjacent two layers of sections were respectively infiltrated with HMy-OH (10 μM, prepared by PBS with pH 7.4) and HCy-OH (10 μM, prepared by PBS with pH 7.4) to fully infiltrate the tissues, and incubated at room temperature for 15 minutes, and then the tissue sections were washed with normal saline, and then infiltrated with ThT (10 μM, prepared by PBS with pH 7.4) to fully infiltrate the tissues, and incubated at room temperature for 15 minutes, and then the tissue sections were washed with normal saline again. The results are shown in Fig. 3, which shows that no fluorescence signal is observed in normal mice; in the adjacent brain sections of AD model mice, the staining areas of ThT and the hydrolyzed fluorescent probes HMy-OH and HCy-OH are coincident. It is proved that the hydrolyzed fluorescent probes HMy-OH and HCy-OH both have the ability of specific targeting of Aβ. Figure 5

[0071] Application Example 5​​​

[0072] The fluorescent probes HCy-Me and HCy-Et prepared in Examples 1-2 were subjected to in vivo imaging. Specifically, the probes HCy-Me (concentration 10 mM, 100 uL, prepared with PBS at pH 7.4) and HCy-Et (10 mM, 100 uL, prepared with PBS at pH 7.4) were respectively injected into the tail vein of mice. Fluorescent images were collected using an IVIS system, with an excitation wavelength of 450 ± 10 nm, an emission wavelength of 620 ± 10 nm, and a collection time of 2 s. The results are shown in Figure 6 From the figure, it can be seen that, 1 h after injection of the fluorescent probes HCy-Me (a) and HCy-Et (b), the fluorescence reached a maximum, while the normal mouse brain of the control group showed no obvious change in fluorescence signal. Figure 6 Figure 6 , a) and HCy-Et (b)​

Claims

1. A responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid protein, characterized in that... The responsive near-infrared fluorescent probe has the following general structural formula: R is selected from methyl or ethyl.

2. The responsive near-infrared fluorescent probe according to claim 1, characterized in that, The responsive near-infrared fluorescent probe includes the following compounds: 。 3. The method for preparing the responsive near-infrared fluorescent probe according to claim 2, characterized in that, The main steps are as follows: 1) 2,3,3-Trimethyl-3H-indole undergoes a substitution reaction with iodides to introduce a substituent onto the nitrogen atom, synthesizing the electron acceptor moiety, i.e., the positively charged hemicyanine, with the general structural formula: R is either methyl or ethyl; 2) 4-Hydroxyphenylboronic acid reacts with 5-bromothiophene-2-carboxaldehyde via a Suzuki coupling reaction to yield the electron-donating moiety, namely 5-(4-hydroxyphenyl)thiophene-2-carboxaldehyde, with the general structural formula: ; 3) The products of steps 1) and 2) are subjected to a Knauvengel condensation reaction to obtain an intermediate compound with the general structural formula: R is methyl or ethyl; 4) The intermediate compound obtained in step 3) undergoes a nucleophilic substitution reaction with cyclopropylformyl chloride to obtain responsive near-infrared fluorescent probes with structures of HCy-Me or HCy-Et, respectively.

4. The method for preparing the responsive near-infrared fluorescent probe according to claim 3, characterized in that, In step 1), the iodide is selected from one or both of iodomethane or iodoethane; the molar ratio between 2,3,3-trimethyl-3H-indole and the iodide is in the range of (1~3):3; the substitution reaction time is 10~16h and the temperature is 55~85°C.

5. The method for preparing the responsive near-infrared fluorescent probe according to claim 3, characterized in that, In step 2), the molar ratio of 4-hydroxyphenylboronic acid to 5-bromothiophene-2-carboxaldehyde is in the range of (1~3):1; the Suzuki coupling reaction time is 10~22h, and the temperature is 50~90°C.

6. The method for preparing a responsive near-infrared fluorescent probe according to claim 3, characterized in that, In step 3), the molar ratio of the product of step 1) to the product of step 2) is 1:(1~3); the reaction time is 1.5~3h, and the temperature is 50~80°C.

7. The method for preparing the responsive near-infrared fluorescent probe according to claim 3, characterized in that, In step 4), the molar ratio of the intermediate compound obtained in step 3) to cyclopropylformyl chloride is (0.5~1.5):2, the reaction time is 1.5~3h, and the temperature is -5~0°C.

8. The use of the responsive near-infrared fluorescent probe according to claim 1 in the preparation of diagnostic reagents for Alzheimer's disease.

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