CN114907222A - TPE (thermoplastic elastomer) -based aggregation-induced emission fluorescent probe and application thereof - Google Patents

TPE (thermoplastic elastomer) -based aggregation-induced emission fluorescent probe and application thereof Download PDF

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CN114907222A
CN114907222A CN202210723297.9A CN202210723297A CN114907222A CN 114907222 A CN114907222 A CN 114907222A CN 202210723297 A CN202210723297 A CN 202210723297A CN 114907222 A CN114907222 A CN 114907222A
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崇辉
王娜
房司雨
王天奕
王赪胤
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Abstract

The invention relates to a TPE-based aggregation-induced emission fluorescent probe and application thereof in the technical field of fluorescence spectrum analysis materials, and the TPE-based aggregation-induced emission fluorescent probe is used for A beta related to Alzheimer's disease 1‑42 Detection and analysis of amyloid, the fluorescent probe of the invention, the chemical name of the compound: (E/Z) -N, N '- ((((1, 2-diphenylethylene-1, 2-dimethyl) di ([1,1' -biphenyl)]-4', 4-diyl)) bis (methylene)) bis (2- (2- (2- (2-hydroxyethoxy) ethoxy) -N, N-dimethylethoxy) 1-amino), referred to as TPE-Q for short. The synthetic route is relatively simple and convenient, the product stability is good, and no specific recognition group exists. For A beta associated with Alzheimer's disease 1‑42 The detection of the aggregate can achieve the same imaging effect as that of a commercial specific fluorescent labeling agent (Th T).

Description

TPE (thermoplastic elastomer) -based aggregation-induced emission fluorescent probe and application thereof
Technical Field
The invention relates to the technical field of fluorescence spectrum analysis materials, in particular to a TPE (thermal plastic elastomer) -based aggregation-induced emission fluorescent probe for A beta related to Alzheimer's disease 1-42 Detection and analysis of amyloid protein.
Background
Fluorescence is a luminescence phenomenon, when a chemical substance is irradiated by incident light with a certain wavelength, molecules absorb external energy and are converted into an excited state, the excited state is converted into a ground state within a short time, and fluorescence is emitted in the conversion process. Based on the fluorescence mechanism, a variety of fluorescent probes have been developed. The fluorescent probe technology has the advantages of high sensitivity, high analysis speed, simple operation and the like. It has become an efficient, convenient and real-time monitoring method for environmental monitoring, biological analysis and clinical diagnosis.
The development of fluorescence technology relies on the design of fluorescent probes. Molecular imaging refers to the implantation of non-invasive fluorescent probes into cells or organs to visualize biological targets that are not convenient for direct observation to assess the physiological processes of living systems. Therefore, the technology of combining fluorescent probe molecules with biological detection is helpful for realizing quantitative and positioning analysis of biological macromolecules or specific organelles in organisms. The fluorescent group has high structural diversity through chemical modification, which is beneficial to functional design and performance modulation of the probe. Compared with fluorescent inorganic nanomaterials, organic fluorophores have higher optical brightness. The use of fluorescent materials in the biomedical field not only provides the ability to directly visualize biological species and activities, but also brings the possibility of using light to perform therapeutic functions. Fluorescence technology has advanced advantages and is the best candidate for microbial detection, such as simplicity of operation, real-time response, in situ sensitivity and specificity.
Alzheimer's Disease (AD) is characterized by language impairment, cognitive decline, disorientation, irreversible memory loss, which causes elderly people to suffer fromSerious health threat. Early diagnosis and intervention of AD have important significance for delaying disease development. Accumulated ap in the cerebrospinal fluid 1-42 Amyloid (a β) plaques are considered to be important biomarkers for early diagnosis of Alzheimer's Disease (AD). Thus, highly specific, non-invasive and rapid determination of A β in the brain 1-42 Amyloid aggregates can facilitate clinically effective diagnosis of AD and can be used to assess potential anti-AD effects of candidate drugs. The small molecular probe for fluorescence imaging can be used for easily detecting A beta in real time 1-42 An amyloid protein. Currently used for detecting Abeta 1-42 Amyloid probes mostly contain specific recognition groups and are relatively complex to synthesize. The TPE-Q fluorescent probe synthesized by the method has a simple synthetic route and low cytotoxicity.
Disclosure of Invention
Aiming at the defects existing in the synthesis and the use of the AD diagnosis early detection probe material in the prior art, the invention provides the TPE-based aggregation-induced emission fluorescent probe which is simple in synthetic route and has smaller cytotoxicity.
The invention aims to realize the fluorescence probe based on the aggregation-induced emission of the TPE, which is characterized in that the fluorescence probe is an isomer TPE-Q with a structural formula of (1):
Figure BDA0003710171510000021
wherein the chemical name of the compound corresponding to formula (1): (E/Z) -N, N ' - ((((1, 2-diphenylethylene-1, 2-dimethyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (2- (2- (2- (2-hydroxyethoxy) ethoxy) -N, N-dimethylethoxy) 1-amino), abbreviated TPE-Q.
Further, the TPE-Q in the above formula (1) is prepared as follows:
1) heating and refluxing 4-bromo-benzophenone in anhydrous THF for reaction, wherein the reaction is Mcmmurry reaction, and the reaction formula is as follows:
Figure BDA0003710171510000022
then separating and purifying to obtain 1, 2-di (4-bromophenyl) -1, 2-diphenylethylene;
2) heating 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene and 4-formylphenyl boric acid in N, N-dimethylacetamide under the condition of reflux reaction, said reaction is Suzuki-Miyaura reaction, and its reaction formula is as follows:
Figure BDA0003710171510000023
then 4',4' - (1, 2-diphenylethylene-1, 2-diyl) di ([1,1' -biphenyl ] -4-formaldehyde) is obtained by separation and purification;
3) heating and refluxing 4',4' - (1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4-formaldehyde) and 1-amino-3, 6, 9-trioxa-11-undecanol in methanol for 12-16, wherein the reaction is a process for eliminating aldehyde-based oxygen by nucleophilic substitution, and then adding sodium borohydride to reduce imine into secondary ammonium, wherein the reaction formula is as follows:
Figure BDA0003710171510000031
finally recrystallizing to obtain 2,2' - (((((((1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azepindiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol);
4) reacting 2,2' - ((((((1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azepindiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) with methyl iodide for 2 to 3 hours, which is an amination reaction in a nucleophilic substitution reaction, according to the following reaction formula:
Figure BDA0003710171510000032
and then separating and purifying to obtain the TPE-Q.
Further, in the step 1), the 4-bromobenzophenone is dissolved in anhydrous THF according to a feeding ratio of 0.8mol/L for reaction, zinc powder and titanium tetrachloride are used as catalysts, the reaction temperature is THF reflux temperature, the reaction time is 20-25 hours, separation and purification are carried out after the reaction, and the steps are sequentially as follows: k 2 CO 3 The solution was quenched, washed with dichloromethane, dried over anhydrous magnesium sulfate, filtered, and column chromatographed [ silica gel; petroleum ether/ethyl acetate; 18: 1(v/v)]Separating to obtain a purified product.
Still further, in step 2), with an aqueous solution of sodium fluoride and potassium carbonate as a catalyst, the reaction product of 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene: 4-formylphenylboronic acid (4.29mmol) is added into N, N-dimethylacetamide according to the molar ratio of 1:3 to react at the temperature of 80 ℃ for 10 hours; separating and purifying the product after reaction, and sequentially comprising the following steps: dichloromethane extraction, drying over anhydrous magnesium sulfate, filtration, column chromatography [ 200-: dichloromethane as eluent ] purified to give the purified product.
Still further, in step 3), the 4',4' - (1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4-carbaldehyde): 1-amino-3, 6, 9-trioxa-11-undecanol in a molar ratio of 1: 2.4; the reaction solvent was methanol (9.25 ml); the reaction temperature is the reflux temperature of the methanol; after the reaction time is 6 hours, cooling the mixture to room temperature, adding sodium borohydride into the reaction solution while stirring within half an hour, reducing the imine into secondary ammonium, and reacting for 5 hours at room temperature; then carrying out separation and purification, comprising the following steps: the purified product was obtained by recrystallization from dichloromethane/deionized water.
Still further, in step 4), with the solvent being methanol, the catalyst being potassium carbonate, 2,2' - (((((((1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azepindiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) (0.111 mmol): the molar ratio of methyl iodide (1.34mmol) is 1:12, the reaction is carried out for 10 to 14 hours at room temperature, and the solvent in the reaction solution is dried after the reaction is finished; then separating and purifying the reactant, and sequentially comprising the following steps: removing impurities from petroleum ether, and carrying out pure crystallization on methanol/dichloromethane to obtain a purified product.
The fluorescence probe based on the TPE has the following advantages: (1) compared with the synthetic method in the prior art, the TPE-based isomer fluorescent molecule synthesized for the first time has the advantages of simple and convenient operation, simple separation method, easy product acquisition and considerable yield;
(2) the TPE is simple to synthesize, good in stability, low in raw material price, free of toxic and harmful substances generated in the synthesis process, and capable of proving that the synthesized product is non-toxic and harmless to cells in a cytotoxicity experiment and being used for newly detecting Abeta 1-42 An analytical probe for protein aggregates;
(3) the molecule does not contain specific recognition groups such as polypeptide and the like.
The invention also provides application of the TPE-Q fluorescent probe to detection of Abeta related to Alzheimer's disease 1-42 In an aggregate. The specific detection mechanism is as follows: in the probe structure, a tetraphenylethylene unit is a molecular rotor, wherein an alkoxy chain and a quaternary ammonium salt group are added to achieve the purpose of increasing the water solubility of the probe, and the introduction of a benzene ring enables the excitation wavelength and the emission wavelength of the molecule to be red-shifted. In A beta 1-42 In the amyloid aggregate system, because the rotation of the molecular rotor is limited, the energy of the excited state of the probe is mainly lost in the form of radiation, and the probe emits stronger fluorescence. In-vitro experiments prove that the TPE-Q fluorescent probe can be used for detecting Abeta 1-42 Protein aggregates. In addition, imaging experiments were performed by using a commercial plaque-specific fluorescent labeling agent Th t as a control. The fluorescence of the two probes in the body of the nematode suffering from Alzheimer's disease was found to overlap well. Secondly, it was suggested laterally that probes could be attached to A.beta.by molecular docking 1-42 The aggregates are bound.
Drawings
FIG. 1 shows TPE-Q probes 1 H NMR spectrum.
FIG. 2 shows TPE-Q probe 13 C NMR spectrum.
FIG. 3 is a high resolution mass spectrum of the TPE-Q probe.
FIG. 4 shows the different incubation times A.beta. 1-42 Morphology change of aggregates
FIG. 5 is a graph of bovine serum albumin and Abeta after addition of TPE-Q 1-42 Fluorescence spectrum of (2).
FIG. 6 is a fluorescent image of nematodes with Alzheimer's disease after addition of TPE-Q.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to these examples.
Example 1
The fluorescent probe is synthesized according to the following steps:
(1) adding zinc powder (2.50g, 37.90mmol) into a 120mL pressure-resistant bottle, charging nitrogen to discharge oxygen in the bottle, adding 25mL anhydrous THF, cooling to 0 deg.C, and slowly dripping 2.2mL titanium tetrachloride TiCl into ice-salt bath via a constant pressure dropping funnel 4 (2.20mL, 20.00mmol) and pyridine (0.10mL, 1.20 mmol). After refluxing the mixture at 75 ℃ for 2h, 25mL of a solution of 4-bromobenzophenone (5.00g, 19.20mmol) in tetrahydrofuran was added and the reaction mixture was further refluxed at 75 ℃ for 20 h. After the reaction is finished, cooling the reaction mixed liquid to room temperature; poured into 40mL of 10% aqueous potassium carbonate solution, stirred vigorously for 5min, and then filtered to give a white solid, which was washed by immersing it in an excess of dichloromethane, and the insoluble solid was removed by filtration. Anhydrous Mg for filtrate 2 SO 4 Drying, filtration and removal of the organic solvent by rotary evaporation gave the crude product which was isolated and purified by column chromatography (silica gel, petroleum ether/ethyl acetate ═ 18: 1) to give the light yellow product 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene (87.2% yield). The reaction formula is as follows:
Figure BDA0003710171510000051
(2) under nitrogen atmosphere, adding K 2 CO 3 (829.26mg, 6.00mmol) and NaF (251.9mg, 6.00mmol) were dissolved in 3mL of deionized water, and the solution was added to a 45mL dry pressure-resistant bottle, followed by the addition of N, N-dimethylacetamide (15mL) and 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene (70 mL)0mg, 1.43mmol), 4-formylphenylboronic acid (643.50mg, 4.29mmol), Pd (PPh) 3 ) 4 (173.4mg, 0.15 mmol). Heating reflux stirring for 10h, pouring the mixture into two times of water, extracting with dichloromethane for 3 times, combining organic layers, and drying with anhydrous magnesium sulfate. The crude product was further purified by column chromatography (200-mesh 300-mesh silica gel) using methylene chloride as eluent to give the yellow solid compound 4',4' - (1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl)]-4-formaldehyde). The reaction formula is as follows:
Figure BDA0003710171510000052
(3) 4',4' "- (1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4-carbaldehyde) (100mg, 0.185mmol), 1-amino-3, 6, 9-trioxa-11-undecanol (85.8mg, 0.444mmol), methanol (9.25mL) were added to a 25mL round bottom flask, and the mixture was heated to reflux overnight. The reaction mixture was cooled to room temperature and further cooled to 0 ℃. Sodium borohydride (63mg, 1.665mmol) was added to the solution stirring in the ice bath in portions over 10min, stirring at room temperature for 5 h. The mixture was filtered, the methanol was removed by cooling the rotary evaporator, the solid mass was dissolved in dichloromethane (600 μ L) and recrystallized from distilled water, and the solid powdery product 2,2' - (((((((((1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azepinyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) was collected by suction filtration. The reaction formula is as follows:
Figure BDA0003710171510000061
(4)2,2' - ((((((1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azepindiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) (100mg, 0.111mmol), iodomethane (83. mu.L, 1.34mmol), potassium carbonate (61.27mg, 0.444mmol), methanol (3mL) were sequentially added to a 15mL pressure-resistant bottle and reacted at room temperature for 10 hours. The reaction mass is dried by spinning off the solvent, and the residue of compound c is washed off with petroleum ether. It was dissolved with a small amount of methanol and then recrystallized in dichloromethane to give the product TPE-Q as a yellow powder (yield 78.2%). The reaction formula is as follows:
Figure BDA0003710171510000062
FIG. 1 shows the NMR spectrum of TPE-Q with a characteristic peak at δ 3.02ppm, corresponding to 4 methyl groups in the quaternary ammonium, and a peak area of 16.
As shown in fig. 2, the nuclear magnetic resonance carbon spectrum of TPE-Q specifically includes: 13 C NMR(101MHz，DMSO-d6)：δ143.44，143.40，141.37，140.87，137.24，134.16，131.86，131.18，128.49，127.51，127.29，127.15，126.53，72.76，70.22，70.20，69.92，67.46，64.31，63.04，60.64，50.29。
FIG. 3 shows the result of high resolution mass spectrometry detection of TPE-Q, which is calculated as [ M (C) 60 H 76 I 2 N 2 O 8 )]1206.3691, test result is [ M-2I] 2+ 476.2799, the theoretical value of TPE-Q is met.
Example 2
TPE-Q in vitro Abeta 1-42 The protein detection is carried out according to the following steps:
(1) in EP0 tube, A beta is added 1-42 A lyophilized powder of the aggregate (1mg) and precooled hexafluoroisopropanol (HPIP, 221.4 μ L) were sealed with a sealing film. The mixture was mixed well and incubated at room temperature for 1 h. The sealing film was removed and left overnight at room temperature. If the solution has not completely evaporated, it is blown dry with nitrogen and dried in a vacuum oven. Drying the obtained Abeta in vacuum 1-42 The membrane was dissolved in DMSO (44.3. mu.L) to give a concentrated 5mM Abeta 1-42 Stock solutions, stored at-20 ℃.
In EP tube 1, the above-mentioned Abeta was added 1-42 Stock solution (12 μ L), pre-cooled PBS (pH 7.4, c 10mM) (588 μ L) was pipetted well to allow Α β to reach equilibrium 1-42 The final concentration of aggregates was 100 mM.
In another EP tube 2, A.beta.was also treated with pre-cooled PBS 1-42 The stock solution was diluted to a final concentration of 100mM, and TPE-Q was added to bring the final concentration of probe in the solution to 10 mM. And marking.
At room temperature, EP tube 1A beta 1-42 And EP tube 2 with addition of TPE-Q Abeta 1-42 And Thioflavin T were incubated for 2, 26, and 50 hours, respectively, and A.beta.was probed by transmission electron microscopy 1-42 Whether the aggregate binds to TPE-Q versus Abeta 1-42 Self-aggregation tendency has an influence. As shown in FIGS. 4(a), (b) and (c), A.beta.without TPE-Q 1-42 The granular morphology is shown in the culture time of 26 h. A beta could be clearly observed until the incubation time reached 50h 1-42 And (3) agglomeration. And the A beta of the TPE-Q fluorescent probe is added 1-42 The morphologies (FIG. 4(d-f)) and those without the addition of the TPE-Q fluorescent probe (FIGS. 4-4(a-c)) did not change significantly, so it can be concluded that the addition of the TPE-Q fluorescent molecule did not significantly alter A.beta. 1-42 The assembly behavior of (1).
(2)Aβ 1-42 Stock solutions were diluted with PBS buffer (pH 7.4, c 10mM) to different gradient concentrations (0-30M). Adding TPE-Q to the Abeta 1-42 The final concentration of the probe in PBS buffer was 10M, the TPE-Q fluorescence emission spectra were measured under 350nm excitation after incubation at room temperature for 30 minutes.
In this example, to test the viscosity fluorescence sensing capability of TPE-Q, TPE-Q (final concentration of 1. mu.M) and A.beta.were measured at 37 deg.C 1-42 (final concentration 3 μ M) was incubated in PBS (pH 7.4) for 48 h. Bovine Serum Albumin (BSA) was used as a control. As shown in FIG. 5(a), TPE-Q (final concentration of 1. mu.M) showed strong fluorescence emission in PBS, which is presumed to be the result of aggregation-induced emission [139] . TPE-Q fluorescence emission intensity was increased by a factor of about 1.35 in the presence of 3. mu.M BSA compared to 1. mu.M PE-Q in PBS. Abeta compared to 1. mu.M TPE-Q in PBS 1-42 In the presence, the fluorescence emission intensity of TPE-Q was enhanced by 3.59 times. Therefore, it can be inferred that A.beta. 1-42 It binds to TPE-Q more readily than BSA.
As shown in FIG. 5(b), A.beta.was increased 1-42 The concentration (eventually increased to 30. mu.M) resulted in TPE-Q (eventuallyConcentration of 10. mu.M), the fluorescence emission intensity increased gradually, with an increase of about 21.60-fold. Maximum fluorescence emission intensity of TPE-Q (488 nm) and Abeta 1-42 Has a good linear relationship (R) 2 0.9948) as shown in fig. 5 (c). This indicates that TPE-Q can be regarded as A beta 1-42 The fluorescent probe of (1).
The amyloid protein can assemble into particle or fiber morphology according to the incubation time. The fluorescence emission intensity of TPE-Q at different incubation times was therefore next examined. As shown in FIG. 5(d), the fluorescence emission intensity of TPE-Q increased (to 90h) with the incubation time. However, a maximum fluorescence enhancement of only 1.26 fold (90h vs.2h) is not sufficient to distinguish A β 1-42 In the assembled form.
Example 3
TPE-Q at Abeta 1-42 The fluorescence imaging test in the nematode body with high protein expression is carried out according to the following steps:
(1) agar powder (500mg) and ultrapure water (25mL) were heated to be completely dissolved using a microwave oven, 200. mu.L of agar solution was immediately taken out with a pipette and dropped into the center of the slide, and then another slide was lightly covered with a glass plate to spread the agar evenly and flatly without any bubble generation, and naturally cooled to solidify. Two agar sheets were prepared in the same manner.
(2) The contemporary nematodes suffering from Alzheimer's disease (in this example, the nematodes suffering from Alzheimer's disease, AD, C.elegans, are incubated for 30min in TPE-Q (5. mu.M) in M9. Levoimidazole solution (5. mu.L) was added to each to anaesthetize the nematodes, and after 3min, no response was observed under the microscope to the nematodes, which were considered to be completely anaesthetized.
(3) One of the slides was gently pushed away so that the agar sheet was only on top of one of the slides, taking care to avoid cracking of the agar sheet. The slide glass is placed upside down on a table, a small amount of the M9 solution containing TPE-Q is dripped, and the slide glass is covered and fixed. In the same manner, M9 solution containing Th T was added dropwise. The fluorescence photographs were taken by inverting (405nm excitation TPE-Q) in a two-photon fluorescence microscope, respectively, as shown in FIG. 5.
FIG. 6 shows ThFluorescence images of AD C.elegans after incubation of T and TPE-Q for 30min, and in order to test the practical application of TPE-Q probes in vivo assay, the fluorescence test was performed with C.elegans (CL4176) as a living body, which is a symptom of Alzheimer's disease, in FIG. 5. In imaging experiments, commercial plaque-specific fluorescent markers (Th T) were selected as controls. As shown in FIG. 6(a), fluorescent spots of Th T were observed in the nematodes after the nematodes were cultured at a concentration of 5. mu.M for 30 min. Likewise, fluorescence of TPE-Q was also observed in Alzheimer's disease nematodes under the same experimental conditions as used for Th T (FIG. 6 b). Both the pooled and BF plots showed good overlap of the two probes (fig. 6c and d). The pearson correlation coefficient was calculated to be 0.926. TPE-Q has good fluorescence overlap in the AD nematode model, which indicates that TPE-Q can also be used as a very potential in vivo Abeta 1-42 A specific imaging agent.

Claims (7)

1. An aggregation-induced emission fluorescent probe based on TPE, which is characterized in that the structural formula of the probe is an isomer TPE-Q of formula (1):
Figure DEST_PATH_IMAGE001
TPE-Q
formula (1)
Wherein the chemical name of the compound corresponding to formula (1): (E/Z) -N, N ' - ((((1, 2-diphenylethylene-1, 2-dimethyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (2- (2- (2- (2-hydroxyethoxy) ethoxy) -N, N-dimethylethoxy) 1-amino), abbreviated TPE-Q.
2. The TPE-based fluorescence probe with aggregation-induced emission (MEL) as claimed in claim 1, wherein the TPE-Q of formula (1) is prepared by the following steps:
1) heating and refluxing 4-bromo-benzophenone in anhydrous THF for reaction, wherein the reaction is Mcmmurry reaction, and the reaction formula is as follows:
Figure 349804DEST_PATH_IMAGE002
then separating and purifying to obtain 1, 2-di (4-bromophenyl) -1, 2-diphenylethylene;
2) heating 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene and 4-formylphenyl boric acid in N, N-dimethylacetamide under the condition of reflux reaction, said reaction is Suzuki-Miyaura reaction, and its reaction formula is as follows:
Figure DEST_PATH_IMAGE003
then 4',4' ' ' - (1, 2-diphenylethylene-1, 2-diyl) di ([1,1' -biphenyl ] -4-formaldehyde) is obtained by separation and purification;
3) heating and refluxing 4',4' ' ' - (1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4-formaldehyde) and 1-amino-3, 6, 9-trioxa-11-undecanol in methanol for 12-16 times, wherein the reaction is a process for eliminating aldehyde-based oxygen by nucleophilic substitution, and then adding sodium borohydride to reduce imine into secondary ammonium, wherein the reaction formula is as follows:
Figure 391578DEST_PATH_IMAGE004
finally recrystallizing to obtain 2,2' - (((((((1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azepindiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol);
4) reacting 2,2' - ((((((1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azepindiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) with methyl iodide for 2 to 3 hours, which is an amination reaction in a nucleophilic substitution reaction, according to the following reaction formula:
Figure DEST_PATH_IMAGE005
and then separating and purifying to obtain the TPE-Q.
3. The TPE-based fluorescence probe capable of inducing aggregation and luminescence according to claim 2, wherein in the step 1), the 4-bromobenzophenone is dissolved in anhydrous THF according to a feeding ratio of 0.8mol/L for reaction, zinc powder and titanium tetrachloride are used as catalysts, the reaction temperature is THF reflux temperature, the reaction time is 20-25 hours, and after the reaction, separation and purification are performed, and the steps are as follows: k 2 CO 3 The solution was quenched, washed with dichloromethane, dried over anhydrous magnesium sulfate, filtered, and column chromatographed [ silica gel; petroleum ether/ethyl acetate; 18: 1(v/v)]Separating to obtain a purified product.
4. The TPE-based fluorescence probe for aggregation-induced Emission (ELL) according to claim 2, wherein in step 2), 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene: adding 4-formylphenylboronic acid into N, N-dimethylacetamide according to the molar ratio of 1:3 to react at 80 ℃ for 10 hours; separating and purifying the product after reaction, and sequentially comprising the following steps: extraction with dichloromethane, drying over anhydrous magnesium sulfate, filtration, column chromatography [200-300 mesh silica gel: dichloromethane as eluent ] to afford the purified product.
5. The TPE-based fluorescence probe of claim 2, wherein in step 3), the 4',4' ' ' - (1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4-carbaldehyde) 1-amino-3, 6, 9-trioxa-11-undecanol molar ratio is 1: 2.4; the reaction solvent is methanol; the reaction temperature is the reflux temperature of the methanol; after the reaction time is 6 hours, cooling the mixture to room temperature, adding sodium borohydride into the reaction solution while stirring within half an hour, reducing the imine into secondary ammonium, and reacting for 5 hours at room temperature; then carrying out separation and purification, comprising the following steps: the purified product is obtained by recrystallization from dichloromethane/deionized water.
6. The TPE-based aggregation-induced emission fluorescent probe according to claim 2, wherein in step 4), the solvent is methanol (8ml), and the catalyst is potassium carbonate, 2,2' - (((((((1, 2-diphenylethylene-1, 2-diyl) bis ([1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azepindiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol): the molar ratio of methyl iodide is 1:12, the reaction is carried out for 10-14h at room temperature, and the solvent in the reaction solution is dried after the reaction is finished; then separating and purifying the reactant, and sequentially carrying out the following steps: removing impurities from petroleum ether, and carrying out pure crystallization on methanol/dichloromethane to obtain a purified product.
7. Use of the TPE-based fluorescence probe for aggregation-induced emission of light according to any one of claims 1-6, wherein the TPE-Q fluorescence probe is used for detecting A β related to Alzheimer's disease 1-42 In an aggregate.
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