CN114907222B - Aggregation-induced emission fluorescent probe based on TPE and application thereof - Google Patents

Aggregation-induced emission fluorescent probe based on TPE and application thereof Download PDF

Info

Publication number
CN114907222B
CN114907222B CN202210723297.9A CN202210723297A CN114907222B CN 114907222 B CN114907222 B CN 114907222B CN 202210723297 A CN202210723297 A CN 202210723297A CN 114907222 B CN114907222 B CN 114907222B
Authority
CN
China
Prior art keywords
bis
reaction
tpe
diyl
diphenylethylene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210723297.9A
Other languages
Chinese (zh)
Other versions
CN114907222A (en
Inventor
崇辉
王娜
房司雨
王天奕
王赪胤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yangzhou University
Original Assignee
Yangzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yangzhou University filed Critical Yangzhou University
Priority to CN202210723297.9A priority Critical patent/CN114907222B/en
Publication of CN114907222A publication Critical patent/CN114907222A/en
Application granted granted Critical
Publication of CN114907222B publication Critical patent/CN114907222B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1007—Non-condensed systems

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Optics & Photonics (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Materials Engineering (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention relates to a fluorescent probe based on aggregation-induced emission of TPE and application thereof in the technical field of fluorescence spectrum analysis materials, which are used for Abeta related to Alzheimer's disease 1‑42 Detection and analysis of amyloid, fluorescent probes of the present invention, chemical name of the compound: (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. The synthetic route is relatively simple and convenient, the product has good stability and no specific recognition group. Aβ for Alzheimer's disease 1‑42 Detection of aggregates can achieve the same imaging effect as commercial specific fluorescent markers (Th T).

Description

Aggregation-induced emission fluorescent probe based on TPE and application thereof
Technical Field
The invention relates to the technical field of fluorescence spectrum analysis materials, in particular to a fluorescence probe based on aggregation-induced emission of TPE (thermoplastic elastomer), which is used for Abeta related to Alzheimer's disease 1-42 Detection and analysis of amyloid.
Background
Fluorescence is a luminescence phenomenon, when a chemical substance is irradiated by incident light with a certain wavelength, the molecule absorbs external energy and is converted into an excited state, the excited state is converted into a ground state in a short time, and fluorescence is emitted in the conversion process. Based on fluorescence mechanisms, 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. The method has become an efficient and convenient real-time monitoring method for environmental monitoring, biological analysis and clinical diagnosis.
The development of fluorescence techniques depends on the design of the fluorescent probes. Molecular imaging refers to the implantation of non-invasive fluorescent probes into cells or organs to visualize biological targets that are inconvenient to directly observe in order to assess the physiological processes of the living system. Thus, techniques of fluorescent probe molecules in combination with biological detection are useful for achieving quantitative and positional analysis of biological macromolecules or specific organelles within an organism. Fluorophores have a high degree of structural diversity through chemical modification, which facilitates functional design and property modulation of the probe. Compared with fluorescent inorganic nano-materials, 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 activity, but also opens the possibility of achieving therapeutic functions with light. Fluorescence techniques have the advanced advantage of being the best candidate for microbial detection, such as simple operation, real-time response, in situ sensitivity and specificity.
Alzheimer's Disease (AD) is characterized by language disorders, cognitive decline, disorientation, irreversible memory loss, which presents serious health risks to elderly people. Early diagnosis and intervention of AD is of great importance in slowing the progression of the disease. Abeta accumulated in cerebral spinal mass 1-42 Amyloid (aβ) plaques are considered as important biomarkers for the early diagnosis of Alzheimer's Disease (AD). Thus, highly specific, noninvasive and rapid determination of aβ in the brain 1-42 Amyloid aggregates may facilitate clinically effective diagnosis of AD and may be used to assess potential anti-AD effects of candidate drugs. By using a small molecular probe for fluorescence imaging, abeta can be easily detected in real time 1-42 Amyloid protein. Currently used for detecting Abeta 1-42 Most of the amyloid probes contain specific recognition groups and the synthesis is relatively complex. The TPE-Q fluorescent probe synthesized by the invention has simple synthesis route and smaller cytotoxicity.
Disclosure of Invention
Aiming at the defects in the synthesis and use of the AD diagnosis early detection probe material in the prior art, the invention provides a fluorescent probe which has simple synthesis route and less cytotoxicity and is based on aggregation-induced emission of TPE.
The invention aims at realizing the following, namely a fluorescent probe based on aggregation-induced emission of TPE, which is characterized in that the structural formula of the fluorescent probe is an isomer TPE-Q of 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.
Further, TPE-Q in the above formula (1) is prepared as follows:
1) The 4-bromo-benzophenone was heated to reflux in anhydrous THF, which was Mcmurry, according to the following formula:
then separating and purifying to obtain 1, 2-di (4-bromophenyl) -1, 2-diphenylethylene;
2) 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene and 4-formylphenylboronic acid were heated to reflux in N, N-dimethylacetamide in the following reaction scheme:
then separating and purifying to obtain 4',4' - (1, 2-diphenylethylene-1, 2-diyl) di ([ 1,1' -biphenyl ] -4-formaldehyde);
3) 4',4' - (1, 2-diphenylethylene-1, 2-diyl) di ([ 1,1' -biphenyl ] -4-formaldehyde) and 1-amino-3, 6, 9-trioxa-11-undecanol are heated and refluxed in methanol for 12-16, the reaction is a nucleophilic substitution process for eliminating aldehyde oxygen, and sodium borohydride is added to reduce imine into secondary ammonium, and the reaction formula is as follows:
finally, the mixture is recrystallized to obtain 2,2' - (((((1, 2-diphenylethylene-1, 2-diyl)) bis ([ 1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azadiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol);
4) 2,2' - (((((1, 2-diphenylethylene-1, 2-diyl) bis ([ 1,1' -biphenyl ] -4', 4-diyl)) bis (azadiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) and methyl iodide) were reacted for 2 to 3 hours, which reaction was an amination reaction in nucleophilic substitution reaction, the reaction formula being as follows:
and separating and purifying to obtain TPE-Q.
Further, in the step 1), the 4-bromobenzophenone is dissolved in anhydrous THF according to the feeding proportion of 0.8mol/L for reaction, zinc powder and titanium tetrachloride are used as catalysts, the reaction temperature is the THF reflux temperature, the reaction time is 20-25 hours, and after the reaction, separation and purification are carried out, wherein the steps are as follows in sequence: k (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), 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene was reacted with an aqueous solution of sodium fluoride and potassium carbonate as a catalyst: 4-formylphenylboric acid (4.29 mmol) is added into N, N-dimethylacetamide as a solvent according to the mol ratio of 1:3 for reaction at 80 ℃ for 10 hours; the product after the reaction is separated and purified, and the steps are as follows: dichloromethane extraction, anhydrous magnesium sulfate drying, filtration, column chromatography [200-300 mesh silica gel: dichloromethane as eluent ] to obtain a 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 methanol; after the reaction time is 6 hours, the mixture is cooled to room temperature, sodium borohydride is added to the reaction solution in a half hour with stirring, imine is reduced to secondary ammonium, and the reaction is carried out for 5 hours at room temperature; then separating and purifying, wherein the steps are as follows: recrystallizing the dichloromethane/deionized water to obtain a purified product.
Still further, in step 4), solvent is methanol, and the catalyst is potassium carbonate, 2' - ((((1, 2-diphenylethylene-1, 2-diyl) bis ([ 1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azadiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) (0.111 mmol): methyl iodide (1.34 mmol) with a molar ratio of 1:12 is reacted at room temperature for 10-14h, and the solvent in the reaction solution is dried after the reaction is finished; the reaction was then purified by the steps of: removing impurities from petroleum ether, and crystallizing methanol/dichloromethane to obtain a purified product.
The aggregation-induced emission fluorescent probe based on TPE has the following advantages: (1) Compared with the synthesis method in the prior art, the method has the advantages of simple operation, simple separation method, easily obtained product and considerable yield;
(2) The method accords with the atomic economy and green chemistry concept, the TPE has simple synthesis, good stability and low raw material price, no toxic or harmful substances are generated in the synthesis process, and the synthesized product can be proved to be nontoxic and harmless to cells in cytotoxicity experiments and can be used as a new detection of Abeta 1-42 An analytical probe for protein aggregates;
(3) The molecule has no specific recognition group 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 the aggregate. The specific detection mechanism is as follows: in the probe structure, the tetraphenyl ethylene unit is a molecular rotor, whereinThe introduction of an 'alkoxy chain' and a quaternary ammonium salt group play a role in increasing the water solubility of the probe, and the introduction of a benzene ring enables the excitation wavelength and the emission wavelength of molecules to be red shifted. At A beta 1-42 In an amyloid aggregate system, the energy of the excited state of the probe is mainly lost in a radiation form due to the limited rotation of a molecular rotor, and the probe emits stronger fluorescence. The TPE-Q fluorescent probe provided by the invention proves that the probe can be used for detecting Abeta through in vitro experiments 1-42 Protein aggregates. In addition, imaging experiments were performed by comparison with commercial plaque-specific fluorescent markers Th t. The fluorescence of the two probes in the nematode with Alzheimer's disease was found to overlap well. Next, the side view of the probe by molecular docking demonstrates that the probe can bind to Abeta 1-42 Aggregate binding.
Drawings
FIG. 1 is a schematic diagram of a probe TPE-Q 1 H NMR spectrum.
FIG. 2 is a schematic diagram of a probe TPE-Q 13 C NMR spectrum.
FIG. 3 is a high resolution mass spectrum of probe TPE-Q.
FIG. 4 shows the various incubation times Abeta 1-42 Morphology change of aggregates
FIG. 5 shows bovine serum albumin and Abeta after TPE-Q addition 1-42 Is a fluorescent spectrum of (3).
FIG. 6 is a fluorescence imaging 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 examples and drawings, but the present invention is not limited to the examples.
Example 1
The synthesis of the fluorescent probe is carried out according to the following steps:
(1) Zinc powder (2.50 g,37.90 mmol) was added to a 120mL pressure-resistant bottle, nitrogen was introduced to vent the oxygen from the bottle, 25mL anhydrous THF was further added, the temperature was cooled to 0℃and then 2.2mL titanium tetrachloride TiCl was slowly added dropwise to the ice salt bath via a constant pressure dropping funnel 4 (2.20 mL,20.00 mmol) and pyridine (0.10 mL,1.20 mmol). After refluxing the mixture at 75℃for 2h, 25mL of 4-bromobenzophenone (5.00 g,19.20 mmol) in tetrahydrogen was addedFuran solution, the reaction mixture was refluxed for a further 20h at 75 ℃. After the reaction is finished, cooling the reaction mixture to room temperature; pouring into 40mL of 10% aqueous potassium carbonate solution, vigorously stirring for 5min, then filtering to obtain a white solid, immersing the obtained white solid into excessive dichloromethane for washing, and removing insoluble solid by filtration. Anhydrous Mg for filtrate 2 SO 4 Drying, filtration, 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 pale yellow product 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene (yield 87.2%). The reaction formula is as follows:
(2) K is carried out under nitrogen atmosphere 2 CO 3 (829.26 mg,6.00 mmol) and NaF (251.9 mg,6.00 mmol) were dissolved in 3mL of deionized water, added to a 45mL dry pressure-resistant bottle, and then N, N-dimethylacetamide (15 mL), 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene (700 mg,1.43 mmol), 4-formylphenylboronic acid (643.50 mg,4.29 mmol), pd (PPh) were added sequentially 3 ) 4 (173.4 mg,0.15 mmol). The mixture was poured into twice the water, extracted 3 times with dichloromethane, the organic layers combined and dried over anhydrous magnesium sulfate. The crude product was further purified by column chromatography (200-300 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:
(3) 4',4' - (1, 2-diphenylethylene-1, 2-diyl) bis ([ 1,1' -biphenyl ] -4-carbaldehyde) (100 mg,0.185 mmol), 1-amino-3, 6, 9-trioxa-11-undecanol (85.8 mg,0.444 mmol), methanol (9.25 mL) 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 continued to cool to 0 ℃. Sodium borohydride (63 mg,1.665 mmol) was added in portions over 10min to the stirring solution in the ice bath and stirred at room temperature for 5h. The mixture was filtered, methanol was removed by cooling the rotary evaporator, the solid object was dissolved in dichloromethane (600 μl) and recrystallized from distilled water, and the solid powdered product 2,2' - (((((1, 2-diphenylethylene-1, 2-diyl)) bis ([ 1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azadiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) was collected by suction filtration. The reaction formula is as follows:
(4) 2,2' - ((((1, 2-diphenylethylene-1, 2-diyl) bis ([ 1,1' -biphenyl ] -4', 4-diyl)) bis (azadiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) (100 mg,0.111 mmol), methyl iodide (83 μl,1.34 mmol), potassium carbonate (61.27 mg,0.444 mmol), and methanol (3 mL) were sequentially added to a 15mL pressure-resistant bottle, and reacted at room temperature for 10 hours. The reaction was dried by spinning a large amount of solvent, and the residue of compound c was washed with petroleum ether. This was dissolved with a small amount of methanol and then recrystallized from methylene chloride to give the product TPE-Q as a yellow powder (yield 78.2%). The reaction formula is as follows:
the nuclear magnetic resonance hydrogen spectrum of TPE-Q is shown in FIG. 1, wherein the characteristic peak is delta 3.02ppm, corresponding to 4 methyl groups in the quaternary ammonium, and the peak area is 16.
The nuclear magnetic resonance carbon spectrum of TPE-Q is shown in FIG. 2, specifically: 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 results of high-resolution mass spectrometry detection of TPE-QCalculated as [ M (C) 60 H 76 I 2 N 2 O 8 )]= 1206.3691, test result is [ M-2I] 2+ = 476.2799, which corresponds to the theoretical value of TPE-Q.
Example 2
TPE-Q Abeta in vitro 1-42 The protein detection is carried out according to the following steps:
(1) In EP0 tube, Aβ was added 1-42 Lyophilized powder of aggregates (1 mg) and pre-chilled hexafluoroisopropanol (HPIP, 221.4 μl) were sealed with a sealing film. The mixture was thoroughly mixed and incubated at room temperature for 1h. The sealing film was removed and left at room temperature overnight. If the solution is left overnight, the solvent is dried with nitrogen without complete evaporation, and then dried in a vacuum oven. Aβ obtained after vacuum drying 1-42 Dissolving the membrane in DMSO (44.3. Mu.L) to obtain concentrated 5mM Abeta 1-42 Stock solutions were stored at-20 ℃.
In EP tube 1, the above Abeta is added 1-42 Stock solution (12 μl), pre-chilled PBS (ph=7.4, c=10 mM) (588 μl) was blown down and mixed well so that aβ 1-42 The final concentration of aggregates was 100mM.
In another EP tube 2, abeta is likewise treated with precooled PBS 1-42 The stock solution was diluted to a final concentration of 100mM, while TPE-Q was added so that the final concentration of probe in the solution was 10mM. Marking is carried out.
EP tube 1A beta at room temperature 1-42 And Abeta with TPE-Q added in EP tube 2 1-42 Aβ was investigated by transmission electron microscopy by incubating with thioflavin T for 2, 26 and 50 hours, respectively 1-42 Whether or not the binding of the aggregate to TPE-Q is to Aβ 1-42 The tendency to aggregate itself has an impact. As shown in FIGS. 4 (a), (b) and (c), abeta of TPE-Q was not added 1-42 The pellet form was observed during the culture time of 26 hours. Aβ was observed significantly until the incubation time reached 50h 1-42 Is not limited, and is not limited. While Aβ with TPE-Q fluorescent probe added 1-42 The morphology of (FIG. 4 (d-f)) and (FIGS. 4-4 (a-c)) without TPE-Q fluorescent probe did not change significantly, so it can be concluded that the addition of TPE-Q fluorescent molecules did not significantly alter Aβ 1-42 Is a group of (2)And (5) assembling.
(2)Aβ 1-42 Stock solutions were diluted with PBS buffer (ph=7.4, c=10 mM) to different gradient concentrations (0-30M). Adding TPE-Q to the above Aβ 1-42 The final probe concentration was 10M in PBS buffer, incubated at room temperature for 30 minutes, and the fluorescence emission spectra of TPE-Q were measured at 350nm excitation.
In this example, to test the viscosity fluorescence sensing ability of TPE-Q, TPE-Q (final concentration of 1. Mu.M) and Abeta were measured at 37 ℃ 1-42 (final concentration 3 μm) was incubated in PBS (ph=7.4) for 48h. 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 was presumed to be a result of aggregation-induced emission [139] . The fluorescence emission intensity of TPE-Q was enhanced by about 1.35-fold in the presence of 3. Mu.M BSA compared to 1. Mu.M MTPE-Q in PBS. Aβ compared to 1 μM TPE-Q in PBS 1-42 In the presence of TPE-Q, the fluorescence emission intensity was enhanced by 3.59 times. Thus, Aβ can be inferred 1-42 Is more easily bound to TPE-Q than BSA.
As shown in FIG. 5 (b), Aβ is increased 1-42 The concentration (eventually increased to 30. Mu.M) resulted in a gradual increase in the fluorescence emission intensity of TPE-Q (final concentration of 10. Mu.M), which was about 21.60-fold. Maximum fluorescence emission intensity (about 488 nm) of TPE-Q versus Abeta 1-42 Is of good linear relation (R 2 =0.9948), as shown in fig. 5 (c). This suggests that TPE-Q may act as Aβ 1-42 Is provided.
As the amyloid proteins can assemble into a particle or fiber form depending on the incubation time. The fluorescence emission intensity of TPE-Q at different incubation times was thus subsequently examined. As shown in FIG. 5 (d), the fluorescence emission intensity of TPE-Q increased (up to 90 h) with the increase of incubation time. However, a maximum fluorescence enhancement of only 1.26 times (90 h vs.2 h) is insufficient to distinguish aβ 1-42 Is a combination of the above.
Example 3
TPE-Q at Abeta 1-42 The fluorescence imaging test of the nematode body with high protein expression is carried out according to the following steps:
(1) Agar powder (500 mg) and ultrapure water (25 mL) were heated to complete dissolution using a microwave oven, 200. Mu.L of the agar solution was immediately taken out with a pipette and dropped into the center of the slide, and then the other slide was gently covered over it to spread the agar uniformly and smoothly without any bubbles, and naturally cooled and solidified. Two agar plates were prepared in the same manner.
(2) The nematodes with Alzheimer's disease after the synchronization (in this example, the AD caenorhabditis elegans with Alzheimer's disease) were incubated for 30min in a solution of TPE-Q (5. Mu.M) in M9, respectively. The nematodes were anesthetized with levamisole solution (5 μl) each, and after 3min, the touch line was observed under a microscope without reaction, and the anesthesia was considered complete.
(3) One of the slides was gently pushed away so that the agarose sheet was only over one of the slides, taking care to avoid cracking of the agarose sheet. The solution was inverted on a table, and a trace of the above-described M9 solution containing TPE-Q was added dropwise, and another slide was covered to fix it. M9 solution containing Th T was added dropwise in the same manner. The two-photon fluorescence microscope was inverted (405 nm excited TPE-Q) and a fluorescence photograph was taken as shown in FIG. 5.
FIG. 6 is a fluorescence image of the AD caenorhabditis elegans after 30min incubation with Th T and TPE-Q, and in FIG. 5 the fluorescence test was performed on caenorhabditis elegans (CL 4176) showing symptoms of Alzheimer's disease as a living body in order to test the practical use of TPE-Q probes in the detection of living bodies. In imaging experiments, commercial plaque-specific fluorescent markers (Th T) were selected as controls. As shown in FIG. 6 (a), after the nematode was cultured at a concentration of 5. Mu.M for 30min, fluorescent spots of Th T were observed in the nematode. Likewise, fluorescence of TPE-Q was also observed in Alzheimer's disease nematodes under the same experimental conditions as Th T (FIG. 6 b). Both the merged and BF panels show that both probes show good overlap (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 suggests that TPE-Q can also act as a very potential in vivo Abeta 1-42 Specific imaging agents.

Claims (4)

1. A fluorescent probe based on aggregation-induced emission of TPE, which is characterized by the structural formula of TPE-Q of formula (1):
；
(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 method for preparing a fluorescent probe based on aggregation-induced emission of TPE according to claim 1, wherein TPE-Q of formula (1) is prepared as follows:
1) The 4-bromo-benzophenone was heated to reflux in anhydrous THF, which was Mcmurry, according to the following formula:
；
then separating and purifying to obtain 1, 2-di (4-bromophenyl) -1, 2-diphenylethylene;
2) Heating and refluxing 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene and 4-formylphenylboric acid in N, N-dimethylacetamide, wherein the reaction is Suzuki-Miyaura reaction, and 1, 2-bis (4-bromophenyl) -1, 2-diphenylethylene is prepared by using aqueous solution of sodium fluoride and potassium carbonate as a catalyst: adding 4-formylphenyl boric acid into N, N-dimethylacetamide solvent according to the mol ratio of 1:3 for reaction at 80 ℃ for 10 hours; the product after the reaction is separated and purified, and the steps are as follows: extracting with dichloromethane, drying with anhydrous magnesium sulfate, filtering, and purifying by column chromatography to obtain purified product 4',4' ' ' - (1, 2-diphenylethylene-1, 2-diyl) di ([ 1,1' -biphenyl ] -4-formaldehyde), wherein the column chromatography is as follows: 200-300 mesh silica gel and methylene dichloride are used as eluent;
3) The molar ratio of 4',4' ' ' - (1, 2-diphenylethylene-1, 2-diyl) bis ([ 1,1' -biphenyl ] -4-carbaldehyde) to 1-amino-3, 6, 9-trioxa-11-undecanol is 1:2.4; the reaction solvent is methanol; the reaction temperature is the reflux temperature of methanol; after the reaction time of 6 hours, the mixture was cooled to room temperature, sodium borohydride was added to the reaction solution in portions with stirring in half an hour, and the imine was reduced to secondary amine, and reacted at room temperature for another 5 hours; then separating and purifying, wherein the steps are as follows: recrystallisation of dichloromethane/deionized water gives a purified product of the formula:
；
finally, the mixture is recrystallized to obtain 2,2' - (((((1, 2-diphenylethylene-1, 2-diyl)) bis ([ 1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azadiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol);
4) 2,2' - (((((1, 2-diphenylethylene-1, 2-diyl) bis ([ 1,1' -biphenyl ] -4', 4-diyl)) bis (azadiyl)) bis (ethane-2, 1-diyl)) bis (oxy)) bis (ethane-1-ol) and methyl iodide) were reacted for 2 to 3 hours, which reaction was an amination reaction in nucleophilic substitution reaction, the reaction formula being as follows:
；
and separating and purifying to obtain TPE-Q.
3. The method for preparing a fluorescent probe according to claim 2, wherein in the step 1), 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 carried out, and the steps are as follows: k (K) 2 CO 3 Quenching the solution, washing with dichloromethane, drying with anhydrous magnesium sulfate, filtering, and separating with column chromatography to obtain purified product, wherein the column chromatography is silica gel, and the eluent is petroleum etherEthyl acetate in a volume ratio of 18:1.
4. the method of preparing a fluorescent probe according to claim 2, wherein in step 4), a solvent is methanol, and a catalyst is potassium carbonate, 2' - (((((1, 2-diphenylethylene-1, 2-diyl)) bis ([ 1,1' -biphenyl ] -4', 4-diyl)) bis (methylene)) bis (azadiyl)) 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 at room temperature for 10-14 and h, and the solvent in the reaction liquid is dried after the reaction is finished; the reaction was then purified by the steps of: removing impurities from petroleum ether, and recrystallizing methanol/dichloromethane to obtain a purified product.
CN202210723297.9A 2022-06-23 2022-06-23 Aggregation-induced emission fluorescent probe based on TPE and application thereof Active CN114907222B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210723297.9A CN114907222B (en) 2022-06-23 2022-06-23 Aggregation-induced emission fluorescent probe based on TPE and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210723297.9A CN114907222B (en) 2022-06-23 2022-06-23 Aggregation-induced emission fluorescent probe based on TPE and application thereof

Publications (2)

Publication Number Publication Date
CN114907222A CN114907222A (en) 2022-08-16
CN114907222B true CN114907222B (en) 2024-03-26

Family

ID=82772306

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210723297.9A Active CN114907222B (en) 2022-06-23 2022-06-23 Aggregation-induced emission fluorescent probe based on TPE and application thereof

Country Status (1)

Country Link
CN (1) CN114907222B (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102279270A (en) * 2011-04-27 2011-12-14 商丘师范学院 Method for monitoring beta amyloid protein aggregation process by aggregation-induced emission
CN104974745A (en) * 2014-04-07 2015-10-14 香港科技大学深圳研究院 Amphiphilic illuminant with aggregation induced emission characteristics and applications thereof
CN110551196A (en) * 2019-08-01 2019-12-10 天津科技大学 Abeta 42 aggregation-induced emission fusion and construction and application thereof
CN112812045A (en) * 2019-11-15 2021-05-18 浙江大学 Onium salt organic catalyst and preparation method and application thereof
CN113788795A (en) * 2021-09-30 2021-12-14 广东省科学院生物与医学工程研究所 Water-soluble aggregation-induced emission nanoparticle and preparation method and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9279806B2 (en) * 2005-04-22 2016-03-08 The Hong Kong University Of Science And Technology Water-soluble AIE luminogens for monitoring and retardation of fibrillation of amyloid proteins

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102279270A (en) * 2011-04-27 2011-12-14 商丘师范学院 Method for monitoring beta amyloid protein aggregation process by aggregation-induced emission
CN104974745A (en) * 2014-04-07 2015-10-14 香港科技大学深圳研究院 Amphiphilic illuminant with aggregation induced emission characteristics and applications thereof
CN110551196A (en) * 2019-08-01 2019-12-10 天津科技大学 Abeta 42 aggregation-induced emission fusion and construction and application thereof
CN112812045A (en) * 2019-11-15 2021-05-18 浙江大学 Onium salt organic catalyst and preparation method and application thereof
CN113788795A (en) * 2021-09-30 2021-12-14 广东省科学院生物与医学工程研究所 Water-soluble aggregation-induced emission nanoparticle and preparation method and application thereof

Also Published As

Publication number Publication date
CN114907222A (en) 2022-08-16

Similar Documents

Publication Publication Date Title
US9714260B2 (en) Asymmetrical Si rhodamine and rhodol synthesis
CN107849441B (en) AIE luminophores for visualization and treatment of cancer
AT405461B (en) LUMINESCENT INDICATOR
CN108219780B (en) Near-infrared fluorescent probe and preparation method and application thereof
CN108191789B (en) Phenothiazine derivative, preparation method and application thereof
CN110373181B (en) Fluorescent probe for detecting fluorine ions
CN108059638A (en) Fluorescence probe of one kind mark amyloid and preparation method and application
CN114907222B (en) Aggregation-induced emission fluorescent probe based on TPE and application thereof
CN113651834A (en) Fluorescent probe based on dithienobenzene derivative and application of fluorescent probe in cell lipid drop imaging
KR101845973B1 (en) compound for detecting β-galactosidase using rearrangement of amin-thiol and Method for manufacturing thereof
KR101845926B1 (en) compound for detecting reactive oxygen species using rearrangement of amin-thiol and Method for manufacturing thereof
CN109796966B (en) Hypochlorous acid ratiometric fluorescent probe and application thereof
CN112812103A (en) Double-emission fluorescent probe for detecting beta-amyloid plaque and preparation method and application thereof
CN114773305B (en) Preparation method and application of 2-cycloarone pH fluorescence ratio probe
CN115160327B (en) Small-molecule fluorescent probe targeting mu opioid receptor and preparation and application thereof
CN115746036A (en) Fluorescent probe SN-BODIPY compound for targeted recognition of Abeta fibers and preparation method thereof
CN113651828B (en) Near-infrared fluorescent probe for detecting chromium ions and aluminum ions as well as preparation method and application of near-infrared fluorescent probe
CN114656447A (en) Near-infrared fluorescence and magnetic resonance Abeta dual-mode imaging probe based on high space-time resolution, and preparation method and application thereof
CN112552901B (en) Ratio type zinc ion fluorescent probe and preparation and application thereof
CN115403475A (en) Preparation method and application of diagnostic reagent for specifically detecting copper ion concentration
CN113666937A (en) Near-infrared fluorescent probe for detecting zinc ions and preparation method and application thereof
Guo et al. One-pot synthesis and applications of two asymmetrical benzoxanthene dyes
CN114230495B (en) Hydrogenated nitrosyl donor and preparation method thereof
CN109810538B (en) Mitochondrial positioning dye and preparation method and application thereof
CN112795377B (en) Preparation and application technical field of novel fluorescent probe with ICT-PET effect and capable of being used for formaldehyde detection

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant