CN117964606A - Photooxidation and imaging Abeta1-42Aggregate difunctional fluorescent probe and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a photooxidation and imaging Abeta 1‑42 aggregate difunctional fluorescent probe as well as a preparation method and application thereof. The structural formula of the fluorescent probe is shown as formula (I). The fluorescent probe synthesized by the invention has longer emission wavelength and larger Stokes displacement, can be specifically combined with Abeta 1‑42 aggregate, has obviously enhanced fluorescence after combination, and can be used for imaging amyloid plaques. Under the irradiation of white light, the fluorescent probe can generate singlet oxygen to inhibit Abeta 1‑42 aggregate. Meanwhile, the fluorescent probe BD-2-3 is also verified on the cell level, so that the uptake of microglia to the Abeta 1‑42 aggregate can be promoted, and the neurotoxicity caused by the Abeta 1‑42 aggregate can be reduced.Formula (I).
Description
Technical Field
The invention belongs to the field of specific therapeutic activity of compounds, and particularly relates to a photooxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe as well as a preparation method and application thereof.
Background
Alzheimer's Disease (AD) is an irreversible neurodegenerative disease, and is manifested clinically by hypomnesis, language disorder, progressive mental retardation, personality and behavioral modification. The main pathogenesis of AD is aβ amyloid deposition, abnormal phosphorylation of tau protein, mitochondrial hypothesis, inflammatory response, cholinergic deficiency, synaptic dysfunction, etc. Month 7 of 2023, FDA fully approved aβ antibodies lecanemab for marketing, lecanemab reduced amyloid markers in early AD patients in double blind phase III clinical trials. Aβ plaques are formed from the hydrolysis of Amyloid Precursor Protein (APP) by α, β and γ secretase enzymes. Specifically, a portion of APP is cleaved by the β -site APP lyase-1 (BACE-1), yielding a C-terminal fragment of 99 amino acids (C99). C99 is then cleaved by gamma secretase to form aβ 1-40 and aβ 42. The denaturation of Abeta amyloid becomes a main therapeutic target, and the acceleration of the clearance of Abeta aggregates and the inhibition of Abeta protein aggregation become main research directions.
Abnormal accumulation of pathological amyloid occurs in AD patients, while intermolecular hydrophobic interactions of polypeptide chains play a critical role in stabilizing β -sheet-rich aggregates. Thus, methods of altering the inherent hydrophilic-hydrophobic state are considered as a potential therapeutic strategy. In the case of photooxidized aβ aggregates, hydrophilic oxygen atoms can be covalently bound to aβ amyloid protein through oxidized amino acid residues, significantly reducing their aggregation propensity. In aβ amyloid fiber, residues such as tyrosine (Tyr), methionine (Met), and histidine (His) are susceptible to photooxidation and can be subsequently converted to imidazoline derivatives, levodopa, and the like, resulting in reduced hydrophobicity of the β chain. It is reported in the literature that the photooxidation of aβ aggregates promotes phagocytosis of aβ protein by microglia and reduces neurotoxicity caused by aβ aggregates.
The molecules of the photo-oxidized Abeta aggregates which are widely studied at present include small organic molecules (rose bengal, methylene blue, thioflavin-T, etc.), gold-based nanomaterials, carbon nanomaterials, metal complexes, self-assembled nanomaterials, etc. Benzothiadiazole (BTD) derivatives have recently been considered as one of the most important classes of fluorescent probes for use in fluorescence technology, for example in biological applications of fluorescent small molecule probes. However, most of these BTDs are used with organic cosolvents. Therefore, it is necessary to specifically modify the BTD structural framework to improve the light stability and water solubility.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provide a double-function fluorescent probe for photooxidation and imaging Abeta 1-42 aggregate.
The invention further aims at providing a preparation method of the photooxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe.
It is still another object of the present invention to provide the use of the photooxidation and imaging aβ 1-42 aggregate bifunctional fluorescent probe.
The aim of the invention is achieved by the following technical scheme:
a double-function fluorescent probe for photo-oxidation and imaging Abeta 1-42 aggregate has a structural formula shown in a formula (I):
Formula (I).
The preparation method of the photooxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe comprises the following steps:
(1) Preparation of intermediate 1
Methyl iodide and 4-methylpyridine are dissolved in an organic solvent and stirred at room temperature for reaction; after the reaction was completed, the reaction solution was cooled, then filtered, and the obtained solid was washed and dried to obtain intermediate 1 (pale yellow solid) having the structural formula shown in formula (II):
Formula (II);
(2) Preparation of intermediate 2
Under the inert gas atmosphere, 3, 6-dibromo-1, 2-phenylenediamine and acetone are dissolved in an organic solvent, and the reaction system is heated to 100 ℃ and stirred for reaction; cooling to room temperature after the reaction is finished, performing rotary evaporation under reduced pressure, and removing a reaction solvent to obtain a crude product;
under the protection of inert gas, adding active manganese dioxide and anhydrous Dichloromethane (DCM) into the obtained crude product, and stirring at room temperature for reaction; after the reaction is finished, filtering the reaction mixture to remove manganese dioxide, extracting the filtrate, further drying an organic phase, preparing sand, and purifying to obtain an intermediate 2 (BD-2, yellow solid); the structural formula is shown as formula (III):
formula (III);
(3) Preparation of intermediate 3
Under the inert gas atmosphere, the intermediate 2 (BD-2) and 4-formylphenyl pinacol borate are dissolved in toluene, an anaerobic potassium carbonate solution is added, and the mixture is heated to 80 ℃ under the catalysis of tetra (triphenylphosphine) palladium to carry out nucleophilic substitution reaction; after the reaction is finished, extracting the reaction liquid, drying an organic phase, performing reduced pressure rotary evaporation, preparing sand, and purifying to obtain an intermediate 3 (yellow solid), wherein the structural formula of the intermediate is shown as the formula (IV):
Formula (IV);
(4) Preparation of a double-functional fluorescent Probe (BD-2-3) for photo-Oxidation and imaging of Abeta 1-42 aggregate
Dissolving intermediate 3 (BD-2-CHO) and intermediate 1 in ethanol, and adding piperidine; heating the reaction system to 80 ℃ and stirring for reaction; and after the reaction is finished, filtering and cleaning the precipitated red solid by using absolute ethyl alcohol, dissolving the obtained solid by using methanol, filtering, collecting filtrate, and concentrating to obtain red solid (BD-2-3), namely the photo-oxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe, wherein the structural formula of the difunctional fluorescent probe is shown in a formula (I).
The organic solvent in step (1) is preferably ethanol.
The molar ratio of 4-methylpyridine to methyl iodide in the step (1) is 1: 1-1.1; preferably 1:1.1.
The amount of the organic solvent in the step (1) is calculated by 1mL of ethanol mixed with every millimole of 4-methylpyridine.
The stirring reaction time in the step (1) is 4 hours.
The organic solvent used for washing the solid in the step (1) is diethyl ether.
The inert gas in the step (2) is nitrogen.
The mixture ratio of the 3, 6-dibromo-1, 2-phenylenediamine and the acetone in the step (2) is that 3mL of acetone is added to every millimole of 3, 6-dibromo-1, 2-phenylenediamine.
The reaction time of heating and stirring at 100 ℃ in the step (2) is 36 hours.
The molar ratio of 3, 6-dibromo-1, 2-phenylenediamine to active manganese dioxide in the step (2) is 1:0.5 to 1.
The reaction time at room temperature was 12 hours as described in step (2).
The purification in the step (2) is purification by column chromatography; the column chromatography conditions are as follows: petroleum ether: the volume ratio of the ethyl acetate is 15:1; 200-300 meshes of silica gel.
The organic solvent of step (2) comprises toluene.
In the step (2), methylene dichloride and distilled water are added to extract the filtrate.
The purity of the active manganese dioxide in the step (2) is 85 percent.
The inert gas in the step (3) is argon.
The molar ratio of intermediate 2 (BD-2), 4-formylphenylboronic acid pinacol ester and tetrakis (triphenylphosphine) palladium in step (3) was 1: 2-4: 0.05.
The concentration of the potassium carbonate solution in the step (3) is 2mol/L, and the dosage of the potassium carbonate is 3-4 mL of the potassium carbonate solution per millimole of the intermediate 2.
The toluene amount described in step (3) was 7.5mL toluene per millimole of intermediate 2.
The heating reaction time in the step (3) is 12 hours.
The purification in the step (3) is column chromatography, and the column chromatography conditions are as follows: petroleum ether: the volume ratio of dichloromethane is 10:3; 200-300 meshes of silica gel.
The molar ratio of intermediate 3 to intermediate 1 in step (4) is 1:2.
The amount of piperidine used in step (4) was 450. Mu.L piperidine per millimole of intermediate 3 and 18mL ethanol per millimole of intermediate 3 (BD-2-CHO).
The stirring reaction time in the step (4) is 2 hours.
The photooxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe can be applied to specifically detecting Abeta 1-42 aggregate. The product comprises a fluorescent probe, a detection (diagnosis) reagent, a kit and the like.
The photooxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe can be applied to preparing products for inhibiting Abeta 1-42 aggregate. The product comprises a fluorescent probe, a detection (diagnosis) reagent, a kit and the like.
The photooxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe can be applied to the preparation of diagnostic products of Alzheimer's Disease (AD). The product comprises a fluorescent probe, a detection (diagnosis) reagent, a kit and the like. Compared with the prior art, the invention has the following advantages and effects:
(1) The invention provides a brand-new photo-oxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe, the synthesis steps of a photosensitizer are simple, the reaction conditions are not harsh, and the final product is easy to obtain. And the fluorescent probe has great application potential in the early diagnosis and treatment of Alzheimer's disease.
(2) The fluorescent probe can specifically detect the Abeta 1-42 aggregate and image the Abeta amyloid plaque.
(3) The fluorescent probe provided by the invention can generate singlet oxygen (1O2) under the excitation of white light, inhibit Abeta 1-42 aggregate, promote microglial cells to phagocytose Abeta 1-42 aggregate, and reduce neurotoxicity caused by Abeta 1-42 aggregate.
Drawings
FIG. 1 is a synthetic scheme for preparing a fluorescent probe (BD-2-3) in the present invention.
FIG. 2 is a 1 H-NMR spectrum of a fluorescent probe (BD-2-3) in an embodiment of the invention.
FIG. 3 is a 13 C-NMR spectrum of a fluorescent probe (BD-2-3) in an embodiment of the invention.
FIG. 4 is a graph showing the evaluation of binding ability of a fluorescent probe (BD-2-3) to Abeta 1-42 aggregate in the examples of the present invention; wherein a is the ultraviolet spectrum of the fluorescent probe (BD-2-3) in different solvents; b is the fluorescence spectrum of the fluorescent probe (BD-2-3) in different solvents; c is the pH stability of the fluorescent probe (BD-2-3); d is a protein titration curve of fluorescent probe (BD-2-3) for Abeta 1-42 aggregate; e is a specific binding diagram of the fluorescent probe (BD-2-3) and Abeta 1-42 aggregate; f is a selective binding diagram of fluorescent probe (BD-2-3) and Abeta 1-42 aggregate.
FIG. 5 is a graph showing fluorescence response of the resulting fluorescent probe (BD-2-3) versus viscosity in examples of the present invention.
FIG. 6 is an image of an amyloid plaque section of the brain of AD mice with the fluorescent probe (BD-2-3) obtained in the examples of the present invention.
FIG. 7 is a graph showing evaluation of singlet oxygen production efficiency of the fluorescent probe (BD-2-3) obtained in the examples of the present invention.
FIG. 8 is a transmission electron microscope image of the fluorescent probe (BD-2-3) photo-oxidized Abeta 1-42 aggregate obtained in the example of the present invention.
FIG. 9 is a confocal image of fluorescent probe (BD-2-3) mediated photooxidation of Abeta 1-42 aggregates to promote microglial uptake as obtained in the examples of the present invention.
FIG. 10 is a graph showing the evaluation of cell by using the fluorescent probe (BD-2-3) obtained in the example of the present invention, wherein a is the cytotoxicity of the fluorescent probe (BD-2-3) against PC12, and b is the evaluation of the reduction of neurotoxicity by the aggregate of photo-oxidized Abeta 1-42.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The reagents and starting materials used in the present invention are commercially available unless otherwise specified. In the following examples, reference is made to the technical specifications for parameters which are not particularly specified.
Example 1: synthetic organic small molecule fluorescent probe (BD-2-3)
The synthetic route of the organic small molecule fluorescent probe (BD-2-3) of the embodiment is shown in FIG. 1, and the specific synthetic steps are as follows:
(1) Synthesis of intermediate 1
① Methyl iodide (11 mmol,1.56 g) and 4-methylpyridine (10 mmol,0.93 g) were added to a round bottom flask and the mixture was completely dissolved in 10mL of ethanol and the reaction was stirred at room temperature for 4h.
② After the reaction solution was cooled, filtration was performed, and the precipitate was repeatedly washed with diethyl ether. The resulting solid was dried in a vacuum oven to give 2.16g of a pale yellow solid, namely intermediate 1, in 92% yield (structural formula shown in formula (II)), 1, 4-lutidine iodide.
1H NMR (400 MHz, DMSO-d6) δ 8.89-8.78 (m, 2 H), 7.97 (d, J = 6.3 Hz, 2 H), 4.29 (s, 3 H), 2.61 (s, 3 H).
(2) Synthesis of intermediate 2
① A mixed solution of 3, 6-dibromo-1, 2-phenylenediamine (1.69 mmol,0.45 g) and acetone (5 mL) in toluene (75 mL) was charged into a two-necked flask. And (3) vacuumizing the reaction liquid, filling nitrogen, placing the reaction liquid in an oil bath, heating to100 ℃, and stirring for reaction for 36h. After the reaction, cooling to room temperature, and removing the redundant reaction solvent by rotary evaporation under reduced pressure to obtain a crude product.
② To the crude product was added active manganese dioxide (85% purity) (1.13 g,1.3 mmol) under inert gas and reacted with 8 mL anhydrous DCM under stirring at room temperature for 12h. After the reaction was completed, the reaction mixture was filtered to remove manganese dioxide, and the filtrate was collected and extracted with DCM and distilled water. The organic phase was dried over anhydrous sodium sulfate, sanded, and purified by column chromatography (200-300 mesh silica gel, petroleum ether: ethyl acetate=15:1 (v/v)) to give 0.25g of yellow solid as intermediate 2 (BD-2) in 48.7% yield. The structural formula of the compound is shown as a formula (III), and the compound is 4, 7-dibromo-2, 2-dimethyl-2H-benzimidazole.
1H NMR (400 MHz, Methanol-d4) δ 7.49 – 7.44 (m, 2H), 1.36 (s, 6H).
(3) Synthesis of intermediate 3
① BD-2 (0.096 g,0.32 mmol) and pinacol 4-formylphenylborate (0.19 g,0.79 mmol) were weighed into a two-necked flask, and catalyst tetrakis (triphenylphosphine) palladium (0.018 g,0.016 mmol) was rapidly added and completely dissolved with stirring with 2.4mL of toluene solution, and then a (2 mol/L,1 mL) anaerobic potassium carbonate aqueous solution was slowly added dropwise with a syringe. Under the protection of argon, the temperature of the reaction system is raised to 80 ℃ and the reflux reaction is carried out for 12 hours.
② After the reaction is cooled, the reaction solution is extracted by using dichloromethane and water, an organic phase is taken for drying, reduced pressure rotary evaporation is carried out, sand is produced, and further column chromatography purification is carried out on dichloromethane and petroleum ether (dichloromethane: petroleum ether=10:3, v/v; silica gel 200-300 meshes) to obtain 0.05g yellow solid which is intermediate 3 (BD-2-CHO) with the yield of 44%, and the structural formula is shown as formula (IV), 4' - (2, 2-dimethyl-2H-benzo [ D ] imidazole-4, 7-diyl) dibenzoaldehyde.
(4) Synthesis of the end product BD-2-3
① BD-2-CHO (0.073 g,0.32 mmol) and intermediate 1 (0.73 g,0.6 mmol) were dissolved in a pressure-resistant tube, and then 144. Mu.L of piperidine was slowly added dropwise thereto;
② The pressure-resistant pipe is put into an oil bath pot, the temperature is raised to 80 ℃, and the reaction is stirred for 2 hours. The wall of the tube was observed to have a red solid precipitated, and the tube was repeatedly washed by filtration with absolute ethanol. The solid obtained was dissolved in methanol, filtered again, and the filtrate was concentrated to give 0.042g of a red solid, which was the final product, namely, a fluorescent probe, designated BD-2-3, in 15.6% yield, having the structural formula shown in formula (I), 4'- ((1E, 1' E) - ((2, 2-dimethyl-2H-benzo [ d ] imidazole-4, 7-diyl) bis (4, 1-phenyl)) bis (ethylene-2, 1-diyl)) bis (1-methylpyridin-1-iodo).
1H NMR (400 MHz, DMSO-d6) δ 8.90 (d, J = 6.5 Hz, 4H), 8.28 (d, J = 6.4 Hz, 4H), 8.18 (d, J = 8.1 Hz, 4H), 8.09 (d, J = 16.3 Hz, 2H), 7.89 (d, J = 8.2 Hz, 4H), 7.71– 7.56 (m, 4H), 4.28 (s, 6H), 1.58 (s, 6H).
Example 2: ultraviolet-fluorescence test of fluorescent probe BD-2-3
BD-2-3 probe prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mmol/L mother liquor, and stored in a refrigerator at-80 ℃. The mother liquor was then diluted to a solution of 10. Mu. Mol/L with various solvents (tetrahydrofuran, toluene, dimethylformamide, methanol, dimethylsulfoxide, PBS, etc.). The ultraviolet absorption spectra of BD-2-3 in different solvents were measured using an ultraviolet spectrophotometer and the maximum absorption wavelength was recorded. The solution was then placed under a fluorescence spectrophotometer to test the fluorescence spectrum and the maximum emission wavelength was recorded.
The results are shown as a, b in fig. 4: BD-2-3 had a maximum absorption wavelength of 433 nm and a maximum emission wavelength of 620 nm in PBS. In solvents of different polarity, the ultraviolet absorption spectrum and the fluorescence emission spectrum remain almost unchanged, and the Stokes shift is 187 nm.
Example 3: pH stability test of fluorescent Probe BD-2-3
PBS solutions (pH: 3-11) with different pH values are prepared, accurately tested by a pH meter, and BD-2-3 mother solution is diluted to 100 mu mol/L and fluorescence spectrum is tested by a fluorescence spectrophotometer.
The results are shown in fig. 4 c: the fluorescence intensity of the fluorescent probe BD-2-3 in solutions with different pH values is not changed obviously, and the better pH stability is presented.
Example 4: evaluation of binding ability of fluorescent Probe BD-2-3 to Abeta 1-42 aggregate
100 Mu mol/L Abeta 1-42 aggregate is diluted to different concentrations (1-10 mu mol/L) by PBS (pH=7.4), and then 1 mu mol/L BD-2-3 solution is added for incubation for 10min in dark place. Fluorescence spectra of Abeta 1-42 aggregates and fluorescent probe (BD-2-3) at different concentrations were measured using a fluorescence spectrophotometer.
Mu.L of Abeta 1-42 aggregate (100. Mu. Mol/L) (purchased from Shanghai blaze), abeta 1-42 oligomer (100. Mu. Mol/L) (purchased from Shanghai blaze), abeta 1-42 monomer (100. Mu. Mol/L) (purchased from Shanghai blaze) solution and 6. Mu.L of BD-2-3 solution (100. Mu. Mol/L) were each prepared into a total volume of 600. Mu.L of PBS solution. After each group of solutions is incubated for 10min under the condition of being at room temperature and avoiding light, the fluorescence spectrum of each group of solutions is tested by a fluorescence spectrophotometer, and data processing is carried out.
The results are shown as d, e in fig. 4: as the concentration of aβ 1-42 aggregates increases, so does the fluorescence intensity. The fluorescence intensity is significantly increased after BD-2-3 binds to Abeta 1-42 aggregates compared to Abeta 1-42 monomers, abeta 1-42 oligomers.
Example 5: evaluation of Selective binding of fluorescent Probe BD-2-3 to Abeta 1-42 aggregate
To PBS buffer (pH=7.4, 10 mmol/L) was added Aβ 1-42 aggregate (purchased from Shanghai blaze organism), common metal ion （Cu+,Ni+,K+,Pd2+,Cd2+,Al3+,Zn2+,Fe3+,Fe2+,Na+,Mg2+）（ was obtained by diluting standard solutions of corresponding chloride salts respectively to obtain stock solutions of metal ions or solutions of various amino acids (Met, glu, cys, arg, GSH, asp, lys, ser, ile, ala) in a total volume of 600. Mu.L, BD-2-3 probe concentration of 1. Mu. Mol/L, Aβ 1-42 aggregate, common metal ion and concentration of various amino acids of 10. Mu. Mol/L, and fluorescence intensities of each group were measured with a microplate reader for data processing.
The results are shown as f in fig. 4: BD-2-3 showed good selectivity for Abeta 1-42 aggregates, compared to Abeta 1-42 aggregates, with substantially no significant change in fluorescence intensity relative to the probe itself after binding to various metal ions and various amino acids.
Example 6: viscosity imaging of fluorescent probe BD-2-3
Different water and glycerol are prepared into solution systems with different viscosities according to different volume ratios (0:100-99:1). After BD-2-3 mother liquor (10 mmol/L) was diluted to 10. Mu. Mol/L with water/glycerol systems of different viscosities, 3mL of each group was placed into a fluorescence spectrophotometer to test the fluorescence spectrum of BD-2-3 under different systems.
As a result, as shown in FIG. 5, the fluorescence intensity of BD-2-3 was continuously increased as the viscosity increased. Abeta 1-42 aggregate and mitochondrial viscosity are closely related to Alzheimer's disease, demonstrating the potential of BD-2-3 for cell imaging.
Example 7: aβ amyloid plaque imaging ability of fluorescent probe BD-2-3
Fluorescence probe BD-2-3 was used for an aβ plaque imaging experiment of AD mouse brain using cerebral cortex from APPsWe/PSEN1 double transgenic male AD mice (C57 BL6, 12 months old). Firstly, dewaxing paraffin model slices, soaking m-xylene for 5 minutes, and repeating twice; soaking in absolute ethanol for 5 minutes, and repeating twice; soaking in 90% ethanol for 5min, and repeating twice; soaking in 80% ethanol for 5min, and repeating twice; soaking in 70% ethanol for 5min, and repeating twice; finally, the mixture was soaked in PBS (pH 7.4) for 5 minutes, and the process was repeated twice. 0.5mL of BD-2-3 in PBS (5. Mu. Mol/L) was slowly added dropwise to the sections and stained for 30min. The photosensitizer on the sections was washed three times with PBS and then again stained a second time with 0.5mL of commercial dye thioflavin-S (5. Mu. Mol/L) PBS solution (Thio-S) for 30 minutes, and after completion washed three times with PBS and blocked slowly with cover slips. Staining of amyloid plaque sections was performed by Leica SP8 laser confocal imaging BD-2-3 with Thio-S. Thios λex=405 nm, λem=425-550 nm; BD-2-3: λex=458 nm, λem=580-650 nm.
As a result, as shown in FIG. 6, the fluorescent probe BD-2-3 was able to image the amyloid plaques of the brain of AD mice well and co-localize well with the commercial dye Thio-S.
Example 8: singlet oxygen generating capability of fluorescent probe BD-2-3
9, 10-Anthryl-bis (methylene) di-malonic acid (ABDA) is selected as an indicator of singlet oxygen (1O2), and ultraviolet absorption at 378nm is gradually weakened under the action of 1O2. The ABDA stock solution (10 mmol/L), BD-2-3 solution (100. Mu. Mol/L) and the reference rose bengal RB solution (100. Mu. Mol/L) were diluted under dark conditions to prepare a total volume of 600. Mu.L PBS solution, such that the probe concentration was 10. Mu. Mol/L and the indicator concentration was 100. Mu. Mol/L. Then, the change of the ultraviolet absorption of different groups of 378nm under the excitation of white light is tested by an enzyme-labeled instrument. The specific experimental group is (1) ABDA (100 mu mol/L) +illumination; (2) RB (10. Mu. Mol/L) +ABDA (100. Mu. Mol/L) +illumination; (3) BD-2-3 (10. Mu. Mol/L) +ABDA (100. Mu. Mol/L) +illumination.
The results are shown in FIG. 7, where the ultraviolet absorption of ABDA is substantially unchanged under illumination, indicating that ABDA can exist stably under white light illumination. When the probe BD-2-3 was added, the ultraviolet absorption at 378nm was significantly reduced, indicating that BD-2-3 had good singlet oxygen generating capability.
Example 9: evaluation of fluorescent Probe BD-2-3 photo-oxidized Abeta 1-42 aggregate
A total volume of 300. Mu.L of Abeta 1-42 aggregate (40. Mu. Mol/L) (purchased from Shanghai blaze) and/or BD-2-3 (10. Mu. Mol/L) solution was prepared with distilled water. All samples were diluted 3-fold after 24h of illumination with white light. And (3) dripping 10 mu L of the solution onto a copper mesh containing a carbon film, carrying out negative dyeing on the solution after the water volatilizes by using 2% (w/v) phosphotungstic acid, and drying the solution. The morphology of aβ 1-42 aggregates under different conditions was observed using a transmission electron microscope of Talos L120c at an accelerating voltage of 120 kv.
As a result, as shown in FIG. 8, the aggregate of Abeta 1-42 in a natural state exhibited a long and thin fibrous shape, and the long and thin fibrous shape was not significantly changed after the BD-2-3 was added for co-incubation. However, when BD-2-3 and Abeta 1-42 aggregates were irradiated with white light, the fibers were substantially disappeared, and only a fine dot shape was observed.
Example 10: fluorescent probe BD-2-3 mediated photooxidation of Abeta 1-42 aggregate to promote microglial uptake
Microglia can improve the pathological features of alzheimer's disease by phagocytosing aβ amyloid fibers. BV2 mouse microglial cells (purchased from cell bank of China academy of sciences) are cultured on a confocal dish until the density is 80-90%. Media for several groups of different samples were prepared: (1) FAM-Abeta1-42aggregate(2.mu.mol/L); (2) FAM-Abeta1-42aggregate(2.mu.mol/L)+BD-2-3(5.mu.mol/L); (3) FAM-Abeta1-42aggregate(2.mu.mol/L)+BD-2-3(5.mu.mol/L)+illumination; (4) FAM-Abeta1-42aggregate(2.mu.mol/L)+BD-2-3(5.mu.mol/L)+chloroquine(CQ)(10.mu.mol/L)+illumination,afteradditiontotheconfocaldish,theilluminationgroupswereeachirradiatedwithwhitelightfor30minandincubatedinanincubatorfor24h. After three washes with PBS, hoechst 33342 (1. Mu. Mol/L) was added for 30min, and washed three times with PBS, and an appropriate amount of cell imaging fluid was added. The uptake of aβ 1-42 aggregates by the individual groups of BV2 mouse microglia was analyzed using laser confocal.
Asaresult,asshowninFIG.9,theFAM-Abeta1-42aggregateandthelightgroupofBD-2-3exhibitedsignificantfluorescencecomparedtotheFAM-Abeta1-42aggregateandtheFAM-Abeta1-42aggregate+BD-2-3. When the lysosome inhibitor chloroquine is added, fluorescence disappears, and the aβ 1-42 aggregate after photooxidation may be degraded by lysosomal phagocytosis. Thus, it was demonstrated that BD-2-3 photooxidation of Abeta 1-42 aggregates promoted phagocytosis of Abeta fibers by microglia.
Example 11: evaluation of cytotoxicity of fluorescent probe BD-2-3 and reduction of neurotoxicity by photooxidation of Abeta 1-42 aggregates.
The effect of fluorescent probe BD-2-3 on PC12 cell (rat adrenal pheochromocytoma cell, purchased from China academy of sciences cell bank) viability was tested by MTT colorimetric method. BD-2-3 solution was diluted to 0, 3.125, 6.25, 12.5 and 25. Mu. Mol/L with DMEM medium, respectively, and then added to 96-well plates containing PC12 cells, followed by further culturing for 24 hours. The MTT-containing medium was added and absorbance at 570nm was measured by an enzyme-labeled instrument.
PC12 cells are also cultured to 80-90% in a 96-well plate, and then culture mediums of 8 groups of different samples are prepared: (a) blank + dark; (B) blank group + illumination; (C) BD-2-3+ darkness; (D) BD-2-3+ illumination; (E) Aβ 1-42 aggregates + darkness; (F) Aβ 1-42 aggregates + illumination; (G) Aβ 1-42 aggregate+BD-2-3+ dark; (H) Aβ 1-42 aggregate+BD-2-3+ light. Wherein the concentration of Abeta 1-42 aggregate is 20 mu mol/L, the concentration of BD-2-3 is 5 mu mol/L, and the culture is carried out for 24 hours by white light irradiation for 30 minutes. Cell viability assay using MTT colorimetric assay the effect of photooxidized aβ 1-42 aggregates on neurotoxicity.
As a result, as shown in FIG. 10a, BD-2-3 probe at a concentration of 25. Mu. Mol/L or less had substantially no effect on the viability of PC12 cells. From fig. 8 b, white light illumination and BD-2-3 probe did not cause significant cell damage. Cell viability was significantly reduced after 24 hours incubation with aβ 1-42 aggregates. However, the addition of BD-2-3 followed by white light excitation can reduce the cell death rate, indicating that BD-2-3 can oxidize Abeta 1-42 aggregates under white light excitation to inhibit neurotoxicity.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A photo-oxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe is characterized in that the structural formula is shown as the formula (I):
Formula (I).
2. The method for preparing the photooxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe according to claim 1, which is characterized by comprising the following steps:
(1) Methyl iodide and 4-methylpyridine are dissolved in an organic solvent and stirred at room temperature for reaction; after the reaction, the reaction solution was cooled, filtered, and the obtained solid was washed and dried to obtain intermediate 1 having a structural formula shown in formula (II):
Formula (II);
(2) Under the inert gas atmosphere, 3, 6-dibromo-1, 2-phenylenediamine and acetone are dissolved in an organic solvent, and the reaction system is heated to 100 ℃ and stirred for reaction; cooling to room temperature after the reaction is finished, performing rotary evaporation under reduced pressure, and removing a reaction solvent to obtain a crude product;
under the protection of inert gas, adding active manganese dioxide and anhydrous dichloromethane into the crude product, and stirring at room temperature for reaction; after the reaction is finished, filtering the reaction mixture to remove manganese dioxide, extracting the filtrate, further drying an organic phase, preparing sand, and purifying to obtain an intermediate 2; the structural formula is shown as formula (III):
formula (III);
(3) Under the inert gas atmosphere, the intermediate 2 and 4-formylphenyl pinacol borate are dissolved in toluene, an anaerobic potassium carbonate solution is added, and nucleophilic substitution reaction is carried out by heating to 80 ℃ under the catalysis of tetra (triphenylphosphine) palladium; after the reaction is finished, extracting the reaction liquid, drying an organic phase, performing reduced pressure rotary evaporation, preparing sand, and purifying to obtain an intermediate 3, wherein the structural formula of the intermediate is shown as the formula (IV):
Formula (IV);
(4) Dissolving intermediate 3 and intermediate 1 in ethanol, and adding piperidine; heating the reaction system to 80 ℃ and stirring for reaction; and after the reaction is finished, filtering and cleaning the precipitated red solid by using absolute ethyl alcohol, dissolving the obtained solid by using methanol, filtering, taking filtrate, and spin-drying to obtain the red solid, namely the photo-oxidation and imaging Abeta 1-42 aggregate difunctional fluorescent probe, wherein the structural formula of the difunctional fluorescent probe is shown as the formula (I).
3. The method according to claim 2, wherein the organic solvent in step (1) is ethanol;
the molar ratio of 4-methylpyridine to methyl iodide in the step (1) is 1: 1-1.1;
The dosage of the organic solvent in the step (1) is calculated by 1mL of ethanol mixed with each millimole of 4-methylpyridine;
The stirring reaction time in the step (1) is 4 hours.
4. The method of claim 2, wherein the inert gas in step (2) is nitrogen;
The mixture ratio of the 3, 6-dibromo-1, 2-phenylenediamine and the acetone in the step (2) is that 3mL of acetone is added into each mole of 3, 6-dibromo-1, 2-phenylenediamine;
The heating and stirring reaction time at 100 ℃ in the step (2) is 36 hours;
the molar ratio of 3, 6-dibromo-1, 2-phenylenediamine to manganese dioxide in the step (2) is 1: 0.5-1;
the stirring reaction time at room temperature in the step (2) is 12 hours;
the organic solvent of step (2) comprises toluene.
5. The method of claim 2, wherein the inert gas in step (3) is argon;
the molar ratio of the intermediate 2, 4-formylphenyl boronic acid pinacol ester to tetrakis (triphenylphosphine) palladium in the step (3) is 1: 2-4: 0.05;
The concentration of the potassium carbonate solution in the step (3) is 2mol/L, and 3-4 mL of the potassium carbonate solution is added into each millimole of the intermediate 2;
The toluene amount in the step (3) is 7.5mL of toluene per millimole of intermediate 2;
the heating reaction time in the step (3) is 12 hours.
6. The process according to claim 2, wherein the molar ratio of intermediate 3 to intermediate 1 in step (4) is 1:2;
the piperidine dosage in the step (4) is 450 mu L piperidine added per millimole of intermediate 3, and 18mL ethanol is added per millimole of intermediate 3;
The stirring reaction time in the step (4) is 2 hours.
7. Use of the photooxidation and imaging aβ 1-42 aggregate bifunctional fluorescent probe of claim 1 for the preparation of a product for detecting aβ 1-42 aggregates.
8. Use of the photooxidation and imaging aβ 1-42 aggregate bifunctional fluorescent probe of claim 1 for the preparation of a product for inhibiting aβ 1-42 aggregates.
9. Use of the photooxidation and imaging aβ 1-42 aggregate bifunctional fluorescent probe of claim 1 for the preparation of a diagnostic product for alzheimer's disease.
10. The use according to any one of claims 7 to 9, wherein the product comprises a fluorescent probe, a detection reagent, a diagnostic reagent or a kit.
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