CN111777603A - Fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaque and peroxynitrite and preparation method and application thereof - Google Patents

Abstract

The invention relates to a preparation method and application of a fluorescent probe capable of simultaneously detecting β amyloid deposition plaque and peroxynitrite, wherein the structural formula of the fluorescent probe is as follows:

the fluorescent probe is named as BTNPO, and the fluorescent probe takes a naphthalene-benzothiazole derivative as a fluorophore and takes a substituted lactam functional group as ONOO^‑By reaction of recognition groups, other than with ONOO^‑Besides quick response, the probe can be combined with A β deposited plaque, and has high sensitivity and good selectivity, and can be used as an excellent imaging tool for living cells and AD model mice to carry out A β deposited plaque and ONOO^‑While simultaneouslyAnd (6) detecting.

Description

Fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaque and peroxynitrite and preparation method and application thereof

Technical Field

The invention belongs to the technical field of fluorescence imaging detection, and particularly relates to a fluorescent probe capable of simultaneously detecting beta amyloid protein deposition plaques and peroxynitrite, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The present study shows that the plaque deposition formed by β amyloid (A β) aggregation is one of the key factors causing the decline of cognitive functions, the neurotoxicity of A β plaque deposition can change the calcium ion homeostasis of mitochondria, induce inflammation and increase the generation of Reactive Oxygen Species (ROS). The A β plaque deposition has been proved to stimulate nerve cells to generate excessive peroxynitrite (ONOO)^-) Accelerating damage to neurons. ONOO^-Researchers found high levels of nitrated proteins in neurons of the brain of Alzheimer's Disease (AD) patients, the fact that A β deposits plaques and ONOO^-Therefore, many small molecule fluorescent probes were developed and used to deposit plaques or ONOO to A β^-However, the inventors found that both A β plaque deposits and ONOO could be detected simultaneously^-The number of the small molecular fluorescent probes is very small.

Disclosure of Invention

To overcome the above problems, it is an object of the present invention to provide a method for simultaneously detecting A β plaque deposits and ONOO^-The fluorescent probe and the preparation method and the application thereof. The fluorescent probe is named as BTNPO, and the probe takes naphthalene-benzothiazole derivative as a fluorophore and takes substituted lactam as ONOO^-Reactive groups capable of reacting with ONOO^-And the probe is capable of binding to A β plaque deposits resulting in an increase in fluorescence the fluorescent probe is capable of passing through the duplexFluorescence channel pairs A β plaque deposits and ONOO^-The fluorescence detection is carried out simultaneously, has the characteristics of high sensitivity and good selectivity, and can realize simultaneous fluorescence imaging of the cell and the living body.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a use of a fluorescent probe for simultaneously detecting β amyloid plaque deposits and peroxynitrite, the fluorescent probe having a structural formula:

the probe has the characteristics of high sensitivity and good selectivity, can realize simultaneous fluorescence imaging of the cell and the living body, and can be used for researching A β plaque deposits and ONOO^-The interrelationship between them.

In a second aspect of the present invention, there is provided a use of a fluorescent probe for simultaneously imaging amyloid-beta plaque deposits and peroxynitrite at a living cell level, the fluorescent probe having a structural formula:

the probe of the invention can deposit plaque and ONOO to A β through double channels at the living cell level^-And (3) fluorescence imaging is carried out, the dyeing effect is good, the dyeing time is short (30min), and the dyeing efficiency is high.

In a third aspect of the present invention, there is provided a use of a fluorescent probe for simultaneous imaging of amyloid-beta plaque deposits and peroxynitrite in a living animal, the fluorescent probe having the structural formula:

the probe can be used for treating A β deposited plaques and ONOO in brain slices of AD model mice through two channels^-To carry outAnd (4) fluorescence imaging.

In a fourth aspect of the invention, there is provided a method for simultaneous imaging detection of amyloid-beta plaque deposits and peroxynitrite at a viable cell level, comprising:

stimulating living cells by using an Abeta aggregate, and then adding the living cells into a solution containing a fluorescent probe for incubation;

after incubation is finished, laser confocal imaging is carried out, and the compound is obtained;

wherein, the structural formula of the fluorescent probe is as follows:

the probe of the invention has very weak autofluorescence, and after the probe is combined with A β deposited plaque, the fluorescence at 418nm is obviously enhanced, and the probe and ONOO^-After reaction, fluorescence at 506nm is significantly enhanced, thus achieving plaque deposition and ONOO to A β through two channels^-Simultaneous detection is performed.

In a fifth aspect of the invention, there is provided a method for simultaneous imaging detection of amyloid beta plaque deposits and peroxynitrite in a living animal comprising:

injecting a fluorescent probe into the abdominal cavity of the living animal, taking out the brain of the living animal, slicing, and carrying out two-photon confocal imaging to obtain the fluorescent probe;

wherein, the structural formula of the fluorescent probe is as follows:

the probe BTNPO provided by the invention is used as an excellent fluorescence imaging tool, and can realize the effects of A β deposited plaque and ONOO in cell level and AD mouse models^-While simultaneously imaging.

In a sixth aspect of the present invention, a fluorescent probe capable of simultaneously detecting β amyloid plaque deposits and peroxynitrite is provided, which has the structural formula:

the invention names the fluorescent probe BTNPO, the probe takes naphthalene-benzothiazole derivative as a fluorophore and takes substituted lactam as ONOO^-Reactive groups capable of reacting with ONOO^-And the probe was able to bind to a β plaque deposits resulting in an increase in fluorescence.

In a seventh aspect of the present invention, there is provided a method for preparing a fluorescent probe capable of simultaneously detecting β amyloid plaque deposits and peroxynitrite, comprising:

dissolving the compound 1 in glacial acetic acid, adding ketomalonic acid diethyl ester, and uniformly mixing to obtain a mixed solution;

reacting the mixed solution to obtain a fluorescent probe;

wherein the structural formula of the compound 1 is

The preparation method is simple, low in cost, strong in practicability and easy to popularize.

The invention has the beneficial effects that:

(1) the probe has very weak autofluorescence, obviously enhanced fluorescence at 418nm after being combined with A β deposited plaque, and the probe and ONOO^-After reaction, fluorescence at 506nm is significantly enhanced, thus achieving plaque deposition and ONOO to A β through two channels^-Simultaneous detection is performed.

(2) The probe can deposit plaque and ONOO to A β through double channels at living cell level^-And (3) fluorescence imaging is carried out, the dyeing effect is good, the dyeing time is short (30min), and the dyeing efficiency is high.

(3) The probe can deposit plaque and ONOO to A β in the brain section of the AD model mouse through two channels^-Fluorescence imaging is performed.

(4) The method is simple, low in cost, strong in practicability and easy to popularize.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows the fluorescent probes of example 1 of the present invention for different concentrations of ONOO^-(A) And A β fluorescence response spectrum of the aggregate (B), wavelength (nm) on the abscissa and fluorescence emission intensity on the ordinate.

FIG. 2 shows fluorescence intensities at 418nm (A) and 506nm (B) of the fluorescent probe of the present invention in the presence of different substances in example 1 of the present invention. The abscissa is the intensity of the fluorescence emission of the probe at the corresponding wavelength, and the ordinate is the intensity of the different species.

FIG. 3 is a sequence of addition of ONOO to the fluorescent probe of example 1 of the present invention^-And A β, and measuring fluorescence intensity of the probe after aggregation, wherein the abscissa is a sample under different conditions, and the ordinate is fluorescence emission intensity of the probe at 418nm (A) or 506nm (B).

FIG. 4 is the alignment of the fluorescent probes of example 1 of the present invention at the PC12 cell level to ONOO^-And a fluorescence image of the deposited plaque of a β.

FIG. 5 is a plot of the fluorescence probe of example 1 of the present invention against ONOO in brain sections from AD model mice of different ages of months ( months 2, 3, 4, 5 and 6)^-And a fluorescence image of the deposited plaque of a β.

FIG. 6 is a three-dimensional fluorescence scanning image of the fluorescent probe of example 1 of the present invention for the brain of a 6-month-old AD mouse.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The invention utilizes the existing raw material intermediate compound 1 to prepare the product probe BTNPO through one-step simple and efficient synthesis and finally silica gel chromatographic column separation. Compound 1 is obtained from 6-amino-2-naphthoic acid and o-aminothiophenol in polyphosphoric acid, heated to 150 deg.C, and reacted overnight.

A method for simultaneously detecting A β plaque and ONOO^-The structural formula of the fluorescent probe is as follows:

the preparation method of the fluorescent probe comprises the following steps:

the reaction equation is:

1) dissolving the compound 1 in glacial acetic acid, adding diethyl ketomalonate into the glacial acetic acid, and uniformly stirring to obtain a mixed solution;

2) and (3) reacting the mixed solution obtained in the step 1) for a set time and temperature to obtain the catalyst.

In some embodiments, in step 1), the ratio of the amounts of the substance of compound 1 and the ketomalonic acid diethyl ester is 1:1-3 to synthesize the target probe BTNPO.

In some embodiments, the reaction temperature in step 2) is 30-118 ℃ and the reaction time is 8-18h, so as to improve the reaction efficiency.

In some examples, the reaction temperature in step 2) is 90-118 ℃ and the reaction time is 12-15h, so that higher reaction rate and yield can be obtained.

In some embodiments, step 2) further comprises a step of spin-drying and purifying the reaction product solution to obtain a high-purity probe, so as to enhance the subsequent fluorescence imaging effect.

In some embodiments, in step 2), the reaction product is purified by silica gel column chromatography, which is simple in operation, large in sample carrying capacity and high in separation purity.

In some embodiments, the eluent for silica gel column chromatography is a mixed solution of dichloromethane, methanol and triethylamine, and the volume ratio of dichloromethane, methanol and triethylamine for reaction product purification is 120-90:1-1.5: 1; preferably 100:1:1, to improve desorption efficiency.

The fluorescent probe can be used for treating A β plaque and ONOO at living cell level^-Application to imaging.

The method for imaging the PC12 cells by the fluorescent probe comprises the following steps: stimulating the cultured PC12 cells by using an Abeta aggregate, adding a dimethyl sulfoxide solution of a probe for incubation, and directly carrying out two-photon confocal imaging after the incubation is carried out for a set time.

Wherein the A beta aggregate is A beta oligomer, fibril, fiber, plaque and the like prepared by adopting A beta monomer.

The stimulation time of the a β aggregates and the probe incubation time are not particularly limited in this application, and in some embodiments, the stimulation time of the a β aggregates is 12h, and the probe incubation time is 30min, which can be adjusted by those skilled in the art according to actual situations.

The fluorescent probe is used as a detection tool for A β deposited plaque and ONOO in an Alzheimer disease model mouse^-The simultaneous imaging detection comprises the following steps:

AD model mice with different ages in months are selected, a probe is injected into the abdominal cavity, and then the brain of the mouse is taken out and sliced to carry out two-photon confocal imaging.

The dosage of the intraperitoneal injection probe is not particularly limited in the present application, and in some embodiments, the dosage of the intraperitoneal injection probe is (20mg kg-1), and the injection amount can be adjusted by a person skilled in the art according to the characteristics of the test animal.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.

Example 1: synthesis of fluorescent probes

Compound 1(274mg, 1mmol) was dissolved in 3mL of glacial acetic acid, and the mixture was heated to reflux. Then, a solution of ketomalonic acid diethyl ester (348mg, 2mmol) in glacial acetic acid (2mL) was added dropwise to the above mixed solution, followed by reaction for 15 hours. After completion of the reaction, the reaction solution was cooled to room temperature, the solvent was concentrated and dried, and the solid was subjected to column chromatography (eluent, dichloromethane: methanol: triethylamine: 100:1:1, V/V, respectively) to give the product solids as 324mg (yield 65%), respectively).

Mass spectrum and nuclear magnetism characterization:

BTNPO：HRMS(ESI):calculated forC₂₂H₁₅N₂O₄S^-(M-H)^-403.0747,found403.0747。

¹H NMR(400MHz,DMSO-d₆)10.92(s,1H),8.69(s,1H),8.22(t,J＝8.3Hz,2H),8.17(d,J＝8.0Hz,1H),8.09(d,J＝8.1Hz,1H),7.96(d,J＝8.8Hz,1H),7.57(t,J＝7.6Hz,1H),7.48(t,J＝7.5Hz,1H),7.33(d,J＝8.6Hz,1H),7.24(s,1H),4.18–4.10(m,1H),4.10–4.02(m,1H),1.01(t,J＝8.0Hz,3H)ppm。

¹³C NMR(100MHz,DMSO)176.22,169.54,167.74,154.11,143.66,134.94,133.08,130.57,129.69,129.23,128.54,127.15,126.11,125.94,123.29,123.21,122.80,121.02,113.46,78.83,61.97,14.38ppm。

effect experiment:

probe pairs A β aggregate and ONOO^-Fluorescence response experiment of (2):

probe stock solution (1.0mM) was added to PBS (pH 7.4) buffer solution, diluted with water, and different concentrations of ONOO were added^-Or aggregate of A β, with a final concentration of 2.0. mu.M for the probe, 50mM for PBS, 0-25. mu.M for aggregate of A β, ONOO^-Concentration of 0-20. mu.M (0-10 eq.) As shown in FIG. 1A, when the probe was excited at 340nm, it had weak fluorescence at 418nm, and after incubation with different concentrations of A β aggregates (the concentration of A β aggregates corresponding in order from bottom to top was 0. mu.M, 4. mu.M, 8. mu.M, 12. mu.M, 16. mu.M, 20. mu.M, 25. mu.M), 41. mu.MThe fluorescence at 8nm is significantly enhanced; in FIG. 1, panel B, the probe had essentially no fluorescence emission at 506nm upon excitation at 380nm, with different concentrations of ONOO^-(ONOO corresponding in sequence from bottom to top^-At a concentration of 0. mu.M, 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 10. mu.M, 12. mu.M, 14. mu.M, 16. mu.M, 18. mu.M, 20. mu.M), a new emission peak at 506nm appeared and fluorescence gradually increased.

Subsequently, probe pairs A β aggregate and ONOO were investigated^-In summary, the selection of different excitation wavelengths can achieve probe pairs of A β aggregates and ONOO^-In FIG. 2, the reference numeral 1 represents 20. mu. MA β aggregates (A in FIG. 2) or ONOO^-(B in FIG. 2), 2 represents blank control, 3 represents 20. mu.M ONOO^-(A in FIG. 2) or A β aggregates (B in FIG. 2), 4 represents 100. mu.MH₂O₂And 5 represents 100. mu. MClO^-And 6 represents 100. mu. M O₂ ^·-And 7 represents 100. mu.M^·OH, 8 represents 100. mu.M¹O₂9 for 100. mu. M t-BuOOH, 10 for 100. mu.M NO, 11 for 5mM GSH, 12 for 5mMCys, 13 for 100. mu. MHcy, 14 for 100. mu. M H₂S, 15 represents 100. mu.M NO₃ ^-And 16 represents 100. mu. MNO₂ ^-And 17 represents 100. mu. MAcO ^-18 represents 100. mu.M SO₄ ^2-And 19 represents 100. mu.M SO₃ ^2-20 stands for 100. mu.M CO ₃ ^2-21 represents 100. mu.M PO₄ ^3-And 22 represents 100. mu.M Na ⁺23 represents 100. mu. M K⁺And 24 represents 100. mu. MCa²⁺And 25 represents 100. mu.M Mg ²⁺26 represents 100. mu.M Zn²⁺And 27 represents 100. mu.M Cu²⁺And 28 represents 100. mu. MFe²⁺And 29 represents 100. mu. MFe³⁺. As can be seen from FIG. 2, none of the other potentially interfering substances is able to cause a change in fluorescence of the probe, including reactive oxygen species/reactive nitrogen species (H)₂O₂，ClO^-，O₂ ^·-，^·OH，¹O₂t-BuOOH, and NO), active sulfur (GSH, Cys, Hcy, and H)₂S), anion (NO)₃ ^-，NO₂ ^-，AcO^-，SO₄ ^2-，SO₃ ^2-，CO₃ ^2-And PO₄ ^3-) Cation (Na)⁺，K⁺，Ca^{2 +}，Mg²⁺，Zn²⁺，Cu²⁺，Fe²⁺And Fe³⁺) In contrast, the probe was at the a β aggregate or ONOO^-(corresponding to reference numeral 1) showed significant fluorescence enhancement in the presence of the probe BTNPO, in summary, the probe BTNPO was directed to the aggregate A β, ONOO^-Has high specificity, and can be used for treating A β aggregate and ONOO in physiological environment by selecting different excitation wavelengths^-And (4) fluorescent visualization research.

Next, the aggregate at A β and ONOO were investigated^-In FIG. 3, excess probe (4. mu.M) was added followed by A β aggregate (20. mu.M) and ONOO^-(20 μ M), setting the excitation wavelength to 340nm, and detecting the change of fluorescence at 418 nm; or sequentially adding ONOO^-(20. mu.M), A β aggregate (20. mu.M), under the same conditions, the excitation wavelength was set to 380nm, and the change in fluorescence emission at 506nm was detected, As shown in FIG. 3A, at excitation of 340nm, fluorescence at 418nm was significantly enhanced as long as A β aggregate was present, and ONOO^-The presence or absence of probe does not affect the detection of the A β aggregate, and as shown in B of FIG. 3, only in ONOO under 380nm excitation^-When existing, a new emission peak appears at 506nm, and the presence of A β aggregates does not influence the probe pair ONOO^-It follows that selecting different excitation wavelengths can achieve the desired effects on the A β aggregates and ONOO^-And simultaneously detecting the two, and the two do not interfere with each other.

Probes at the PC12 cell level on A β plaque deposits and ONOO^-Simultaneous imaging experiments of (1):

as shown in FIG. 4, we verified the probe BTNPO on the cell level against A β plaque deposits and ONOO^-Stimulation of PC12 cells with different concentrations of A β aggregates (0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M and 40. mu.M) to produce ONOO^-12h later, the cells were incubated with 10 μ M probe for 30min, and then plaques deposited with A β (Ex:700 nm;em:380-430nm) and intracellularly produced ONOO^-(Ex:750 nm; Em:490-550 nm.) two-photon fluorescence imaging the results of the experiments are shown in FIG. 4, with increasing concentration of A β aggregates, the ONOO in PC12 cells^-The concentration of the probe is increased, and the probe can realize the plaque deposition and ONOO of A β by selecting different excitation wavelengths and signal receiving ranges^-The two channels of (2) are imaged simultaneously.

Probes in Alzheimer's disease model mice (APPswe/PS1-dE9) against A β plaque and ONOO^-Simultaneous imaging experiments of (1):

in FIG. 5, AD model mice of different ages of months (2, 3, 4, 5 and 6 months) were selected and injected intraperitoneally with a probe (20 mgkg)^-1) And taking out the mouse brain after 2h, slicing, carrying out two-photon confocal imaging, exciting 700nm, collecting the range of 380-450nm, detecting A β deposited plaques, exciting 750nm, collecting the range of 490-550nm, and detecting ONOO^-. The results are shown in FIG. 5, which shows the ONOO in the brains of AD mice with increasing age^-The concentration is gradually increased, and A β plaque deposits begin to appear in the brains of AD mice with the age of 4 months and gradually increase along with the increase of the age of the months.

In FIG. 6, three-dimensional fluorescence scans of 6-month-old AD mouse brain sections from FIG. 5 show that the probes detect A β plaque deposits and ONOO^-Is about 100 μm. The upper left is ONOO^-The upper right is the fluorescence scan of the A β plaque deposit, and the lower is the overlay of the two.

In conclusion, the probe BTNPO can be used as an excellent fluorescence imaging tool to realize the plaque deposition of A β and ONOO in a cell level and AD mouse model^-While simultaneously imaging.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (10)

1. The application of the fluorescent probe in simultaneously detecting the beta amyloid protein deposition plaque and the peroxynitrite is characterized in that the structural formula of the fluorescent probe is as follows:

2. the application of the fluorescent probe in the simultaneous imaging of beta amyloid deposition plaque and peroxynitrite at the living cell level is characterized in that the structural formula of the fluorescent probe is as follows:

3. the use of claim 2, wherein the living cells are PC12 cells.

4. The application of the fluorescent probe in the simultaneous imaging of beta amyloid plaque deposits and peroxynitrite in living animals is characterized in that the structural formula of the fluorescent probe is as follows:

5. a method for simultaneous imaging detection of amyloid beta plaque deposits and peroxynitrite at a viable cell level, comprising:

stimulating living cells by using an Abeta aggregate, and then adding the living cells into a solution containing a fluorescent probe for incubation;

after incubation is finished, laser confocal imaging is carried out, and the compound is obtained;

wherein, the structural formula of the fluorescent probe is as follows:

6. the method for simultaneous imaging detection of amyloid beta plaque deposits and peroxynitrite at the viable cell level of claim 5, wherein said stimulation time is 12 hours;

or the incubation time is 30 min.

7. A method for simultaneous imaging detection of amyloid beta plaque deposits and peroxynitrite in a living animal comprising:

injecting a fluorescent probe into the abdominal cavity of the living animal, taking out the brain of the living animal, slicing, and carrying out two-photon confocal imaging to obtain the fluorescent probe;

wherein, the structural formula of the fluorescent probe is as follows:

8. the method for simultaneous imaging and detection of β amyloid plaques and peroxynitrite in a living animal of claim 7, wherein the intraperitoneal injection of the probe is at a dose of 20mg kg^-1。

9. A fluorescent probe capable of simultaneously detecting amyloid beta plaque deposits and peroxynitrite, which is characterized in that: the structural formula is as follows:

10. a preparation method of a fluorescent probe capable of simultaneously detecting beta amyloid plaque deposits and peroxynitrite is characterized by comprising the following steps:

dissolving the compound 1 in glacial acetic acid, adding ketomalonic acid diethyl ester, and uniformly mixing to obtain a mixed solution;

reacting the mixed solution to obtain a fluorescent probe;

wherein the structural formula of the compound 1 is as follows:

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