A near-infrared chemiluminescent probe responsive to tau protein, its preparation method and application

Abstract

This invention belongs to the field of biochemistry technology, and relates to a near-infrared chemiluminescent probe responsive to tau protein, its preparation method, and its application. The preparation method of the near-infrared chemiluminescent probe includes the following steps: adding compound (I), compound (II), a catalyst, and an alkaline additive to a first solvent for reaction to obtain compound (III); adding compound (III), compound (IV), and piperidine to a second solvent for reaction to obtain compound (V); adding compound (V) and trifluoroacetic acid to a third solvent for reaction to obtain compound (VI); and adding compound (VI), compound (VII), and an acid compound to a fourth solvent for reaction to obtain the near-infrared chemiluminescent probe. The chemiluminescent probe of this invention exhibits high selectivity and efficient chemiluminescence capability, providing a promising tool for the diagnosis of Alzheimer's disease (AD). Its structural formula is: [Insert structural formula here].

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a near-infrared chemiluminescent probe that responds to tau protein, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly, and its main neuropathological features include brain lesions. -Amyloid protein (A) Senile plaques formed by tau deposition and neurofibrillary tangles (NFTs) formed by the aggregation of hyperphosphorylated tau protein. Recent studies have shown that the pathological burden and distribution of tau protein are more correlated with the degree of cognitive impairment in Alzheimer's disease (AD) patients than with Alzheimer's disease (ADS). Higher correlation. Therefore, accurate detection and imaging of tau protein in the brain is of vital importance for the early diagnosis, disease staging, treatment evaluation, and pathological mechanism research of Alzheimer's disease (AD).

[0003] Chemiluminescence is a process in which an excited-state intermediate is generated through a chemical reaction, thereby releasing photons. Compared to fluorescence imaging, chemiluminescence does not require external excitation light, eliminating autofluorescence and light scattering caused by excitation light at the source, thus achieving extremely high signal-to-noise ratio and sensitivity. Currently, chemiluminescence imaging technology is widely used in in vitro immunoassays, but its application in in vivo imaging, especially for targeting specific brain protein sites, is still in its early stages. Previous studies have reported on chemiluminescent probes based on systems such as peroxalate or luciferase for detecting reactive oxygen species or reporter gene imaging, but designing and constructing chemiluminescent probes that specifically target the key pathological tau protein in Alzheimer's disease (AD) remains a challenging area with very few reports.

[0004] Based on the current research status, the development of chemiluminescent probes for imaging tau protein in the brains of AD model mice faces the following challenges: 1) targeting ability with high specificity and high affinity for tau protein; 2) construction of near-infrared chemiluminescent probes; 3) design of probes that respond to the "on" response of tau protein.

[0005] Therefore, there is an urgent need to propose a near-infrared chemiluminescent probe that responds to tau protein, as well as its preparation method and application. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a near-infrared chemiluminescent probe responsive to tau protein, its preparation method, and its applications. The probe designed in this invention achieves near-infrared chemiluminescence capability through a conjugated structure extended by double bonds. (600nm); at the same time, by introducing pyridine groups, the nitrogen atoms of which are protonated, will form electrostatic interactions with tau proteins, thus achieving high selectivity for tau proteins.

[0007] To achieve the above objectives, a first aspect of the present invention provides a near-infrared chemiluminescent probe that responds to tau protein, the structural formula of which is:

[0008] .

[0009] A second aspect of the present invention provides a method for preparing the aforementioned tau protein-responsive near-infrared chemiluminescent probe, comprising the following steps:

[0010] (1) Compound (I), compound (II), catalyst, and basic additive are added to the first solvent to carry out the first reaction to obtain compound (III);

[0011] (2) Compound (III), compound (IV), and piperidine are added to a second solvent to carry out a second reaction to obtain compound (V);

[0012] (3) The compound (V) and trifluoroacetic acid are added to a third solvent to carry out a third reaction to obtain compound (VI);

[0013] (4) The compounds (VI), (VII), and acid compounds are added to the fourth solvent to carry out the fourth reaction, thereby obtaining the near-infrared chemiluminescent probe;

[0014] The reaction process is as follows:

[0015] .

[0016] A third aspect of the invention provides the use of the aforementioned tau protein-responsive near-infrared chemiluminescent probe in the preparation of in vivo brain tau protein imaging agents.

[0017] The beneficial effects of the technical solution of the present invention are as follows: The chemiluminescent probe designed in this invention achieves near-infrared chemiluminescence capability through an extended conjugated structure (obtained through a second reaction, where carbon-carbon double bonds form a large conjugated structure). (600 nm); Simultaneously, high selectivity for tau protein was achieved by introducing a pyridine group (obtained through the first reaction). Combined with chemiluminescence imaging, the near-infrared chemiluminescent probe designed in this invention can detect tau protein in the brain of AD mice, providing a reliable chemical tool for the early diagnosis of tau protein. Specifically:

[0018] 1. The chemiluminescent probe of this invention has high selectivity; compared to A 40 and -syn aggregates; the fluorescence intensity of the probe of this invention is enhanced 10-fold after binding to tau aggregates, exhibiting specificity and selectivity; simultaneously, the chemiluminescent probe of this invention has near-infrared chemiluminescence capability, with a maximum emission wavelength of 605 nm in dimethyl sulfoxide; the chemiluminescent probe of this invention binds to A... 40 After aggregation, the chemiluminescence intensity is enhanced by nearly 7 times, which can be used for in vivo "on-screen" chemiluminescence imaging; it has good stability under physiological pH conditions, which is conducive to in vivo in situ A Imaging.

[0019] 2. The preparation method of the near-infrared chemiluminescence probe provided by the present invention uses readily available raw materials, mild and easily controllable reaction conditions, saves reaction costs, and ensures the yield of the target product.

[0020] 3. The near-infrared chemiluminescent probe provided by this invention has a high efficiency of chemiluminescence and can be used to detect tau protein in the brain of AD mice, providing a potential tool for the diagnosis of AD disease.

[0021] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0022] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the invention.

[0023] Figure 1 The NMR spectrum of compound (VI) obtained by the method for preparing a tau protein-responsive near-infrared chemiluminescent probe provided in Example 1 of the present invention is shown.

[0024] Figure 2 The liquid chromatography-mass spectra of the near-infrared chemiluminescent probe that responds to tau protein obtained in Example 1 of the present invention are shown.

[0025] Figure 3 The chemiluminescence spectrum and fluorescence intensity change spectrum of the near-infrared chemiluminescent probe responding to tau protein obtained in Example 1 of the present invention are shown, wherein... Figure 3 In Figure 'a', the chemiluminescence spectrum of the luminescent probe is shown. Figure 3 Figure b shows the fluorescence intensity changes of the chemiluminescent probe before and after binding to different protein aggregates in the test solution.

[0026] Figure 4The chemiluminescence imaging spectrum and intensity quantification analysis diagram of the near-infrared chemiluminescent probe responding to tau protein obtained in Example 1 of the present invention are shown, wherein, Figure 4 Figure 'a' shows the chemiluminescence imaging spectra of the luminescent probe before and after binding to different protein aggregates in the test solution; Figure 4 Figure b shows a quantitative analysis of the intensity of the chemiluminescent probe before and after binding to different protein aggregates in the test solution.

[0027] Figure 5 The image shows the staining pattern of the tau protein-responsive near-infrared chemiluminescent probe obtained in Example 1 of the present invention in a brain tissue section of an AD mouse, wherein... Figure 5 In Figure 'a', we see the staining pattern of Tht in a section of brain tissue from an AD mouse. Figure 5 b in the image shows the staining pattern of the luminescent probe in a slice of brain tissue from an AD mouse. Figure 5 c in the figure shows the overlap staining pattern of Thrt and the luminescent probe in brain tissue sections of AD mice.

[0028] Figure 6 The following diagram shows a chemiluminescence imaging image and intensity quantification analysis image of the tau protein-responsive near-infrared chemiluminescent probe obtained in Example 1 of the present invention in the brain of an AD mouse. Figure 6 Figure 'a' shows a chemiluminescent imaging image of the chemiluminescent probe in the brain of an AD mouse. Figure 6 Figure b shows a quantitative analysis of the intensity of the chemiluminescent probe in the brain of AD mice. Detailed Implementation

[0029] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0030] To achieve the above objectives, a first aspect of the present invention provides a near-infrared chemiluminescent probe that responds to tau protein, the structural formula of which is:

[0031] .

[0032] A second aspect of the present invention provides a method for preparing the aforementioned tau protein-responsive near-infrared chemiluminescent probe, comprising the following steps:

[0033] (1) Compound (I), compound (II), catalyst, and basic additive are added to the first solvent to carry out the first reaction to obtain compound (III);

[0034] (2) Compound (III), compound (IV), and piperidine are added to a second solvent to carry out a second reaction to obtain compound (V);

[0035] (3) The compound (V) and trifluoroacetic acid are added to a third solvent to carry out a third reaction to obtain compound (VI);

[0036] (4) The compounds (VI), (VII), and acid compounds are added to the fourth solvent to carry out the fourth reaction, thereby obtaining the near-infrared chemiluminescent probe;

[0037] The reaction process is as follows:

[0038] .

[0039] According to the present invention, preferably, the first solvent, the second solvent, the third solvent and the fourth solvent are each independently selected from at least one of toluene, dioxane, ethanol, dichloromethane and ethanol.

[0040] According to the present invention, preferably, in step (1):

[0041] The catalyst is selected from at least one of 1,1'-bis(diphenylphosphine)ferrocene-palladium(II) dichloromethane complex and tris(dibenzylacetone)dipalladium;

[0042] The alkaline additive is cesium carbonate and / or potassium tert-butoxide;

[0043] The molar ratio of the compounds (I), (II), catalyst, and alkaline additive is 1:(1.05-1.5):(0.05-0.15):(2-4).

[0044] With 1 mmol of the compound (I), the volume of the first solvent is 20-50 mL.

[0045] According to the present invention, preferably, in step (1):

[0046] The conditions for the first reaction include: a reaction temperature of 90-120℃ and a reaction time of 8-12h.

[0047] According to the present invention, preferably, in step (2):

[0048] The molar ratio of compound (III), compound (IV) and piperidine is 1:(1.05-1.2):(0.3-0.6).

[0049] The volume of the second solvent is 5-30 mL, based on 1 mmol of compound (III).

[0050] In this invention, the piperidine, as a weak base, can promote the second reaction (i.e., the condensation reaction).

[0051] According to the present invention, preferably, in step (2):

[0052] The conditions for the second reaction include: a reaction temperature of 70-90℃ and a reaction time of 2-4 hours.

[0053] According to the present invention, preferably, in step (3):

[0054] The molar ratio of the compound (V) to trifluoroacetic acid is 1:(20-40).

[0055] Based on a concentration of 1 mmol of the compound (V), the volume of the third solvent is 5-20 mL;

[0056] The conditions for the third reaction include: a reaction temperature of 20-35℃ and a reaction time of 4-7h.

[0057] According to the present invention, preferably, in step (4):

[0058] The acid compound is hydrochloric acid;

[0059] The molar ratio of the compounds (VI), (VII) and acid compounds is 1:(3-4):(4-5).

[0060] Based on 1 mmol of the compound (VII), the volume of the fourth solvent is 5-10 mL;

[0061] The conditions for the fourth reaction include: a reaction temperature of 70-90℃ and a reaction time of 3-5h.

[0062] In this invention, the reaction solution is evaporated at the end of the fourth reaction to obtain the near-infrared chemiluminescent probe.

[0063] In this invention, the hydrochloric acid is used to improve the acidic environment for the fourth reaction and promote the reaction. From the reaction mechanism, under acidic conditions, compounds (VI) and (VII) first form a Schiff base intermediate and then obtain the final product.

[0064] A third aspect of the invention provides the use of the aforementioned tau protein-responsive near-infrared chemiluminescent probe in the preparation of in vivo brain tau protein imaging agents.

[0065] Preferably, the application of the near-infrared chemiluminescent probe that responds to tau protein in tau protein imaging of the live mouse brain.

[0066] Preferably, the near-infrared chemiluminescent probe that responds to tau protein is used for the detection of tau protein in the brain of a live mouse.

[0067] For any experimental steps or conditions not specified in the examples, the operation or conditions of conventional experimental steps described in the literature in this field can be followed; for any reagents or instruments whose manufacturers are not specified, they are all conventional reagent products that can be obtained commercially.

[0068] In the following embodiments, the device used for chemiluminescence imaging is an IVIS. ® Spectrum CT.

[0069] Example 1

[0070] This embodiment provides a method for preparing a near-infrared chemiluminescent probe that responds to tau protein, comprising the following steps:

[0071] (1) Synthesis of Compound III: Under nitrogen protection, Compound I (1 mmol), Compound II (1.1 mmol), tris(dibenzylacetone)palladium (0.1 mmol), and cesium carbonate (3 mmol) were added to a 50 mL Schlenk tube. Then, 25 mL of anhydrous and oxygen-free toluene solvent was added to the Schlenk tube, and the mixture in the Schlenk tube was stirred at 110 °C and reacted for 12 h. After the reaction was completed, the toluene solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a white oily substance, namely Compound (III) (the eluent was n-hexane:ethyl acetate = 3:1). v / v (Yield: 53%)

[0072] (2) Synthesis of compound V: Under nitrogen protection, compound III (1 mmol) and compound IV (1.1 mmol) were added to a 50 mL Schlenk tube, followed by the addition of piperidine (0.5 mmol) and 10 mL of anhydrous ethanol. The Schlenk tube was then placed in an 80 °C oil bath and stirred for 4 h. After the reaction was complete, the mixture was extracted with dichloromethane, washed three times with saturated brine, dried over anhydrous MgSO4, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain an orange solid, namely compound (V) (eluent: hexane: ethyl acetate = 3:1). v / v (Yield: 80%)

[0073] (3) Synthesis of compound VI: Under nitrogen protection, compound V (1 mmol) and trifluoroacetic acid (30 mmol) were added to a 50 mL Schlenk tube, and then 8 mL of anhydrous dichloromethane solvent was added to the Schlenk tube. The mixture in the Schlenk tube was stirred at 35 °C and reacted for 6 h. After the reaction was completed, the dichloromethane solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a black solid, namely compound (VI) (the eluent was n-hexane:ethyl acetate = 2:1). v / v (Yield: 72%)

[0074] The NMR spectrum of compound (VI) is as follows: Figure 1 As shown, from Figure 1 It can be seen that, 1 H NMR (500 MHz, ) δ / ppm 8.12 (s, 1H), 8.02 (d, J = 5.8 Hz, 1H), 7.94 (s, 1H), 7.83 (d, J = 11.4 Hz, 1H), 7.55 (d, J = 8.3 Hz, 2H), 7.27 – 7.18 (m, 3H), 7.08 (d, J = 15.3 Hz, 1H), 6.85 (d, J = 7.4 Hz, 2H), 2.38 (s, 3H); thus the structure of compound (VI) can be verified.

[0075] (4) Synthesis method of chemiluminescent probe: Under nitrogen protection, compound (VI) (1 mmol) and compound (VII) (3.6 mmol) were added to a 10 mL pressure-resistant tube, and then 3 mL of ethanol and 6 M hydrochloric acid solution (1 mmol) were added to the pressure-resistant tube. The pressure-resistant tube was placed in an oil bath at 70 °C, stirred and reacted for 3 h; after the reaction was completed, the reaction solution was centrifuged and filtered to obtain a black solid, which is the chemiluminescent probe (yield: 62%).

[0076] The liquid chromatography-mass spectra of the chemiluminescent probe obtained in this embodiment are as follows: Figure 2 As shown, from Figure 2 It can be seen that mass spectrometry (ESI positive ion mode):

[0077] The theoretical value (calculated value) is C. 24 H 20 N6O's [M+H] + =409.18;

[0078] The measured value is [M+H]. +=409.20.

[0079] This can verify the purity of the chemiluminescent probe.

[0080] Test Example 1

[0081] This test example demonstrates the fluorescence, chemiluminescence, and selectivity of the chemiluminescent probe obtained in Example 1.

[0082] The chemiluminescent probe obtained in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. A mixture of DMSO and PBS buffer solution (the volume ratio of DMSO to PBS buffer solution was 10 / 90; the concentration of PBS in the PBS buffer solution (phosphate buffer) was 10 mM; the pH of the mixture was 7.0) was used as the test solution. The probe test concentration was 10 μM (i.e., the stock solution was diluted 1000 times with the test solution).

[0083] The chemiluminescence spectral detection results of the chemiluminescence probe obtained in Example 1 are as follows: Figure 3 As shown, from Figure 3 As can be seen from 'a', the maximum emission wavelength of the probe's chemiluminescence is at 605 nm. Figure 3 From b, we can see that compared to A 40 and -syn aggregates, the chemiluminescent probe obtained in Example 1 showed a 10-fold increase in fluorescence intensity after binding with tau aggregates, exhibiting specificity and selectivity.

[0084] The chemiluminescence imaging spectra of the luminescent probe of this invention before and after binding to different protein aggregates in the test solution are shown below. Figure 4 As shown in Figure a, the quantitative analysis of the intensity of the chemiluminescent probe binding to different protein aggregates in the test solution before and after binding is shown in Figure a. Figure 4 As shown in b, by Figure 4 From b, we can see that compared to A 40 and -syn aggregates, the chemiluminescence intensity of the chemiluminescence probe obtained in Example 1 was enhanced by 6.6 times after binding with tau aggregates. Therefore, the chemiluminescence probe of the present invention can be used for in vivo "on-screen" chemiluminescence imaging.

[0085] Figure 5 a in Figure 5 b and Figure 5 c in the diagram shows the staining patterns of Thrt, the luminescent probe, Thrt, and the luminescent probe in brain tissue sections from AD mice, respectively. Figure 5 As shown in 'a', Tht, as a commercial dye, can bind to tau protein in brain tissue slices from AD mice and emit green fluorescence; Figure 5As can be seen from b in Example 1, the chemiluminescent probe obtained in Example 1 exhibits red fluorescence. From... Figure 5 As shown in 'c', the tau proteins in the green and red channels can overlap well, indicating that the probe has a good binding ability to tau proteins in brain tissue slices.

[0086] Test Example 2

[0087] This test case demonstrates the chemiluminescent imaging of the brain of AD mice using the chemiluminescent probe from Example 1.

[0088] Weigh 1 mg of the chemiluminescent probe and dissolve it in 1 mL of injection solution (the injection solution comprises 15% DMSO, 15% castor oil, and 70% PBS buffer solution, based on the total volume) to obtain the probe solution. Inject the probe solution via the tail vein into AD mice at a dose of 4 mg / kg. Approximately 10 minutes later, administer IVIS... ® Spectrum CT for in vivo imaging of mice, such as Figure 6 As shown in 'a', based on the imaging results, we can see that the chemiluminescence signal in the brains of AD mice is significantly stronger than that in wild-type control mice. Figure 6 As shown in b, the intensity of the luminescent probe was increased by 1.7 times in Alzheimer's mice compared to wild-type mice, indicating that the probe has the ability to detect and image tau protein in the brain of live mice.

[0089] The results of tests 1 and 2 above demonstrate that the near-infrared chemiluminescence probe of the present invention has high selectivity and can be used in combination with chemiluminescence imaging to detect tau protein in the brain of live mice, showing great promise for application in the biomedical field.

[0090] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A near-infrared chemiluminescent probe responsive to tau protein, characterized in that, The structural formula of the near-infrared chemiluminescent probe is: 。 2. The method of producing a near-infrared chemiluminescent probe responsive to tau protein according to claim 1, characterized by, Includes the following steps: (1) Compound (I), compound (II), catalyst, and basic additive are added to the first solvent to carry out the first reaction to obtain compound (III); (2) Compound (III), compound (IV), and piperidine are added to a second solvent to carry out a second reaction to obtain compound (V); (3) The compound (V) and trifluoroacetic acid are added to a third solvent to carry out a third reaction to obtain compound (VI); (4) The compounds (VI), (VII), and acid compounds are added to the fourth solvent to carry out the fourth reaction, thereby obtaining the near-infrared chemiluminescent probe; The reaction process is as follows: 。 3. The production method according to claim 2, wherein, The first solvent, the second solvent, the third solvent, and the fourth solvent are each independently selected from at least one of toluene, dioxane, dichloromethane, and ethanol.

4. The preparation method according to claim 2, wherein, In step (1): The catalyst is selected from at least one of 1,1'-bis(diphenylphosphine)ferrocene-palladium(II) dichloromethane complex and tris(dibenzylacetone)dipalladium; The alkaline additive is cesium carbonate and / or potassium tert-butoxide; The molar ratio of the compounds (I), (II), catalyst, and alkaline additive is 1:(1.05-1.5):(0.05-0.15):(2-4). With 1 mmol of the compound (I), the volume of the first solvent is 20-60 mL.

5. The preparation method according to claim 2, wherein, In step (1): The conditions for the first reaction include: a reaction temperature of 90-120℃ and a reaction time of 8-12h.

6. The preparation method according to claim 2, wherein, In step (2): The molar ratio of compound (III), compound (IV) and piperidine is 1:(1.05-1.2):(0.3-0.6). The volume of the second solvent is 5-30 mL, based on 1 mmol of compound (III).

7. The preparation method according to claim 2, wherein, In step (2): The conditions for the second reaction include: a reaction temperature of 70-90℃ and a reaction time of 2-4 hours.

8. The preparation method according to claim 2, wherein, In step (3): The molar ratio of the compound (V) to trifluoroacetic acid is 1:(20-40). Based on a concentration of 1 mmol of the compound (V), the volume of the third solvent is 5-20 mL; The conditions for the third reaction include: a reaction temperature of 20-35℃ and a reaction time of 4-7h.

9. The preparation method according to claim 2, wherein, In step (4): The acid compound is hydrochloric acid; The molar ratio of the compounds (VI), (VII) and acid compounds is 1:(3-4):(4-5). Based on 1 mmol of the compound (VII), the volume of the fourth solvent is 5-10 mL; The conditions for the fourth reaction include: a reaction temperature of 70-90℃ and a reaction time of 3-5h.

10. The use of the tau protein-responsive near-infrared chemiluminescent probe of claim 1 in the preparation of a live brain tau protein imaging agent.

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