A chemiluminescent probe responsive to beta amyloid and preparation method and application thereof

Abstract

The application belongs to the technical field of biochemistry, and discloses a chemiluminescent probe responding to beta amyloid protein, a preparation method and application. The structural formula of the probe is as follows: the probe designed in the application realizes near-infrared chemiluminescence capability (>650nm) through an extended conjugated structure; and realizes high selectivity to A beta by introducing a heteroatom conjugated pi bridge.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry, and more specifically, relates to a method for... Amyloid protein-responsive chemiluminescent probes, their preparation methods, and applications. Background Technology

[0002] Optical imaging has been widely used in preclinical and clinical research. Among the many optical imaging modalities, near-infrared fluorescence imaging is one of the most commonly used techniques in preclinical research. However, fluorescence imaging has some inherent limitations, primarily caused by the excitation light. First, for biological samples, the excitation light excites not only the fluorescent probe but also other fluorophore-containing molecules in the sample, resulting in troublesome tissue autofluorescence. Second, due to the Stokes shift, the excitation wavelength is always much shorter than the emission wavelength, and the relatively short excitation wavelength is detrimental to the penetration of biological tissues. Finally, due to the inadequacy of filters in the imaging system, fluorescent probes with small Stokes shifts are interfered with by excitation light leakage in actual imaging. Therefore, these excitation light-related limitations lead to a lower signal-to-noise ratio (SNR) and poor tissue penetration. In contrast, chemiluminescence imaging can partially solve the problems of fluorescence imaging because it does not require external excitation light. Furthermore, compared to fluorescence imaging, chemiluminescence imaging can provide better tissue penetration at the same emission wavelength. Recent studies have shown that chemiluminescence imaging at a wavelength of 800 nm can achieve tissue penetration of 4 cm, while fluorescence imaging cannot achieve this penetration depth at the same emission wavelength.

[0003] Although chemiluminescence imaging has been extensively studied in a variety of diseases, including cancer and diabetes, its application in Alzheimer's disease (AD) remains limited. amyloid-β (abbreviated as A) However, applications of A are rare. Ran's research group has done pioneering work in this field, developing a method for... The probes ADLumin-1 and ADLumin-5 are protein-activated chemiluminescent probes and are used for three-dimensional imaging of the brain in Alzheimer's disease (AD) mice. However, it should be noted that current probes for brain chemiluminescence imaging still face problems such as poor specificity and low imaging resolution. Furthermore, the main challenges in designing chemiluminescent probes include: 1) the short wavelength of small-molecule chemiluminescence leads to limited penetration depth into biological tissues, thus affecting imaging resolution; 2) [The text abruptly shifts to a different topic:] ...a protein-activated chemiluminescent probe ADLumin-1 and ADLumin-5 are used for three-dimensional imaging of the brain in Alzheimer's disease (AD) mice. Poor selectivity, unable to accurately identify A Spatial distribution information.

[0004] Therefore, there is an urgent need to propose a method for... Amyloid protein-responsive chemiluminescent probes, their preparation methods, and applications. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a solution that... Amyloid protein-responsive chemiluminescent probes, their preparation methods, and applications. The probes designed in this invention achieve near-infrared chemiluminescence capabilities through an extended conjugated structure. 650nm); and simultaneously by introducing heteroatom conjugation The bridge enables access to A High selectivity.

[0006] To achieve the above objectives, the first aspect of the present invention provides a method for... A chemiluminescent probe responsive to amyloid protein, the structural formula of which is:

[0007] .

[0008] A second aspect of this invention provides a method for preparing the chemiluminescent probe, comprising the following steps:

[0009] (1) Compound (I), compound (II) and catalyst were added to the first solvent to carry out the first reaction to obtain compound (III);

[0010] (2) Compound (IV), compound (V), and sodium hydroxide were added to the second solvent to carry out the second reaction, yielding compound (VI);

[0011] (3) The compound (VI), the compound (III), and the catalyst are added to a third solvent to carry out a third reaction to obtain compound (VII);

[0012] (4) Compound (VII), compound (VIII), and hydrochloric acid are added to the fourth solvent to carry out the fourth reaction, thereby obtaining the chemiluminescent probe;

[0013] The reaction process is as follows:

[0014] .

[0015] According to the present invention, preferably, the first solvent, the second solvent, the third solvent and the fourth solvent are each independently at least one selected from dioxane, methanol, toluene and ethanol.

[0016] According to the present invention, preferably, in step (1): the catalyst is 1,1'-bis(diphenylphosphine)ferrocene-palladium(II)dichloromethane complex (CAS No. 95464-05-4) and / or tetra(triphenylphosphine)palladium;

[0017] The molar ratio of compound (I), compound (II) and catalyst is 1:(1.1-1.5):(0.05-0.1).

[0018] Based on 1 mol of the compound (I), the volume of the first solvent is 50-100 mL, meaning that when the amount of the compound (I) is 1 mol, the volume of the first solvent is 50-100 mL.

[0019] The conditions for the first reaction include: a reaction temperature of 90-120℃ and a reaction time of 8-12h.

[0020] According to the present invention, preferably, in step (2):

[0021] The molar ratio of the compounds (IV), (V), and sodium hydroxide is 1:(1.1-1.2):(3-4).

[0022] Based on 1 mol of the compound (IV), the volume of the second solvent is 40-60 mL;

[0023] The conditions for the second reaction include: a reaction temperature of 40-60℃ and a reaction time of 15-24h.

[0024] In this invention, the sodium hydroxide, as a strong base, can promote the second reaction (i.e., the condensation reaction).

[0025] According to the present invention, preferably, in step (3):

[0026] The catalyst is a 1,1'-bis(diphenylphosphine)ferrocene-palladium(II)dichloromethane complex and / or tetra(triphenylphosphine)palladium;

[0027] The molar ratio of compound (VI), compound (III) and catalyst is 1:(1.0-1.5):(0.05-0.1).

[0028] Based on 1 mol of the compound (VI), the volume of the third solvent is 40-60 mL;

[0029] The conditions for the third reaction include: a reaction temperature of 90-120℃ and a reaction time of 6-10h.

[0030] According to the present invention, preferably, in step (4):

[0031] The molar ratio of the compounds (VII), (VIII) and hydrochloric acid is 1:(3-4):(4-5).

[0032] Based on 1 mol of the compound (VII), the volume of the fourth solvent is 5-10 mL;

[0033] The conditions for the fourth reaction include: a reaction temperature of 70-90℃, a reaction time of 3-5h, and after the reaction is completed, the reaction solution is dried by rotary evaporation to obtain the chemiluminescent probe.

[0034] 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 (VII) and (VIII) first form a Schiff base intermediate and then obtain the final product.

[0035] A third aspect of the present invention provides the chemiluminescent probe described above for the preparation of living brain tissue. Applications of amyloid imaging agents.

[0036] The fourth aspect of the present invention provides the chemiluminescent probe in the brain of a live mouse. Applications in amyloid imaging.

[0037] According to the present invention, preferably, the chemiluminescent probe is applied to the brain of a live mouse. Amyloid protein tracing.

[0038] In this invention, the AD mouse refers to a mouse model of Alzheimer's disease (AD).

[0039] 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). 650nm); and simultaneously by introducing heteroatom conjugation The bridge (obtained through a second reaction, where heteroatoms refer to nitrogen, oxygen, and sulfur atoms) enables the connection of A... High selectivity. Combined with chemiluminescence three-dimensional imaging, the chemiluminescent probe designed in this invention can target A in the brain of AD mice. Visualizing the spatial distribution information of A to clarify A This provides a reliable chemical tool for understanding the pathogenesis of Alzheimer's disease. Specifically:

[0040] 1. The chemiluminescent probe of this invention has high selectivity; compared with Tau and -syn aggregates, the probe of this invention and A 40The fluorescence intensity increased 42-fold after aggregation, demonstrating specificity and selectivity; simultaneously, the chemiluminescent probe of this invention exhibits near-infrared chemiluminescence capability with a maximum emission wavelength of 660 nm; the chemiluminescent probe of this invention binds to A... 40 After the aggregates bind, the chemiluminescence intensity is enhanced by nearly 10 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.

[0041] 2. The chemiluminescent probe preparation method provided by this invention uses readily available raw materials, has mild and easily controllable reaction conditions, saves reaction costs, and ensures the yield of the target product.

[0042] 3. The chemiluminescent probe provided by this invention has highly efficient chemiluminescence capability and can be used for A in the brain of AD mice. Tracing. Combined with three-dimensional imaging, the chemiluminescent probe of this invention can provide A in the brain of AD mice. Spatial distribution information for A It provides a reliable means for imaging and detection, and offers a promising tool for the diagnosis of AD disease.

[0043] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0044] 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.

[0045] Figure 1 The fluorescence spectrum of the chemiluminescent probe obtained in Example 1 of the present invention is shown.

[0046] Figure 2 The chemiluminescence spectrum of the chemiluminescent probe obtained in Example 1 of the present invention is shown.

[0047] Figure 3 The fluorescence intensity changes of the chemiluminescent probe obtained in Example 1 of the present invention before and after binding to different protein aggregates in the test solution are shown.

[0048] Figure 4 The chemiluminescent probe obtained in Example 1 of the present invention and Aβ are shown. 40 Chemiluminescence imaging images before and after aggregate binding.

[0049] Figure 5 The image shows a 3D chemiluminescent image of the chemiluminescent probe obtained in Example 1 of the present invention in the brain of an AD mouse. Detailed Implementation

[0050] 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. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0051] 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.

[0052] In the following embodiments, the apparatus used for chemiluminescence imaging and in vivo 3D imaging is an IVIS. ® SpectrumCT.

[0053] Example 1

[0054] This embodiment provides a method for... The preparation method of amyloid protein-responsive chemiluminescent probe includes the following steps:

[0055] (1) Synthesis of Compound III: Under nitrogen protection, Compound I (1 mmol), Compound II (1.1 mmol), and tetrakis(triphenylphosphine)palladium (0.1 mmol) were added to a 50 mL Schlenk tube. Then, 20 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 16 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 = 5:1). v / v (Yield: 63%)

[0056] (2) Synthesis of compound VI: Under nitrogen protection, compound IV (1 mmol) and compound V (1.1 mmol) were added to a 50 mL Schlenk tube. Then, sodium hydroxide (3 mmol) and anhydrous methanol (10 mL) were added to the Schlenk tube. The Schlenk tube was placed in an oil bath at 50 °C and stirred for 20 h. After the reaction was completed, the mixture was extracted with dichloromethane, washed three times with saturated brine, dried with anhydrous MgSO4, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain an orange solid, namely compound (VI) (eluent: hexane: ethyl acetate = 3:1). v / v (Yield: 63%)

[0057] (3) Synthesis of compound VII: Under nitrogen protection, compound VI (1 mmol), compound III (1.5 mmol), and tetrakis(triphenylphosphine)palladium (0.1 mmol) were added to a 50 mL Schlenk tube. Then, 20 mL of anhydrous and oxygen-free toluene solvent was added to the Schlenk tube, and the mixture in the Schlenk tube was stirred at 100 °C and reacted for 6 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 black solid, namely compound (VII) (the eluent was n-hexane:ethyl acetate = 2:1). v / v (Yield: 78%)

[0058] The NMR data for compound (VII) are as follows: 1 H NMR (500 MHz, CDCl3) δ / ppm 8.08 (s, 1H), 8.03 (s, 1H), 7.97 – 7.91 (m, 2H), 7.66 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 15.9 Hz, 1H), 7.40–7.29 (m, 3H), 7.11 (d, J = 15.9 Hz, 1H), 6.77 (s, 1H), 6.73 (s, 1H), 6.68 (d, J = 8.2 Hz, 1H), 4.65 (s, 2H), 3.43 (q, J = 6.8 Hz, 4H), 1.22 (t, J = 7.0 Hz, 6H); This verifies the structure of compound (VII).

[0059] (4) Synthesis method of chemiluminescent probe: Under nitrogen protection, compound (VII) (1 mmol) and compound (VIII) (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: 52%).

[0060] As can be seen from the liquid chromatography-mass spectrometry (LC-MS) spectrometry of the chemiluminescent probe obtained in this embodiment, the mass spectrometry (ESI positive ion mode) shows:

[0061] The theoretical value (calculated value) is C. 30 H 27 [M+H] of N5O2S + =522.20;

[0062] The measured value is [M+H]. +=522.30.

[0063] This can verify the purity of the chemiluminescent probe.

[0064] Test Example 1

[0065] This test example demonstrates the fluorescence, chemiluminescence, and selectivity of the chemiluminescent probe obtained in Example 1.

[0066] 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).

[0067] The fluorescence and chemiluminescence spectral detection results of the chemiluminescent probe obtained in Example 1 are as follows: Figure 1 and Figure 2 As shown, from Figure 1 and Figure 2 It can be seen that the maximum emission wavelengths of the probe's fluorescence and chemiluminescence are at 635 and 660 nm, respectively.

[0068] The selective spectrum of the chemiluminescent probe obtained in Example 1 is shown below. Figure 3 As shown, from Figure 3 It can be seen that, compared to Tau and a-syn aggregates, the chemiluminescent probe obtained in Example 1 is similar to Aβ. 40 The fluorescence intensity increased 42-fold after aggregation, demonstrating specificity and selectivity; moreover, the chemiluminescent probe obtained in Example 1 was compatible with Aβ. 40 The response changes of aggregate binding are as follows Figure 4 As shown, from Figure 4 As can be seen from the above, the chemiluminescent probe obtained in Example 1 is compatible with Aβ. 40 After the aggregates bind, the chemiluminescence intensity increases by nearly 10 times, indicating that the chemiluminescence probe of the present invention can be used for in vivo "on-screen" chemiluminescence imaging.

[0069] Test Example 2

[0070] This test case demonstrates 3D imaging of the chemiluminescent probe in the brain of AD mice using Example 1.

[0071] 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 is used for in vivo three-dimensional imaging of mice, such as... Figure 5 As shown, from Figure 5 As can be seen from the 3D imaging results, the chemiluminescent signals in the cerebral cortex and hippocampus of AD mice are stronger, indicating that these two regions have a greater accumulation of Ab aggregates. Furthermore, from... Figure 5 Furthermore, different distributions of chemical signals can be observed directly within the brain contour, demonstrating that the chemiluminescent probe of this invention has high resolution.

[0072] The results of tests 1 and 2 above demonstrate that the chemiluminescent probe of the present invention has high selectivity and, combined with 3D imaging, can be used for in vivo mouse brain A. ( Tracing amyloid protein has great potential applications in the biomedical field.

[0073] 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 chemiluminescent probe responsive to β-amyloid protein, characterized in that, The structural formula of the probe is: 。 2. The method for preparing the chemiluminescent probe according to claim 1, characterized in that, Includes the following steps: (1) Compound (I), compound (II) and catalyst were added to the first solvent to carry out the first reaction to obtain compound (III); (2) Compound (IV), compound (V), and sodium hydroxide were added to the second solvent to carry out the second reaction, yielding compound (VI); (3) The compound (VI), the compound (III), and the catalyst are added to a third solvent to carry out a third reaction to obtain compound (VII); (4) Compound (VII), compound (VIII), and hydrochloric acid are added to the fourth solvent to carry out the fourth reaction, thereby obtaining the chemiluminescent probe; The reaction process is as follows: 。 3. The method for preparing the chemiluminescent probe according to claim 2, wherein, The first solvent, the second solvent, the third solvent, and the fourth solvent are each independently at least one of dioxane, methanol, toluene, and ethanol.

4. The method for preparing the chemiluminescent probe according to claim 2, wherein, In step (1): The catalyst is a 1,1'-bis(diphenylphosphine)ferrocene-palladium(II)dichloromethane complex and / or tetra(triphenylphosphine)palladium; The molar ratio of compound (I), compound (II) and catalyst is 1:(1.1-1.5):(0.05-0.1). Based on 1 mol of the compound (I), the volume of the first solvent is 50-100 mL; The conditions for the first reaction include: a reaction temperature of 90-120℃ and a reaction time of 8-12h.

5. The method for preparing the chemiluminescent probe according to claim 2, wherein, In step (2): The molar ratio of the compounds (IV), (V), and sodium hydroxide is 1:(1.1-1.2):(3-4). Based on 1 mol of the compound (IV), the volume of the second solvent is 40-60 mL; The conditions for the second reaction include: a reaction temperature of 40-60℃ and a reaction time of 15-24h.

6. The method for preparing the chemiluminescent probe according to claim 2, wherein, In step (3): The catalyst is a 1,1'-bis(diphenylphosphine)ferrocene-palladium(II)dichloromethane complex and / or tetra(triphenylphosphine)palladium; The molar ratio of compound (VI), compound (III) and catalyst is 1:(1.0-1.5):(0.05-0.1). Based on 1 mol of the compound (VI), the volume of the third solvent is 40-60 mL; The conditions for the third reaction include: a reaction temperature of 90-120℃ and a reaction time of 6-10h.

7. The method for preparing the chemiluminescent probe according to claim 2, wherein, In step (4): The molar ratio of the compounds (VII), (VIII) and hydrochloric acid is 1:(3-4):(4-5). Based on 1 mol 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℃, a reaction time of 3-5h, and the reaction solution being evaporated at the end of the reaction to obtain the chemiluminescent probe.

8. The use of the chemiluminescent probe of claim 1 in the preparation of an in vivo brain β-amyloid imaging agent.

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