A near-infrared fluorescent probe specifically targeting Aβ42, its preparation method and application

Abstract

This invention belongs to the field of biochemistry and relates to a near-infrared fluorescent probe specifically targeting A42, its preparation method, and its application. The preparation method of the near-infrared fluorescent probe specifically targeting A42 includes the following steps: adding compound (I), compound (II), and piperidine to a solvent and reacting to obtain the near-infrared fluorescent probe. The fluorescent probe preparation method provided by this invention uses readily available raw materials, requires only one reaction step, and employs mild and easily controllable reaction conditions, saving reaction costs and ensuring the yield of the target product. The fluorescent probe provided by this invention possesses highly efficient microscopic imaging capabilities and can be used for staining and imaging of A42 aggregates in ex vivo tissue sections, providing a reliable means for A42 detection and a promising tool for the diagnosis of Alzheimer's disease (AD).

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically, it relates to a method for treating A 42. Targeted near-infrared fluorescent probes, their preparation methods and applications. Background Technology

[0002] -Amyloid protein (A) Plaques, a key pathological feature of Alzheimer's disease (AD), have been studied for over a century since their first discovery in autopsy brain tissue of AD patients. However, regarding A... The exact role of plaques in the progression of Alzheimer's disease remains highly controversial in the academic community, mainly because there is a lack of significant correlation between plaque deposition burden (including number and area) and the severity of cognitive decline in patients. Although the composition of plaques is relatively clear, mainly composed of A... 40 and A 42. Composed of two subtypes, but with significant differences in pathological effects: Unlike the plaque as a whole, whose role is still controversial, the academic community almost unanimously agrees that A The neurotoxicity of 42 is much higher than that of A. 40.

[0003] Therefore, it can be inferred that if A can be achieved 40 and A The precise distinction of 42 will greatly advance our understanding of A. Understanding the role of plaques in the pathological progression of Alzheimer's disease (AD). However, small molecule probes with this distinguishing ability are currently extremely rare. Because A... 40 and A 42 differ only slightly in their amino acid sequences, and developing probes capable of specifically recognizing both has long been considered a near-impossible task. In previous studies, researchers were hampered by the challenge of antibody recognition of soluble and insoluble A... Inspired by this mechanism, a series of methods capable of selectively detecting soluble A have been successfully designed. Fluorescent molecules provide an important tool for early Alzheimer's disease (AD) pathology research. Based on the antibody's ability to accurately distinguish A... 40 and A Based on the capability of 42, we further hypothesize that it might be possible to achieve control over these two A molecules by designing appropriate small molecule probes. The differentiation of subtypes opens up new research avenues for revealing the relationship between plaque heterogeneity and the pathological mechanisms of Alzheimer's disease (AD). Summary of the Invention

[0004] The purpose of this invention is to solve the aforementioned problems existing in the prior art. The purpose of this invention is to address the shortcomings of the prior art by proposing a solution for A. 42. Specific Targeted Near-Infrared Fluorescent Probes, Preparation Methods, and Applications. The probes designed in this invention achieve near-infrared fluorescence emission through extended conjugated structures and strong intramolecular charge transfer interactions. 700nm).

[0005] To achieve the above objectives, the first aspect of the present invention provides a method for dealing with A. 42. A specifically targeted near-infrared fluorescent probe, the structural formula of which is: .

[0006] The second aspect of the invention provides the aforementioned method for A. 42. A method for preparing a specifically targeted near-infrared fluorescent probe, characterized by comprising the following steps: Compound (I), compound (II), and piperidine were added to a solvent and reacted to obtain the near-infrared fluorescent probe. The reaction process is as follows: .

[0007] The third aspect of the invention provides the aforementioned A 42 Application of specific-targeting near-infrared fluorescent probes in the preparation of imaging agents for brain tissue sections of AD mice.

[0008] The fourth aspect of the present invention provides the aforementioned A 42 Application of specific-targeting near-infrared fluorescent probes in imaging brain tissue sections of AD mice.

[0009] The present invention has the following beneficial effects: The beneficial effects of the technical solution of this invention are as follows: The near-infrared fluorescent probe designed in this invention achieves near-infrared luminescence capability through its extended conjugated structure and strong intramolecular charge transfer (obtained through a reaction, where carbon-carbon double bonds form a large conjugated structure, and a strong intramolecular charge transfer interaction is formed between the benzofuran group and the 2-cyanomethylbenzothiazole group). (700nm); Meanwhile, this fluorescent probe showed efficacy against A in in vitro spectroscopy experiments. The 42 aggregates exhibit high selectivity. Combined with tissue section staining experiments, the fluorescent probe designed in this invention can specifically target A. 42 aggregates, specifically targeting A Antibodies with 42 aggregates exhibit excellent co-localization effects, which helps distinguish A. 40 and A 42 provides reliable chemical tools. Specifically: 1. The near-infrared fluorescent probe of this invention has high selectivity; compared to A 40 oligomers, A 40 aggregates and A 42 oligomers, the probe of this invention and A The fluorescence intensity increased nearly 15-fold after binding to the 42 aggregates, exhibiting specificity and selectivity. It can also be used for in vivo "on-screen" fluorescence imaging. Furthermore, the chemiluminescent probe of this invention possesses near-infrared chemiluminescence capability with a maximum emission wavelength of 730 nm. It exhibits good stability under physiological pH conditions, facilitating in-situ in vivo A... Imaging.

[0010] 2. The fluorescent probe preparation method provided by the present invention uses readily available raw materials, requires only one reaction step, and has mild and easily controllable reaction conditions, saving reaction costs and ensuring the yield of the target product.

[0011] 3. The fluorescent probe provided by this invention has efficient microscopic imaging capabilities and can be used for A in isolated tissue sections. 42 aggregates were stained and imaged, for A The detection of 42 provides a reliable means and offers a promising tool for the diagnosis of AD disease.

[0012] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0013] 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.

[0014] Figure 1 The near-infrared fluorescent probe obtained in Example 1 of the present invention is shown in the 1H NMR spectrum.

[0015] Figure 2 This illustrates the near-infrared fluorescent probe obtained in Example 1 of the present invention reacting with different A molecules in the test solution. Fluorescence intensity changes before and after protein isoform binding.

[0016] Figure 3 The near-infrared fluorescent probe obtained in Example 1 of the present invention and A are shown. 40 aggregates and A Simulated molecular docking diagram after 42-component bonding.

[0017] Figure 4 The following is a fluorescence imaging image of the near-infrared fluorescent probe obtained in Embodiment 1 of the present invention, wherein, Figure 4 The left image shows a specific target A. Image showing the staining effect of antibody aggregate 42 in brain tissue sections of AD mice (green fluorescent channel). Figure 4 The middle image shows the staining and imaging effect of the near-infrared fluorescent probe obtained in Example 1 in a brain tissue section of an AD mouse (red fluorescent channel). Figure 4 The right image in the image shows a specific target A. Image showing the effect of antibody and near-infrared fluorescent probe overlapping staining on 42 aggregates. Detailed Implementation

[0018] 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.

[0019] The first aspect of the present invention provides a method for dealing with A 42. A specifically targeted near-infrared fluorescent probe, the structural formula of which is: .

[0020] The second aspect of the invention provides the aforementioned method for A. 42. A method for preparing a specifically targeted near-infrared fluorescent probe, characterized by comprising the following steps: Compound (I), compound (II), and piperidine were added to a solvent and reacted to obtain the near-infrared fluorescent probe. The reaction process is as follows: .

[0021] According to the present invention, preferably, the solvent is anhydrous ethanol and / or anhydrous toluene.

[0022] According to the present invention, preferably, the molar ratio of compound (I), compound (II) and piperidine is 1:(1.1-1.2):(0.3-0.5).

[0023] According to the present invention, preferably, the volume of the solvent is 8-30 mL, based on 1 mol of the compound (I).

[0024] According to the present invention, preferably, the reaction conditions include: a reaction temperature of 70-90°C and a reaction time of 2-8 hours.

[0025] According to the present invention, preferably, the preparation method further includes: after the reaction is completed, extracting with an extractant to separate the organic phase, and then washing and drying the organic phase to obtain the near-infrared fluorescent probe.

[0026] According to the present invention, preferably, the extractant is dichloromethane and / or ethyl acetate; The washing process uses a saturated sodium chloride solution; The drying process employs a desiccant, which is anhydrous magnesium sulfate and / or anhydrous sodium sulfate.

[0027] The third aspect of the invention provides the aforementioned A 42 Application of specific-targeting near-infrared fluorescent probes in the preparation of imaging agents for brain tissue sections of AD mice.

[0028] The fourth aspect of the present invention provides the aforementioned A 42 Application of specific-targeting near-infrared fluorescent probes in imaging brain tissue sections of AD mice.

[0029] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0030] The device used for fluorescence imaging in the microscopy example is a Zeiss Axio Vert.A1.

[0031] Example 1

[0032] This embodiment provides a method for A The preparation method of 42 specific-targeting near-infrared fluorescent probes includes the following steps: Synthesis of Compound III: Under nitrogen protection, Compound I (1 mmol) and Compound II (1.1 mmol) were added to a 50 mL Schlenk tube. Then, piperidine (0.5 mmol) and 10 mL of anhydrous ethanol were added to the Schlenk tube. The tube was 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, and then dried over anhydrous MgSO4. The resulting orange solid, compound (III), was obtained by filtration (yield: 80%). Its 1H NMR spectrum is shown below. Figure 1 As shown: The NMR data for compound (III) are as follows: 1 H NMR (500 MHz, CDCl3) δ / ppm 8.02 (d, J = 8.2 Hz, 1H), 7.94 (d, J =12.1 Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.51–7.47 (m, 1H), 7.37 (ddd, J=14.5, 7.8, 2.5 Hz, 3H), 6.99 (d, J = 14.7 Hz, 1H), 6.84 (s, 1H), 6.71 (s,1H), 6.67 (dd, J = 8.8, 2.2 Hz, 1H), 3.44 (q, J = 7.1 Hz, 4H), 1.22 (t, J =7.1 Hz, 6H); this verifies the structure of compound (III).

[0033] Test Example 1

[0034] This test example demonstrates the fluorescence and selectivity of the chemiluminescent probe obtained in Example 1: The near-infrared fluorescent 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).

[0035] The fluorescence spectrum detection results of the near-infrared fluorescent probe obtained in Example 1 are as follows: Figure 2 As shown, from Figure 2 It can be seen that the maximum fluorescence emission wavelength of the probe is at 730 nm. Compared to A 40 oligomers, A 40 aggregates and A 42 oligomers, the probe of this invention and A The fluorescence intensity increased nearly 15-fold after binding to the 42 aggregates, exhibiting specificity and selectivity, and can also be used for "on-screen" fluorescence imaging; moreover, the near-infrared fluorescent probe obtained in Example 1 is compatible with A 40 and A 42. Simulated conformation diagram and predicted bonding force values ​​during bonding are as follows: Figure 3 As shown, from Figure 3 As can be seen from this, the probe and A When 42 is combined with A 40 has a smaller predicted binding force value, indicating that the probe binds to A. 42 has a stronger binding force.

[0036] Test Example 2

[0037] This test case demonstrates the fluorescence imaging of the near-infrared fluorescent probe from Example 1 in brain tissue sections from AD mice.

[0038] Weigh 0.5 mg of the near-infrared fluorescent probe and dissolve it in 10 mL of incubation solution (based on the total volume of the incubation solution, including 5% DMSO and 95% PBS buffer solution) to obtain the probe solution. Co-incubate the probe solution with AD mouse brain tissue sections. After approximately 15 minutes, perform fluorescence imaging using a Zeiss Axio Vert. A1 fluorescence microscope, as shown... Figure 4 As shown, where, Figure 4 The left image shows a specific target A. Image showing the staining effect of antibody aggregate 42 in brain tissue sections of AD mice (green fluorescent channel). Figure 4 The middle image shows the staining and imaging effect of the near-infrared fluorescent probe obtained in Example 1 in a brain tissue section of an AD mouse (red fluorescent channel). Figure 4 The right image in the image shows a specific target A. Image showing the effect of antibody-near-infrared fluorescent probe overlap staining of 42 aggregates. Figure 4 As shown in the right image, based on the imaging results, we can see that the red signal channel is related to A. The 42 antibodies (green signal channel) almost completely overlap, indicating that the fluorescent probe of this invention has high A... 42. Targeting and imaging capabilities.

[0039] The results of tests 1 and 2 above demonstrate that the near-infrared fluorescent probe of the present invention exhibits good resistance to A in both in vitro verification and tissue imaging experiments. With its high selectivity and fluorescence imaging capabilities, it has great potential for application in the biomedical field.

[0040] 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 method for dealing with A 42. A specifically targeted near-infrared fluorescent probe, characterized in that, The structural formula of the probe is: 。 2. The method for A as described in claim 1 42. A method for preparing a specifically targeted near-infrared fluorescent probe, characterized in that, Includes the following steps: Compound I, compound II, and piperidine were added to a solvent and reacted to obtain the near-infrared fluorescent probe. The reaction process is as follows: 。 3. The preparation method according to claim 2, wherein, The solvent is anhydrous ethanol and / or anhydrous toluene.

4. The preparation method according to claim 2, characterized in that, The molar ratio of compound I, compound II and piperidine is 1:(1.1-1.2):(0.3-0.5).

5. The preparation method according to claim 2, characterized in that, Based on 1 mol of compound I, the volume of the solvent is 8-30 mL.

6. The preparation method according to claim 2, characterized in that, The reaction conditions include: a reaction temperature of 70-90℃ and a reaction time of 2-8h.

7. The preparation method according to claim 2, characterized in that, The preparation method further includes: after the reaction is completed, extracting with an extractant to separate the organic phase, and then washing and drying the organic phase to obtain the near-infrared fluorescent probe.

8. The preparation method according to claim 7, characterized in that, The extractant is dichloromethane and / or ethyl acetate; The washing process uses a saturated sodium chloride solution; The drying process employs a desiccant, which is anhydrous magnesium sulfate and / or anhydrous sodium sulfate.

9. The A according to claim 1 42 Application of specific-targeting near-infrared fluorescent probes in the preparation of imaging agents for brain tissue sections of AD mice.

10. The A according to claim 1 42 Application of specific-targeting near-infrared fluorescent probes in imaging brain tissue sections of AD mice.

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