Blue water-soluble probe for marking A beta plaque as well as preparation method and application of blue water-soluble probe

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a blue water-soluble probe for marking A beta plaques as well as a preparation method and application of the blue water-soluble probe. According to the present invention, after the probe is combined with the A beta fiber, the fluorescence intensity is substantially improved, and the probe almost does not emit light in the aqueous solution so as to avoid the interference caused by the false positive signal of the traditional probe; the probe disclosed by the invention can realize high-resolution in-situ labeling of the A beta plaque, has good imaging performance, can be applied to the aspect of accurate imaging of the whole-brain A beta plaque, and can help researchers to accurately and conveniently detect the existence and distribution of the A beta plaque, so that the probe plays an important role in AD research, diagnosis and treatment development.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a blue water-soluble probe for marking Aβ plaques, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is the main cause of dementia (60%-80%) and the most common neurodegenerative disease, which brings serious economic burden to individuals and society. Amyloid β-protein (Aβ), as the most important pathological marker of AD, is closely related to the pathological development of AD. There are various brain structures such as neurons, glia, nerve fibers and blood vessels around Aβ plaques. Various pathological lesions such as amyloid angiopathy, white matter lesions, ventricular enlargement and brain atrophy related to these brain tissue structures are commonly seen in the clinical images of AD patients. Previous studies have shown that Aβ, as the earliest pathological feature in the AD pathological circuit, has an extremely close correlation with these brain tissue structure lesions. Therefore, in-depth investigation of the interaction between Aβ plaques and various surrounding brain tissue structures is of great significance to understanding the pathological mechanism and progression of AD.

[0003] In the past few decades, various fluorescent materials with near-infrared emission wavelengths (>650nm) have been well developed, such as rare earth-doped upconversion luminescent nanoparticles, quantum dots and organic small molecule phosphors. At the same time, the emerging aggregation-induced emission luminogens (AIEgens) can effectively avoid the aggregation-induced quenching effect of traditional fluorescent dyes. Through mechanisms such as restriction of intramolecular motions (RIM), AIEgens can achieve efficient emission in the aggregated state. However, the current fluorescent probes for Aβ plaques and surrounding related structures are mainly located in the green, red and near-infrared emission ranges, which can easily cause spectral overlap of fluorophores and crossover of fluorescence emission when performing multi-channel labeling.

[0004] Related studies have shown that under laser irradiation at wavelengths of 488nm, 561nm, and 631nm, the somatosensory cortex, auditory cortex, and visual cortex of unstained 3-month-old mice will produce lipofuscin autofluorescence. This autofluorescence phenomenon will overlap with the signal of the fluorescently labeled probe, interfering with the accuracy of the signal, especially when detecting specific proteins in cells or tissue samples, which may affect the accuracy and repeatability of experimental data. In recent years, fluorescent dyes for Aβ imaging have been widely developed, but most of them are concentrated in the above-mentioned bands. Under specific experimental requirements and technical conditions, the presence of autofluorescence may cause these dyes to encounter problems when imaging Aβ, mainly including:

[0005] 1) Reduced signal specificity: When the relevant probes work at wavelengths of 488nm, 561nm, and 631nm, due to the strong autofluorescence of lipofuscin, the detected signal may contain both the fluorescence signal from the probe and the background autofluorescence from lipofuscin, making it difficult for researchers to distinguish between the true Aβ signal and background noise. Especially in brain tissue, the point-like distribution of lipofuscin autofluorescence further increases the possibility of "false positives" in the detection, making the specific detection of the probe more challenging.

[0006] 2) The accuracy of quantitative detection is limited: the presence of autofluorescence will cause the fluorescence signal of the probe to be mixed with more background noise, which cannot accurately reflect the content and distribution of Aβ. In addition, the intensity of autofluorescence varies with age, tissue type and pathological state, further increasing the difficulty of quantitative analysis.

[0007] 3) Reduced sensitivity: Lipofuscin autofluorescence will reduce the overall detection sensitivity when overlapping with the probe signal, especially when Aβ levels are low. The probe signal is weak and can be easily masked by the background fluorescence of lipofuscin, which makes it difficult to detect Aβ in the early stages of the disease or when Aβ aggregation is low.

[0008] Therefore, the development of blue Aβ fluorescent probes is of great significance. It can provide more tools and strategies for the study of Alzheimer's disease in terms of improving imaging resolution, reducing background interference, and realizing multi-channel fluorescent labeling of Aβ plaques and the surrounding environment. This will help promote a deeper understanding of the pathogenesis of AD and may have an important impact on drug development and early diagnosis. Summary of the invention

[0009] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a blue water-soluble probe for marking Aβ plaques and a preparation method and application thereof.

[0010] The object of the present invention is achieved by the following technical solution: A blue water-soluble probe for marking Aβ plaques, wherein the probe has a general structural formula as shown in formula (I):

[0011]

[0012] Wherein, A is any one of the following substituents:

[0013]

[0014] B is any one of the following substituents:

[0015]

[0016]

[0017] n is 2, 3 or 4, Indicates the connection between groups.

[0018] The preparation method of the above-mentioned blue water-soluble probe for marking Aβ plaques has a synthetic route as follows:

[0019]

[0020] Wherein, compound C is any one of the following structures:

[0021]

[0022] Compound E is any one of the following structures:

[0023] Wherein, Y is Cl, Br or I; A is any one of the following substituents:

[0024]

[0025] B is any one of the following substituents:

[0026]

[0027] n is 2, 3 or 4, Indicates the connection between groups.

[0028] As a preferred technical solution, the reaction temperature of the base-catalyzed condensation is 30-60°C, and the reaction time is 5-10 hours.

[0029] As a preferred technical solution, the reaction temperature of the Suzuki coupling is 70-100° C., and the reaction time is 13-20 h.

[0030] As a preferred technical solution, the reaction temperature of the ionization is 60-90°C, and the reaction time is 6-12h.

[0031] The application of the above-mentioned water-soluble probes in obtaining accurate imaging of Aβ plaques in the whole brain.

[0032] As a preferred technical solution, the water-soluble probe is used to fluorescently label Aβ plaques in the brain of AD mice.

[0033] As a preferred technical solution, the probe is a blue water-soluble probe.

[0034] In the structure of the probe of the present invention, the ionization position of the pyridine nitrogen atom is the meta position. In the case of meta-position ionization, the electronic interaction between the pyridine nitrogen atom and other functional groups is weakened. Since the distance between the nitrogen atom and other functional groups at the meta position is relatively far, the electron transferability is relatively weak. The ionization of this structure will cause the electron distribution in the molecule to be more localized, changing the energy difference between the excited state and the ground state of the molecule, which will cause the fluorescence emission of the molecule to be blue-shifted, thereby showing a completely different luminescence phenomenon from the para-ionized structure. At the same time, due to the localization of the electron distribution, the meta-ionized molecule will show a relatively narrow and intense fluorescence spectrum, so that the target molecule obtains fluorescence emission in the blue wavelength range, thereby avoiding the interference of the organism's spontaneous fluorescence and achieving high-performance imaging of Aβ.

[0035] The present invention has the following advantages: The present invention discloses a blue water-soluble probe for labeling Aβ plaques, and a preparation method and application thereof. The fluorescence performance of the probe is optimized by adjusting the structure of its electron-donating and electron-withdrawing groups, and at the same time, by introducing different hydrophilic groups, it has suitable hydrophilic properties, ensuring that it has low background interference during imaging. After such probes are combined with Aβ fibers, the fluorescence intensity is greatly improved, and they hardly emit light in aqueous solution, avoiding the interference caused by the "false positive" signal of traditional probes. In selective experiments, it exhibits excellent anti-interference performance, can achieve specific targeting of Aβ, and has a high affinity for Aβ. In addition, the in vitro imaging results show that the probe of the present invention can achieve high-resolution in situ labeling of Aβ plaques and has good imaging performance. This blue water-soluble probe can help researchers accurately and conveniently detect the presence and distribution of Aβ plaques, thereby playing an important role in the research, diagnosis and treatment development of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Partial fluorescence imaging of Aβ plaques in AD mouse brain slices; (A)(B)(C)(D) are AD-1 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; (E)(F)(G)(H) are AD-4 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; (I)(J)(K)(L) are AD-9 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; (M)(N)(O)(P) are AD-12 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; (Q)(R)(S)(T) are AD-15 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; probe concentration: 5μM, scale bar: 50μm.

[0037] Figure 2This is another part of the fluorescent imaging of Aβ plaques in AD mouse brain slices; in the figure, (A)(B)(C)(D) are AD-19 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; (E)(F)(G)(H) are AD-24 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; (I)(J)(K)(L) are AD-28 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; (M)(N)(O)(P) are AD-32 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; (Q)(R)(S)(T) are AD-36 staining, PD-NA-OH staining, superimposed images and Pearson correlation coefficient diagrams, respectively; probe concentration: 5μM, scale bar: 50μm. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The protection scope of the present invention is not limited to the following: Embodiment 1:

[0039] The blue water-soluble probe described in formula AD1-AD9 has the following synthesis route:

[0040]

[0041] The synthesis method comprises the following steps:

[0042] Weigh compound a and compound b in a molar ratio of 1:1, dissolve in methanol, add a catalytic amount of tetrahydropyrrole catalyst, heat to 40°C for reaction for 8 hours, cool and filter to obtain yellow-green solid c. Weigh c (1 eq.) and compound d (1 eq.), compound e (1 eq.) and compound f (1 eq.) respectively, dissolve in a mixed solvent of water and dioxane (water: dioxane = 1:9), add anhydrous potassium carbonate (2 eq), and then add catalyst bis(triphenylphosphine) palladium dichloride (0.05 eq) under nitrogen protection, heat to 90°C for reaction for 16 hours, concentrate in vacuo, and separate and purify the crude product by silica gel chromatography to obtain brown-gray solid g, yellow-green solid h and yellow-green solid i. Weigh g, h, i (1 eq.) and dissolve them in acetonitrile, add j, k, l, m, n, o, p, q, r (2 eq.) respectively, heat to 80°C and react for 8 hours, cool and filter, and recrystallize and wash with acetonitrile / ethyl acetate to obtain yellow-green compounds AD-1 to AD-9. AD-1: MS (ESI, m / z): 475.13; AD-2: MS (ESI, m / z): 396.18; AD-3: MS (ESI, m / z): 398.16; AD-4: MS (ESI, m / z): 475.13; AD-5: MS (ESI, m / z): 396.18; AD-6: MS (ESI, m / z): 398.16; AD-7: MS (ESI, m / z): 447.13; AD-8: MS (ESI, m / z): 382.19; AD-9: MS (ESI, m / z): 384.17.

[0043] Embodiment 2:

[0044] The blue water-soluble probe described in formula AD10-AD18 has the following synthesis route:

[0045]

[0046] The synthesis method comprises the following steps:

[0047] Weigh compound a and compound b in a molar ratio of 1:1, dissolve in methanol, add a catalytic amount of tetrahydropyrrole catalyst, heat to 40°C for reaction for 8 hours, cool and filter to obtain yellow-green solid c. Weigh c (1 eq.) and compound d (1 eq.), compound e (1 eq.) and compound f (1 eq.) respectively, dissolve in a mixed solvent of water and dioxane (water: dioxane = 1:9), add anhydrous potassium carbonate (2 eq), and then add catalyst bis(triphenylphosphine) palladium dichloride (0.05 eq) under nitrogen protection, heat to 90°C for reaction for 16 hours, concentrate in vacuo, and separate and purify the crude product by silica gel chromatography to obtain brown-gray solid g, yellow-green solid h and yellow-green solid i. Weigh g, h, i (1 eq.) and dissolve them in acetonitrile, add j, k, l, m, n, o, p, q, r (2 eq.) respectively, heat to 80°C and react for 8 hours, cool and filter, and recrystallize and wash with acetonitrile / ethyl acetate to obtain yellow-green compounds AD-10 to AD-18. AD-10: MS (ESI, m / z): 489.15; AD-11: MS (ESI, m / z): 410.20; AD-12: MS (ESI, m / z): 412.18; AD-13: MS (ESI, m / z): 489.15; AD-14: MS (ESI , m / z): 410.20; AD-15: MS (ESI, m / z): 412.18; AD-16: MS (ESI, m / z): 461.14; AD-17: MS (ESI, m / z): 396.21; AD-18: MS (ESI, m / z): 398.19.

[0048] Embodiment 3:

[0049] The blue water-soluble probe described in formula AD19-AD27 has the following synthesis route:

[0050]

[0051] The synthesis method comprises the following steps:

[0052] Weigh compound a and compound b in a molar ratio of 1:1, dissolve in methanol, add a catalytic amount of tetrahydropyrrole catalyst, heat to 40°C for reaction for 8 hours, cool and filter to obtain yellow-green solid c. Weigh c (1 eq.) and compound d (1 eq.), compound e (1 eq.) and compound f (1 eq.) respectively, dissolve in a mixed solvent of water and dioxane (water: dioxane = 1:9), add anhydrous potassium carbonate (2 eq), and then add catalyst bis(triphenylphosphine) palladium dichloride (0.05 eq) under nitrogen protection, heat to 90°C for reaction for 16 hours, concentrate in vacuo, and separate and purify the crude product by silica gel chromatography to obtain brown-gray solid g, yellow-green solid h and yellow-green solid i. Weigh g, h, i (1 eq.) and dissolve them in acetonitrile, add j, k, l, m, n, o, p, q, r (2 eq.) respectively, heat to 80°C and react for 8 hours, cool and filter, and recrystallize and wash with acetonitrile / ethyl acetate to obtain yellow-green compounds AD-19 to AD-27. AD-19: MS(ESI,m / z): 501.15; AD-20: MS(ESI,m / z): 422.20; AD-21: MS(ESI,m / z): 424.18; AD-22: MS(ESI,m / z): 501.15; AD-23: MS(ESI , m / z): 422.20; AD-24: MS (ESI, m / z): 424.18; AD-25: MS (ESI, m / z): 473.14; AD-26: MS (ESI, m / z): 408.20; AD-27: MS (ESI, m / z): 410.19.

[0053] Embodiment 4:

[0054] The blue water-soluble probe described in formula AD28-AD36 has the following synthesis route:

[0055]

[0056] The synthesis method comprises the following steps:

[0057] Weigh compound a and compound b in a molar ratio of 1:1, dissolve in methanol, add a catalytic amount of tetrahydropyrrole catalyst, heat to 40°C for reaction for 8 hours, cool and filter to obtain yellow-green solid c. Weigh c (1 eq.) and compound d (1 eq.), compound e (1 eq.) and compound f (1 eq.) respectively, dissolve in a mixed solvent of water and dioxane (water: dioxane = 1:9), add anhydrous potassium carbonate (2 eq), and then add catalyst bis(triphenylphosphine) palladium dichloride (0.05 eq) under nitrogen protection, heat to 90°C for reaction for 16 hours, concentrate in vacuo, and separate and purify the crude product by silica gel chromatography to obtain brown-gray solid g, yellow-green solid h and yellow-green solid i. Weigh g, h, i (1 eq.) and dissolve them in acetonitrile, add j, k, l, m, n, o, p, q, r (2 eq.) respectively, heat to 80°C and react for 8 hours, cool, filter, and recrystallize and wash with acetonitrile / ethyl acetate to obtain yellow-green compounds AD-28 to AD-36. AD-28: MS (ESI, m / z): 515.16; AD-29: MS (ESI, m / z): 436.22; AD-30: MS (ESI, m / z): 438.19; AD-31: MS (ESI, m / z): 515.16; AD-32: MS (ESI , m / z): 436.22; AD-33: MS (ESI, m / z): 438.19; AD-34: MS (ESI, m / z): 487.16; AD-35: MS (ESI, m / z): 422.22; AD-36: MS (ESI, m / z): 424.20.

[0058] Embodiment 5:

[0059] Brain slices of 5×FAD transgenic AD mice (9 months old, 100 μm thick) were used and rinsed 3 times with PBS buffer. The slices were placed in 1 μM PD-NA-OH solution, incubated at room temperature for 5 minutes, and then rinsed 3 times with PBS solution. Then, probe AD-1, AD-4, AD-9, AD-12, AD-15, AD-19, AD-24, AD-28, AD-32 and AD-36 solutions (1 μM) were added and incubated for 5 minutes. After probe staining, the slices were rinsed 3 times with PBS, sealed, and observed under a confocal microscope. The probes AD-1, AD-4, AD-9, AD-12, AD-15, AD-19, AD-24, AD-28, AD-32 and AD-36 of the present invention are excited by a 405nm laser and collect fluorescence signals in the 420-460nm band, and PD-NA-OH is excited by a 488nm laser and collects fluorescence signals in the 560-660nm band.

[0060] The experimental results are as follows Figure 1 and Figure 2As shown, in vitro fluorescence co-localization imaging shows that the AD mouse brain tissue slice image stained with the blue Aβ probe of the present invention can see clear Aβ plaques in the blue channel, and the position of the Aβ plaques observed in the image stained with the red Aβ probe PD-NA-OH basically coincides, and the overlap of the two can be clearly observed in the superimposed image. The image was further correlated and the Pearson correlation coefficient was calculated. The Pearson correlation coefficient (RP) of the stained image was around 0.80, which verified the co-localization effect of the probe. These results show that the water-soluble blue AIE probe designed by this method has good targeting to the Aβ plaques in the AD mouse brain tissue slices, and can achieve accurate imaging of the Aβ plaques.

[0061] In summary, compared with the existing Aβ probes, the probe of the present invention has the following advantages:

[0062] 1) Lower background interference: The emission wavelength of blue fluorescence is shorter, which can effectively avoid the interference of tissue autofluorescence. In biological imaging, tissues themselves are prone to produce green and red background fluorescence, while the background interference of blue light is lower, so the blue Aβ probe helps to improve the signal-to-noise ratio and image clarity of imaging.

[0063] 2) Possibility of multimodal and multichannel imaging: The short wavelength characteristics of blue fluorescent probes provide new combination possibilities for multimodal imaging. By combining with fluorescent dyes or imaging techniques of other wavelengths, multi-color or multi-type imaging can be achieved to more comprehensively observe the distribution and pathological changes of Aβ in the body.

[0064] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A blue water-soluble probe for labeling Aβ plaques, characterized in that: The probe has a general structural formula as shown in formula (1): Wherein, A is any one of the following substituents: B is any one of the following substituents: n is 2, 3 or 4, Indicates the connection between groups.

2. The method for preparing a blue water-soluble probe for marking Aβ plaques according to claim 1, characterized in that: The synthetic route is: Wherein, compound C is any one of the following structures: Compound E is any one of the following structures: A is any one of the following substituents: B is any one of the following substituents: n is 2, 3 or 4, Indicates the connection between groups.

3. The method for preparing a blue water-soluble probe for marking Aβ plaques according to claim 2, characterized in that: The reaction temperature of the base-catalyzed condensation is 30-60° C., and the reaction time is 5-10 hours.

4. The method for preparing a blue water-soluble probe for marking Aβ plaques according to claim 2, characterized in that: The reaction temperature of the Suzuki coupling is 70-100° C., and the reaction time is 13-20 hours.

5. The method for preparing a blue water-soluble probe for marking Aβ plaques according to claim 2, characterized in that: The reaction temperature of the ionization is 60-90° C., and the reaction time is 6-12 hours.

6. Use of the water-soluble probe according to claim 1 in obtaining accurate imaging of Aβ plaques in the whole brain.

7. The use according to claim 6, characterized in that: The water-soluble probe is used to fluorescently label Aβ plaques in the brain of AD mice.

8. The use according to claim 6, characterized in that: The probe is a blue water-soluble probe.

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