Blue water-soluble probe for labeling abeta plaque and preparation method and application thereof

Abstract

This invention belongs to the field of fluorescent probe technology, specifically relating to a blue water-soluble probe for labeling Aβ plaques, its preparation method, and its applications. The probe has the following general structural formula: After binding to Aβ fibers, the fluorescence intensity of the probe is significantly increased, and it emits almost no light in aqueous solution, avoiding interference from false positive signals of traditional probes. The probe of this invention can achieve high-resolution in-situ labeling of Aβ plaques, exhibiting excellent imaging performance. It can be applied to obtaining precise imaging of Aβ plaques throughout the brain. This probe helps researchers accurately and conveniently detect the presence and distribution of Aβ plaques, thus playing an important role in the research, diagnosis, and treatment development of Alzheimer's disease (AD).

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a blue water-soluble probe for labeling Aβ patches, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is the leading cause of dementia (60%-80%) and the most common neurodegenerative disease, imposing a severe economic burden on individuals and society. Amyloid-β-protein (Aβ), as the most important pathological marker of AD, is closely related to its pathological development. Aβ plaques are surrounded by various brain structures, including neurons, glial cells, nerve fibers, and blood vessels. Various pathological lesions related to these brain structures, such as amyloid angiopathy, white matter lesions, ventricular enlargement, and brain atrophy, are commonly seen in the clinical imaging of AD patients. Previous studies have shown that Aβ, as the earliest pathological feature in the AD pathological circuitry, is extremely closely correlated with lesions in these brain structures. Therefore, in-depth investigation of the interaction between Aβ plaques and the various surrounding brain structures is of great significance for understanding the pathological mechanisms and progression of AD.

[0003] Over the past few decades, various fluorescent materials with near-infrared emission wavelengths (>650 nm) have been well developed, such as rare-earth-doped upconversion luminescent nanoparticles, quantum dots, and organic small molecule phosphors. Meanwhile, emerging aggregation-induced emission (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, current fluorescent probes targeting Aβ patches and surrounding structures are mainly located in the green, red, and near-infrared emission ranges, which easily leads to spectral overlap and fluorescence emission crossover of fluorophores during multi-channel labeling.

[0004] Related studies have shown that lipofuscin autofluorescence occurs in the somatosensory, auditory, and visual cortices of unstained 3-month-old mice under laser irradiation at wavelengths of 488 nm, 561 nm, and 631 nm. This autofluorescence can overlap with the signal of fluorescently labeled probes, interfering with the accuracy of the signal, especially when detecting specific proteins in intracellular or tissue samples, potentially affecting the accuracy and reproducibility of experimental data. In recent years, fluorescent dyes for Aβ imaging have been widely developed, but most are concentrated in the aforementioned wavelength ranges. Under specific experimental requirements and technical conditions, the presence of autofluorescence may cause these dyes to encounter problems in Aβ imaging, mainly including:

[0005] 1) Reduced signal specificity: When the probes operate at wavelengths of 488nm, 561nm, and 631nm, the strong autofluorescence of lipofuscin means that the detected signal may contain both the fluorescent signal from the probe and the background autofluorescence from lipofuscin, making it difficult for researchers to distinguish the true Aβ signal from background noise. Especially in brain tissue, the dotted distribution of lipofuscin autofluorescence further increases the possibility of false positives, making specific detection of the probes even more challenging.

[0006] 2) Limited accuracy of quantitative detection: The presence of autofluorescence introduces significant background noise into the probe's fluorescence signal, making it difficult to accurately reflect the content and distribution of Aβ. Furthermore, the intensity of autofluorescence varies with age, tissue type, and pathological condition, further complicating quantitative analysis.

[0007] 3) Reduced sensitivity: The autofluorescence of lipofuscin reduces the overall detection sensitivity when it overlaps with the probe signal. In particular, when the Aβ level is low, the probe signal is weak and can be easily masked by the background fluorescence of lipofuscin, making it difficult to detect Aβ in the early stages of the disease or when there is less Aβ accumulation.

[0008] Therefore, the development of blue Aβ fluorescent probes is of paramount importance. Their ability to improve imaging resolution, reduce background interference, and achieve multi-channel fluorescent labeling of Aβ plaques and their surrounding environment can provide more tools and strategies for Alzheimer's disease research. This will contribute to a deeper understanding of the pathogenesis of AD and may have a significant impact on drug development and early diagnosis. Summary of the Invention

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

[0010] The objective of this invention is achieved through the following technical solution: a blue water-soluble probe for labeling Aβ plaques, the probe having a general structural formula as shown in formula (I):

[0011]

[0012] Wherein, A can be any of the following substituents:

[0013]

[0014] B can be any of the following substituents:

[0015]

[0016]

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

[0018] The synthetic route for preparing the blue water-soluble probe labeled with Aβ plaques is as follows:

[0019]

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

[0021]

[0022] Compound E has any of the following structures:

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

[0024]

[0025] B can be any of the following substituents:

[0026]

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

[0028] As a preferred technical solution, the reaction temperature of the alkali-catalyzed condensation is 30-60℃, and the reaction time is 5-10h.

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

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

[0031] The above-mentioned water-soluble probes are used in obtaining precise 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 brains of AD mice.

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

[0034] In the probe structure of this invention, the pyridine nitrogen atom is ionized at the meta position. In meta-ionization, the electronic interaction between the pyridine nitrogen atom and other functional groups is weakened. Due to the greater distance between the meta-nitrogen atom and other functional groups, electron transfer is relatively weak. This ionization leads to a more localized electron distribution within the molecule, altering the energy difference between the excited and ground states. This results in a blue shift in the molecule's fluorescence emission, exhibiting a completely different luminescence phenomenon compared to para-ionized structures. Simultaneously, due to the localized electron distribution, meta-ionized molecules exhibit a narrower and stronger fluorescence spectrum, enabling the target molecule to emit fluorescence in the blue wavelength range. This avoids interference from biological autofluorescence, achieving high-performance imaging of Aβ.

[0035] This invention offers the following advantages: It discloses a blue water-soluble probe for labeling Aβ plaques, its preparation method, and its applications. By adjusting the structure of its electron-donating and electron-withdrawing groups, the probe's fluorescence performance is optimized. Simultaneously, by introducing different hydrophilic groups, it acquires suitable hydrophilic properties, ensuring low background interference during imaging. When this probe binds to Aβ fibers, its fluorescence intensity is significantly enhanced, and it exhibits almost no luminescence in aqueous solution, avoiding interference from false-positive signals of traditional probes. In selectivity experiments, it demonstrates excellent anti-interference performance, achieving specific targeting of Aβ and exhibiting high affinity for Aβ. Furthermore, in vitro imaging results show that the probe of this invention can achieve high-resolution in-situ labeling of Aβ plaques with excellent imaging performance. This blue water-soluble probe can help researchers accurately and conveniently detect the presence and distribution of Aβ plaques, thus playing an important role in the research, diagnosis, and treatment development of Alzheimer's disease (AD). Attached Figure Description

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

[0037] Figure 2This is a fluorescence imaging image of another part of the Aβ plaque in an AD mouse brain slice; in the image, (A)(B)(C)(D) are AD-19 staining, PD-NA-OH staining, overlay images and Pearson correlation coefficient maps, respectively; (E)(F)(G)(H) are AD-24 staining, PD-NA-OH staining, overlay images and Pearson correlation coefficient maps, respectively; (I)(J)(K)(L) are AD-28 staining, PD-NA-OH staining, overlay images and Pearson correlation coefficient maps, respectively; (M)(N)(O)(P) are AD-32 staining, PD-NA-OH staining, overlay images and Pearson correlation coefficient maps, respectively; (Q)(R)(S)(T) are AD-36 staining, PD-NA-OH staining, overlay images and Pearson correlation coefficient maps, respectively; probe concentration: 5 μM, scale bar: 50 μm. Detailed Implementation

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

[0039] The synthetic route for the blue water-soluble probes described in formulas AD1-AD9 is as follows:

[0040]

[0041] The synthesis method includes the following steps:

[0042] Compounds a and b, in a molar ratio of 1:1, were weighed and dissolved in methanol. A catalytic amount of tetrahydropyrrole catalyst was added, and the mixture was heated to 40°C and reacted for 8 hours. After cooling and filtration, a yellow-green solid c was obtained. c (1 eq.) was weighed and dissolved in a mixed solvent of water and dioxane (water:dioxane = 1:9) with compounds d (1 eq.), e (1 eq.), and f (1 eq.), respectively. Anhydrous potassium carbonate (2 eq.) was added, and then bis(triphenylphosphine)palladium dichloride catalyst (0.05 eq.) was added under nitrogen protection. The mixture was heated to 90°C and reacted for 16 hours. The mixture was concentrated under vacuum, and the crude product was purified by silica gel chromatography to obtain a brownish-gray solid g, a yellow-green solid h, and a 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 and heat to 80 °C for 8 hours. Cool, filter, and recrystallize 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] Example 2:

[0044] The synthetic route for the blue water-soluble probes described in formulas AD10-AD18 is as follows:

[0045]

[0046] The synthesis method includes the following steps:

[0047] Compounds a and b, in a molar ratio of 1:1, were weighed and dissolved in methanol. A catalytic amount of tetrahydropyrrole catalyst was added, and the mixture was heated to 40°C and reacted for 8 hours. After cooling and filtration, a yellow-green solid c was obtained. c (1 eq.) was weighed and dissolved in a mixed solvent of water and dioxane (water:dioxane = 1:9) with compounds d (1 eq.), e (1 eq.), and f (1 eq.), respectively. Anhydrous potassium carbonate (2 eq.) was added, and then bis(triphenylphosphine)palladium dichloride catalyst (0.05 eq.) was added under nitrogen protection. The mixture was heated to 90°C and reacted for 16 hours. The mixture was concentrated under vacuum, and the crude product was purified by silica gel chromatography to obtain a brownish-gray solid g, a yellow-green solid h, and a 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 and heat to 80 °C for 8 hours. Cool, filter, and recrystallize with acetonitrile / ethyl acetate to obtain yellow-green compounds AD-10~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] Example 3:

[0049] The synthetic route for the blue water-soluble probes described in formulas AD19-AD27 is as follows:

[0050]

[0051] The synthesis method includes the following steps:

[0052] Compounds a and b, in a molar ratio of 1:1, were weighed and dissolved in methanol. A catalytic amount of tetrahydropyrrole catalyst was added, and the mixture was heated to 40°C and reacted for 8 hours. After cooling and filtration, a yellow-green solid c was obtained. c (1 eq.) was weighed and dissolved in a mixed solvent of water and dioxane (water:dioxane = 1:9) with compounds d (1 eq.), e (1 eq.), and f (1 eq.), respectively. Anhydrous potassium carbonate (2 eq.) was added, and then bis(triphenylphosphine)palladium dichloride catalyst (0.05 eq.) was added under nitrogen protection. The mixture was heated to 90°C and reacted for 16 hours. The mixture was concentrated under vacuum, and the crude product was purified by silica gel chromatography to obtain a brownish-gray solid g, a yellow-green solid h, and a 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 and heat to 80 °C for 8 hours. Cool, filter and recrystallize with acetonitrile / ethyl acetate to obtain yellow-green compounds AD-19~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] Example 4:

[0054] The synthetic route for the blue water-soluble probes described in formulas AD28-AD36 is as follows:

[0055]

[0056] The synthesis method includes the following steps:

[0057] Compounds a and b, in a molar ratio of 1:1, were weighed and dissolved in methanol. A catalytic amount of tetrahydropyrrole catalyst was added, and the mixture was heated to 40°C and reacted for 8 hours. After cooling and filtration, a yellow-green solid c was obtained. c (1 eq.) was weighed and dissolved in a mixed solvent of water and dioxane (water:dioxane = 1:9) with compounds d (1 eq.), e (1 eq.), and f (1 eq.), respectively. Anhydrous potassium carbonate (2 eq.) was added, and then bis(triphenylphosphine)palladium dichloride catalyst (0.05 eq.) was added under nitrogen protection. The mixture was heated to 90°C and reacted for 16 hours. The mixture was concentrated under vacuum, and the crude product was purified by silica gel chromatography to obtain a brownish-gray solid g, a yellow-green solid h, and a 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 and heat to 80 °C for 8 hours. Cool, filter, and recrystallize with acetonitrile / ethyl acetate to obtain yellow-green compounds AD-28~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] Example 5:

[0059] Brain slices from 5×FAD transgenic AD mice (9 months old, 100 μm thick) were washed three times with PBS buffer. The brain slices were then incubated in 1 μM PD-NA-OH solution at room temperature for 5 minutes, followed by three washes with PBS. Subsequently, 1 μM solutions of probes AD-1, AD-4, AD-9, AD-12, AD-15, AD-19, AD-24, AD-28, AD-32, and AD-36 were added and incubated for 5 minutes. After probe staining, the brain slices were washed three times with PBS, mounted, 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 this invention are excited by a 405nm laser and fluorescence signals are collected in the 420-460nm band. PD-NA-OH is excited by a 488nm laser and fluorescence signals are collected in the 560-660nm band.

[0060] Experimental results are as follows Figure 1 and Figure 2As shown, in vitro fluorescence colocalization imaging revealed clear Aβ plaques in the blue channel of AD mouse brain tissue slices stained with the blue Aβ probe of this invention. These plaques largely overlapped with those observed in images stained with the red Aβ probe PD-NA-OH, and the superimposed images clearly showed overlap. Further correlation analysis of the images, calculating the Pearson correlation coefficient, showed that the Pearson correlation coefficient (RP) for all stained images was around 0.80, validating the probe's colocalization effect. These results demonstrate that the water-soluble blue AIE probe designed using this method exhibits good targeting specificity for Aβ plaques in AD mouse brain tissue slices, enabling precise imaging of Aβ plaques.

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

[0062] 1) Lower background interference: Blue fluorescence has a shorter emission wavelength, which can effectively avoid interference from tissue autofluorescence. In bioimaging, tissues themselves tend to produce green and red background fluorescence, while blue light has lower background interference. Therefore, blue Aβ probes help improve the signal-to-noise ratio and image clarity of imaging.

[0063] 2) Possibilities for multimodal and multichannel imaging: The short-wavelength characteristics of blue fluorescent probes offer new possibilities for multimodal imaging. By combining with other wavelengths of fluorescent dyes or imaging techniques, multiple colors or types of imaging can be achieved, allowing for a more comprehensive observation of the distribution and pathological changes of Aβ in vivo.

[0064] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection 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 equation (I): ； Formula (1) Wherein, A can be any of the following substituents: ； B can be any of the following substituents: ； ； n is 2, 3, or 4. It indicates the connection between groups.

2. The method for preparing a blue water-soluble probe for labeling Aβ plaques according to claim 1, characterized in that, The synthetic route is as follows: Wherein, compound C is any one of the following structures: ； Compound E has any of the following structures: Where Y is Cl, Br, or I; D can be any of the following substituents: ； A can be any of the following substituents: ； B can be any of the following substituents: ； n is 2, 3, or 4. It indicates the connection between groups.

3. The method for preparing a blue water-soluble probe for labeling Aβ plaques according to claim 2, characterized in that, The reaction temperature for the alkali-catalyzed condensation is 30~60℃, and the reaction time is 5~10h.

4. The method for preparing a blue water-soluble probe for labeling Aβ plaques according to claim 2, characterized in that, The reaction temperature for the Suzuki coupling is 70~100℃, and the reaction time is 13~20h.

5. The method for preparing a blue water-soluble probe for labeling Aβ plaques according to claim 2, characterized in that, The ionization reaction temperature is 60~90℃, and the reaction time is 6~12h.

6. The application of the water-soluble probe of claim 1 in obtaining precise imaging of Aβ plaques in the whole brain, characterized in that, The application described is not related to disease diagnosis and treatment.

7. The application according to claim 6, characterized in that, The water-soluble probe was used to fluorescently label Aβ plaques in the brains of AD mice.

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

Images