Double-response fluorescent probe for simultaneously imaging myelin sheath and A beta aggregate in AD brain as well as preparation method and application of double-response fluorescent probe

Abstract

The invention discloses a dual-response fluorescent probe for simultaneously imaging myelin sheath and A beta aggregate in AD brain and a preparation method and application thereof. The structure of the probe is shown as any one of the following structures. A series of amphiphilic fluorescent probes NQPI1-6 with pyridinium salt structures aiming at the myelin sheath response of a central nervous system and a peripheral nervous system are prepared, wherein the probes NQPI6 can be used for specifically and simultaneously imaging the myelin sheath integrity and A beta aggregation in mouse brain tissues. The probe can carry out detailed three-dimensional imaging on a lipid-rich myelin sheath structure and can also be effectively combined with the A beta plaque to generate a strong red fluorescence signal, and the bifunctional fluorescent probe provides a powerful chemical tool for synchronous imaging of the myelin sheath and the A beta plaque. And a tool and a method with wide prospects are provided for researching the interaction between A beta plaque formation and demyelination in the AD progress process. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and in particular relates to a dual-response fluorescent probe for simultaneously imaging myelin sheath and Aβ aggregates in AD brains, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by progressive cognitive dysfunction and memory impairment. Due to the complexity of the pathogenesis of AD, effective intervention remains a daunting challenge. Initially, AD was thought to affect mainly the cortical region of the brain, with obvious pathological changes in the gray matter, such as senile plaques formed by the deposition of β-amyloid protein (Aβ) and neurofibrillary tangles (NFTs) caused by hyperphosphorylation of tau protein. However, recent studies have shown that changes in the structure of central nervous system myelin (produced by oligodendrocytes) are considered to be closely related to AD, which leads to changes in the white matter (WM) structure in AD patients. Histological and functional imaging studies have found that the microstructure of the brain white matter is severely damaged not only in patients with advanced AD, but also in patients with early symptoms, including preclinical symptoms, subjective cognitive impairment (SCI), and mild cognitive impairment (MCI). The observed changes in WM are mainly myelin shedding and axonal damage, resulting in a reduction in the volume of nerve fiber bundles and WM, especially affecting the integrity of the myelin sheath.

[0003] Myelin is a lipid-rich structure that wraps around nerve axons and plays a vital role in insulating nerve fibers, maintaining axonal action potentials, and facilitating rapid and precise electrical signal conduction. These key features have conferred evolutionary advantages to the vertebrate nervous system. Given the key role of myelin in regulating axonal conduction velocity and integrity, myelin lesions are likely to be one of the causes of systemic cognitive decline in AD patients; at the same time, WM degeneration and demyelination are also important warning signs of AD progression. Comparative analysis of brain tissues from patients without dementia and AD patients showed that total protein content and myelin-related proteins (such as myelin basic protein (MBP), myelin proteolipoprotein (PLP), cyclic nucleotide phosphohydrolase (CNP), and cholesterol) were significantly reduced, indicating that myelin loss exists in the pathological process of AD. Some studies have suggested that the abnormal accumulation of Aβ plaques triggers a series of abnormal cytoskeletal changes, leading to the loss of dendritic spines, the formation of atrophic axons, and ultimately neuronal dysfunction. In AD mouse models, focal demyelination was observed near Aβ plaques in addition to altered oligodendrocyte features near amyloid plaques, suggesting that Aβ pathology can have downstream effects in late disease stages. In mouse AD models, age-related defects in myelin structure promote the accumulation of Aβ-producing machinery within axonal swellings, which in turn promote the cleavage of cortical amyloid precursor protein and the formation of Aβ plaques. Therefore, loss of myelin integrity may be an upstream risk factor for neuronal Aβ deposition. Therefore, studying the interplay between demyelination, WM damage, and Aβ plaque formation is crucial to understanding the pathogenesis of AD and has important clinical implications for the prevention, diagnosis, and treatment of AD.

[0004] Simultaneous detection of Aβ and myelin can provide insight into the complex interrelationships between these key biomarkers. While it is possible to image them separately using two single-response fluorescent probes, this approach is complex, the spatial and temporal distribution of the probes may be different, and they may interfere with each other. However, there is currently no single probe or chemical tool that can simultaneously detect and image these markers in brain tissue. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a dual-response fluorescent probe for simultaneously imaging myelin and Aβ aggregates in AD brains, and a preparation method and application thereof. The probes described in the present invention are all amphiphilic fluorescent probes with a pyridinium salt structure, which can selectively stain the myelin regions of the central nervous system and the peripheral nervous system, and can be used for three-dimensional (3D) myelin imaging of mouse brain tissue, and have good tissue penetration and high specificity. Among them, the representative probe NQPI6 can also specifically label Aβ aggregates at the same time, thereby realizing dual-function imaging of Aβ plaques and myelin in mouse brain slices, revealing the close relationship between myelin damage and Aβ plaque distribution in different brain regions and developmental stages of AD model mice, effectively solving the dilemma that there are currently no probes or chemical tools that can simultaneously detect and image Aβ and myelin in AD brain tissue, and providing a new perspective for understanding the complex relationship between myelin degradation and Aβ pathology.

[0006] The present invention also provides a preparation method and application of the dual-response fluorescent probe for simultaneously imaging myelin sheath and Aβ aggregates in AD brain.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a myelin-responsive amphiphilic fluorescent probe having a pyridinium salt structure, the structure of which is shown in any one of the following:

[0008]

[0009] The R is selected from any one of the following:

[0010]

[0011] Preferably, the present invention provides a myelin-responsive amphiphilic fluorescent probe having a pyridinium salt structure and a plurality of control probes, the structure of the probe being as follows:

[0012]

[0013] Preferably, the present invention proposes a series of amphiphilic fluorescent probes NQPI1-6 with pyridinium salt structures that respond to the myelin sheath of the central nervous system and peripheral nervous system, wherein the probe NQPI6 can specifically label Aβ plaques at the same time.

[0014] Among them, the Chinese name of the structural formula NPPI is 4-(4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-methylpyridin-1-ium iodide.

[0015] The Chinese name of the structural formula PAP-1 is 2-(3-(2-(4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)vinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile.

[0016] The Chinese name of the structural formula PAP-2 is 2-(5,5-dimethyl-3-(2-(4'-(4-methylpiperazine-1-yl)-[1,1'-biphenyl]-4-yl)vinyl)cyclohex-2-ene-1-ylidene)malononitrile

[0017] The Chinese name of the structural formula NQPI1 is 4-(2-(6-(9H-carbazol-9-yl)quinolin-2-yl)vinyl)-1-methylpyridin-1-ium iodide.

[0018] The Chinese name of the structural formula NQPI2 is 4-(2-(6-(bis(4-methoxyphenyl)amino)quinolin-2-yl)vinyl)-1-methylpyridin-1-ium iodide.

[0019] The Chinese name of the structural formula NQPI3 is 4-(2-(6-(10H-phenothiazin-10-yl)quinolin-2-yl)vinyl)-1-methylpyridin-1-ium iodide.

[0020] The Chinese name of the structural formula NQPI4 is 4-(2-(6-(dimethylamino)quinolin-2-yl)vinyl)-1-methylpyridin-1-ium iodide.

[0021] The Chinese name of the structural formula NQPI5 is 4-(2-(6-(azetidin-1-yl)quinolin-2-yl)vinyl)-1-methylpyridin-1-ium iodide.

[0022] The Chinese name of the structural formula NQPI6 is 1-methyl-4-(2-(6-(pyrrolidin-1-yl)quinolin-2-yl)vinyl)pyridin-1-ium iodide.

[0023] The method for preparing the myelin-responsive amphiphilic fluorescent probe having a pyridinium salt structure of the present invention comprises the following steps:

[0024] N,N-dimethyl-p-toluidine and (E)-2-butenal were added to HCl solution, stirred at room temperature, and an organic solvent was added, heated and stirred, and after the reaction, extraction, washing, drying, and concentration under reduced pressure were performed to obtain a crude product, and the crude product was purified to obtain a yellow-brown solid compound 1;

[0025] SeO2 was dissolved in 1,4-dioxane / H2O, heated and stirred, then compound 1 was added, heated and stirred, after the reaction was completed, the reactant was cooled to room temperature, filtered and concentrated under reduced pressure to obtain a crude product, and the crude product was purified by chromatography to obtain yellow solid compound 2;

[0026] Compound 2 and 1,4-dimethylpyridinium iodide were dissolved in an organic solvent, piperidine was added dropwise to the mixture, and heated and stirred. After the reaction was completed, the reactants were cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The crude product was dissolved and recrystallized to obtain a red solid NQPI4;

[0027] The reaction formula is as follows:

[0028]

[0029] The method for preparing the myelin-responsive amphiphilic fluorescent probe having a pyridinium salt structure of the present invention comprises the following steps:

[0030] Cesium carbonate, 2,2'-bis(diphenylphosphine)-1,1'-binaphthyl and palladium (II) acetate are dissolved in an organic solvent, and then 6-bromo-2-methylquinoline and different secondary amines are added, heated and stirred, cooled to room temperature after the reaction, the solution is filtered, extracted, and concentrated under reduced pressure to obtain the residue, and the desired compound 1R is purified, wherein the secondary amine required for preparing NQPI1 is carbazole; the secondary amine required for preparing NQPI2 is 4,4'-dimethoxydiphenylamine; the secondary amine required for preparing NQPI3 is phenothiazine; the secondary amine required for preparing NQPI5 is azocyclobutane; and the secondary amine required for preparing NQPI6 is tetrahydropyrrole;

[0031] SeO2 is dissolved in 1,4-dioxane / H2O, heated and stirred, and then compound 1R is added, and the mixture is heated and stirred. After the reaction is completed, the reactant is cooled to room temperature and filtered. The filtrate is concentrated under reduced pressure to obtain a crude product, and the crude product is purified to obtain the corresponding aldehyde 2R;

[0032] Compound 2R and 1,4-dimethylpyridinium iodide were dissolved in anhydrous ethanol, piperidine was added dropwise to the mixture, and the mixture was heated and stirred. After the reaction was completed, the reactants were cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The crude product was dissolved and recrystallized to obtain red solid NQPI1, 2, 3, 5 and 6;

[0033] The reaction formula is as follows:

[0034]

[0035] Preferably, the method for preparing the probe NQPI1-6 targeting myelin sheath of the present invention comprises the following steps:

[0036] To a solution of N,N-dimethyl-p-toluidine (10mmol, 1.36g) in 6N HCl (22ml) solution was added (E)-2-butenal (20mmol, 0.7g), stirred at room temperature for 1h, then 10ml of toluene was added, and the reaction mixture was stirred at 110°C for 12h. After extraction with dichloromethane three times, the organic phase was washed with saturated brine 2-3 times, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA=40:1, v / v) to obtain yellow-brown solid compound 1 (1.0g, 72%).

[0037] Cesium carbonate (3.4 g, 10.4 mmol), 2,2'-bis(diphenylphosphine)-1,1'-binaphthyl (340 mg, 0.52 mmol) and palladium (II) acetate (58 mg, 0.26 mmol) were dissolved in toluene (10 ml) solution, and then 6-bromo-2-methylquinoline (1.76 g, 8 mmol) and different secondary amines (RH, 9.6 mmol) were added, and the reaction mixture was further refluxed at 110 ° C for 8 h. After cooling to room temperature, the solution was filtered through diatomaceous earth, extracted with ethyl acetate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain the desired compound 1R (yield of about 54%).

[0038] SeO2 (1.66 g, 15 mmol) was dissolved in 1,4-dioxane / H2O (10 ml: 1 ml, v / v) and heated at 60°C for 30 min. Compound 1 or compound 1R (10 mmol) was then added and the mixture was stirred at 80°C for another 4 h. After the reaction was completed, the reactants were cooled to room temperature. The mixture was filtered through diatomaceous earth, and the filtered residue was rinsed several times with a small amount of dichloromethane to remove the precipitate. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to obtain the corresponding compound aldehyde 2 or aldehyde 2R as a yellow solid (yield of about 76%).

[0039] Compound 2R (5 mmol) and 1,4-dimethylpyridinium iodide (117 mg, 5 mmol) were dissolved in anhydrous ethanol. Piperidine (49 μL, 0.5 mmol) was added dropwise to the mixture and stirred at 80°C for 8 h. The crude product was dissolved in a small amount of anhydrous ethanol and recrystallized by adding ether dropwise to obtain a red solid NQPI1-6 (yield of about 60%).

[0040] The invention discloses an application of the myelin-responsive amphiphilic fluorescent probe with a pyridinium salt structure in the preparation of a drug or reagent for detecting myelin.

[0041] Wherein, the myelin sheath is the myelin sheath of the central nervous system and the peripheral nervous system.

[0042] The myelin sheaths are myelin sheaths in different brain regions of brain slices, myelin sheaths in spinal cord slices, myelin sheaths in sciatic nerve slices and myelin sheaths in right occipital lobe slices.

[0043] Furthermore, the probes NQPI1-6 label myelin sheaths in different brain regions of mouse brain slices, including probe NQPI6 labeling myelin sheaths in different brain regions of mouse brain slices, mouse spinal cord slices, mouse sciatic nerve slices and right occipital lobe slices of human brain, and the probe NQPI6 performs three-dimensional mapping imaging of myelin sheaths in mouse brain tissue.

[0044] Furthermore, the probe detects myelin for analyzing myelin density and mapping the trajectory of myelinated nerve fiber bundles.

[0045] The myelin-responsive amphiphilic fluorescent probe with a pyridinium salt structure of the present invention is used in the preparation of drugs or reagents for simultaneous detection and imaging of myelin and Aβ aggregates in AD brains.

[0046] Among them, the amphiphilic fluorescent probe is used in the preparation of drugs or reagents for imaging myelin integrity and Aβ aggregation in brain tissue.

[0047] The amphiphilic fluorescent probe is used in the preparation of drugs or reagents for three-dimensional mapping and imaging of brain tissue myelin sheath and for marking Aβ plaques in brain slices.

[0048] Furthermore, the probe NQPI6 that dual-responds to myelin and Aβ aggregates in AD brains is used to detect Aβ aggregates in AD brains, the probe NQPI6 marks Aβ plaques in brain sections of 5xFAD mice, and the probe NQPI6 performs simultaneous dual-response imaging of the pathology of myelin and Aβ plaques in brain sections of 5xFAD mice and WT mice of the same age.

[0049] The amphiphilic fluorescent probe of the present invention is used in preparing a reagent or a tool capable of simultaneously imaging myelin sheath and Aβ plaques.

[0050] The present invention discloses a series of amphiphilic fluorescent probes NQPI1-6 with a pyridinium salt structure for myelin response in the central nervous system and peripheral nervous system, and their preparation methods and applications, wherein the probe NQPI6 can simultaneously detect and image myelin and Aβ aggregates in AD brains. Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline and memory loss. Although β-amyloid protein (Aβ) plaques in gray matter are recognized pathological features, significant damage to white matter, including myelin destruction and axonal damage, can also be observed in Alzheimer's patients. However, due to the lack of powerful imaging research tools, the intrinsic connection between myelin damage and Aβ plaque deposition during the development of AD remains unclear. The present invention shows a series of amphiphilic fluorescent probes NQPI1-6 with a pyridinium salt structure for myelin response in the central nervous system and peripheral nervous system, wherein the novel dual-response fluorescent probe NQPI6 can specifically and simultaneously image myelin integrity and Aβ aggregation in mouse brain tissue. NQPI6 emits a unique green fluorescence signal with a high signal-to-noise ratio, enabling detailed three-dimensional imaging of lipid-rich myelin structures. Importantly, the probe can also effectively bind to Aβ plaques, generating a strong red fluorescence signal. Using the synchronous imaging of NQPI6, it is possible to detect different degrees of demyelination in different brain regions of AD model mice of different ages, which is correlated with the distribution of Aβ plaques. This dual-functional fluorescent probe provides a powerful chemical tool for the simultaneous imaging of myelin and Aβ plaques, and provides a promising product and method for studying the interaction between Aβ plaque formation and demyelination during the progression of AD.

[0051] The present invention develops a bifunctional fluorescent probe that can simultaneously detect Aβ and myelin. Based on the fragment assembly strategy of quinoline molecules, the present invention designs and develops a series of amphiphilic two-photon fluorescent probes with pyridinium salt structures, which can selectively stain the myelin regions of the central nervous system and peripheral nervous system with a high signal-to-noise ratio, and can be used for three-dimensional (3D) myelin imaging of mouse brain tissue, with good tissue penetration and high specificity. Through probe screening, it was found that the representative probe NQPI6 can also specifically label Aβ aggregation at the same time, thereby achieving bifunctional imaging of Aβ plaques and myelin in mouse brain slices. Using NQPI6, the present invention reveals the close relationship between myelin damage and Aβ plaque distribution in different brain regions and developmental stages of AD model mice, providing a new perspective for understanding the complex relationship between myelin degradation and Aβ pathology. The innovatively designed two-in-one probe of the present invention will help to better understand the pathogenesis of AD.

[0052] Design principle of fluorescent probe NQPI1-6: The design of the probe of the present invention imitates the amphiphilic structure of phospholipids in the membrane, balances the hydrophobic donor and hydrophilic receptor molecules, and the hydrophilic and positively charged pyridinium salt molecules face the surface of the lipid bilayer, interacting with the negatively charged phospholipid phosphate groups through strong electrostatic effects, thereby enhancing the plasma membrane targeting of the probe. In addition, a quinoline ring with good fluorescence properties is added as a fluorophore in the present invention. In order to optimize these dyes for the complex brain environment, various cyclic aliphatic amines are introduced as electron donors to adjust the properties of the probe, including fat solubility and fluorescence properties. A series of new myelin-targeted fluorescent probes were designed and synthesized, named NQPIs. The present invention has developed a new dual-response fluorescent probe that can simultaneously image myelin integrity and Aβ aggregates in AD brains, with a low signal-to-noise ratio and good selectivity.

[0053] The present invention proposes a two-photon probe with a completely new structure, which has dual responses to myelin and Aβ plaques, and performs two-photon imaging in myelin detection in brain slices. Figure 1 The importance of the amphiphilic structure in myelin detection was verified by changing the functional groups; various cyclic aliphatic amines were introduced as electron donors to replace N, N'-dimethylamine, thereby adjusting the properties of the probe, including lipid solubility and fluorescence properties. Through this iterative process, NQPI6, which uses tetrahydropyrrole as an electron donor, was determined to be the best probe.

[0054] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0055] The present invention constructs a bifunctional molecular probe that can simultaneously image myelin and Aβ plaques. The probe can efficiently label myelin fibers and promote deep imaging of the mouse central nervous system (CNS) and peripheral nervous system (PNS). The images obtained thereby have a high signal-to-noise ratio, providing a valuable chemical tool for analyzing myelin density and mapping the trajectory of myelinated nerve fiber bundles. Among them, the environmentally sensitive probe NQPI6 can also effectively identify Aβ plaques in different brain regions in an Alzheimer's disease (AD) mouse model. The present invention utilizes dual-channel synchronous imaging technology, and NQPI6 can simultaneously observe Aβ plaques and myelin in different brain slices of Alzheimer's disease model mice, revealing the direct correlation between demyelination in the cortex and hippocampus and the presence of Aβ plaques. The probe constructed by the present invention not only provides a new tool and strategy for studying the intricate relationship between Aβ plaque formation and demyelination during the pathological development of AD, but also promotes the development of innovative diagnostic and therapeutic strategies for both myelin and Aβ plaques.

[0056] The preparation and synthesis method of the fluorescent probe for NQPI of the present invention has a novel synthesis route, is simple and easy to operate, has low cost, has high raw material utilization rate and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The chemical structures of PAP-1, PAP-2, NPPI, and NQPI1-6 listed in the present invention and the sagittal whole brain sections of 6-week-old C57BL / 6J mice stained with probes and their fluorescence intensity ratios;

[0058] Figure 2 This is the cytotoxicity MTT graph of NQPI6 listed in the present invention;

[0059] Figure 3 A is a confocal fluorescence image of SH-SY5Y cells with different cholesterol concentrations co-incubated with the NQPI6 listed in the present invention; B is the relative ratio of fluorescence intensity of SH-SY5Y cells with different cholesterol concentrations after incubation with the NQPI6 listed in the present invention;

[0060] Figure 4 A and B are representative images of double staining of NQPI6 and Plp1 antibodies in the cerebellum (CB), cortex, and corpus callosum (CC) of control and Akt cTKO mice at P14; C and D are representative images of double staining of NQPI6 and Mbp antibodies in the cerebellum (CB), cortex, and corpus callosum (CC) of control and Akt cTKO mice at P14;

[0061] Figure 5 A is LFB staining of sciatic nerve fiber sections; B is a confocal image of the sciatic nerve fiber sections of the peripheral nervous system of 6-week-old C57BL / 6J mice after incubation of NQPI6 listed in the present invention;

[0062] Figure 6 This is a confocal image of the NQPI6 listed in the present invention after incubation with spinal cord slices of the peripheral nervous system of 6-week-old C57BL / 6J mice;

[0063] Figure 7 A is a LFB staining of a sagittal slice of the left occipital lobe of a human being adjacent to a NQPI6-stained slice; B is a confocal image of the NQPI6 listed in the present invention after incubation with a sagittal slice of the left occipital lobe of a human being;

[0064] Figure 8 This is a mosaic image of sagittal whole brain sections of 6-week-old C57BL / 6J mice stained with NQPI6;

[0065] Fig. 9 A is a 3D two-photon fluorescence image of the striatum of a sagittal mouse brain slice stained with NQPI6 listed in the present invention at different depths; B is a 3D two-photon fluorescence image of the striatum of a cross-sectional mouse brain slice stained with NQPI6 listed in the present invention at different depths;

[0066] Fig.10A is the fluorescence spectrum of NQPI6 listed in the present invention after reacting with Aβ aggregates in PBS buffer; B is the linear fitting diagram of the fluorescence intensity at 640nm and the concentration of Aβ aggregates after NQPI6 listed in the present invention reacted with different concentrations of Aβ aggregates; C is the fluorescence intensity at 640nm and the linear fitting diagram of the fluorescence intensity at 640nm after adding to 5μM Aβ 1-42 A graph showing the relationship between the concentration of NQPI6 in the PBS solution of the aggregates; D is a schematic diagram showing the selectivity of the NQPIs listed in the present invention for different substances in vitro;

[0067] Fig.11 The NQPI and Aβ listed in the present invention 1-42 Schematic diagram of molecular docking of the cryo-EM structure of the fiber (PDB ID: 5OQV);

[0068] Fig.12 Fluorescence imaging of cortical paraffin sections of 5xFAD mice after double staining with specific β-amyloid antibodies and NQPI6;

[0069] Fig.13 Confocal microscopy images of different brain regions and fluorescence intensity values ​​of myelin channels after incubation of NQPI listed in the present invention with sagittal whole brain slices of 6-month-old 5xFAD mice and wild-type WT mice of the same age; Fig.13 AC in the middle is the confocal imaging result, which shows the changes in tissue structure. A is the cortex, B is the corpus callosum, and C is the hippocampus. Fig.13 EG is right Fig.13 Data analysis of fluorescence intensity of AC myelin loss;

[0070] Fig.14 Immunofluorescence imaging of antibody-labeled Aβ protein and surrounding myelin in 5xFAD mice and age-matched wild-type controls. DETAILED DESCRIPTION

[0071] The present invention is further described below in conjunction with specific embodiments and drawings.

[0072] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0073] Example 1

[0074] A method for preparing a probe NPPI, the preparation process of which is as follows:

[0075]

[0076] 4-Dimethylaminocinnamaldehyde (875 mg, 5 mmol) and 1,4-dimethylpyridinium iodide (117 mg, 5 mmol) were dissolved in 10 mL of anhydrous ethanol. Piperidine (49 μL, 0.5 mmol) was added dropwise to the mixture and stirred at 80°C for 8 h. The crude product was dissolved in a small amount of anhydrous ethanol and recrystallized by adding ether dropwise to obtain a red solid NPPI (1.30 mg, 3.3 mmol, 66.3%). 1 H NMR (600MHz, DMSO-d6) δ8.69(d,J=6.8Hz,2H),8.02(d,J=6.9Hz,2H),7.79(dt,J=15.3,5.2Hz ,1H),7.47(d,J=8.9Hz,2H),7.04-6.97(m,2H),6.76-6.70(m,3H),4.18(s,3H),2.99(s,6H). 13 C NMR (151MHz, DMSO-d6) δ153.18,151.55,144.95,143.71,142.48,129.56,123.99,123.73,123.54,122.88,112.49,46.97,40.51.MS(ESI):C 18 H 21 N2 + [M] + ,calcd 265.17,found 265.1700.

[0077] Example 2

[0078] A method for preparing a probe NQPI4, the preparation process of which is as follows:

[0079]

[0080] N, N-dimethyl-p-toluidine (10 mmol, 1.36 g) and 2-butenal (20 mmol, 0.7 g) were added to a 6N HCl (22 mL) solution, stirred at room temperature for 1 h, 10 mL of toluene was added, and the reaction mixture was stirred at 110°C for 12 h. After extraction with dichloromethane three times, the organic phase was washed with saturated brine three times, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (PE: EA = 40: 1, v / v) to obtain a yellow-brown solid compound 1 (1.0 g, 72%).

[0081] SeO2 (1.66 g, 15 mmol) was dissolved in 1,4-dioxane / H2O (10 mL: 1 mL, v / v) and heated at 60°C for 30 min. Compound 1 (10 mmol) was then added and the mixture was stirred at 80°C for another 4 h. After the reaction was completed, the reactants were cooled to room temperature. The mixture was filtered through diatomaceous earth and the filtered residue was rinsed 3 times with a small amount of dichloromethane. The filtrate and the rinse were combined and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (PE: EA = 60: 1, v / v) to obtain a yellow solid compound 2 (76%).

[0082] Compound 2 (5 mmol) and 1,4-dimethylpyridinium iodide (117 mg, 5 mmol) were dissolved in 10 mL of anhydrous ethanol. Piperidine (49 μL, 0.5 mmol) was added dropwise to the mixture and stirred at 80 °C for 8 h. After the reaction was completed, the reactants were cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in a small amount of anhydrous ethanol and recrystallized by adding ether dropwise to obtain a red solid NQPI4 (60%). 1 H NMR (600MHz, Methanol-d4) δ8.74(d,J=6.5Hz,2H),8.19(d,J=6.5Hz,2H),8.12(d,J=8.6Hz,1H),7.94-7.86(m,2H),7.8 0(d,J=8.6Hz,1H),7.75(d,J=16.2Hz,1H),7.50(dd,J=9.4,2.9Hz,1H),6.92(d,J=2.8Hz,1H),4.33(s,3H),3.13(s,6H). 13 C NMR(151MHz,Methanol-d4)δ153.12,149.65,148.62,144.84,141.64,140.31,134.7 8,130.34,129.15,125.42,124.04,120.50,119.99,104.05,46.55,39.23.MS(ESI):C 19 H 20 N + [M] + ,calcd 290.17,found 290.1651.

[0083] Example 3

[0084] A method for preparing probes NQPI1, 2, 3, 5 and 6, the preparation process is as follows:

[0085]

[0086] Cesium carbonate (3.4 g, 10.4 mmol), 2,2'-bis(diphenylphosphine)-1,1'-binaphthyl (340 mg, 0.52 mmol) and palladium (II) acetate (58 mg, 0.26 mmol) were dissolved in toluene (10 ml) solution, and then 6-bromo-2-methylquinoline (1.76 g, 8 mmol) and different secondary amines (RH, 9.6 mmol) were added, and the reaction mixture was further refluxed at 110 ° C for 8 h. After cooling to room temperature, the solution was filtered through diatomaceous earth, extracted with ethyl acetate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (PE: EA = 20: 1, v / v) to obtain the desired compound 1R (yield of about 54%). The secondary amine required for the preparation of NQPI1 is carbazole; the secondary amine required for the preparation of NQPI2 is 4,4'-dimethoxydiphenylamine; the secondary amine required for the preparation of NQPI3 is phenothiazine; the secondary amine required for the preparation of NQPI5 is azocyclobutane; and the secondary amine required for the preparation of NQPI6 is tetrahydropyrrole.

[0087] SeO2 (1.66 g, 15 mmol) was dissolved in 1,4-dioxane / H2O (10 ml: 1 ml, v / v) and heated at 60°C for 30 min. Compound 1R (10 mmol) was then added and the mixture was stirred at 80°C for another 4 h. After the reaction was completed, the reactants were cooled to room temperature. The mixture was filtered through diatomaceous earth, and the filtered residue was rinsed several times with a small amount of dichloromethane to remove the precipitate. The filtrate and the rinse were combined and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (PE: EA = 40: 1, v / v) to obtain the corresponding aldehyde 2R as a yellow solid (yield of about 76%).

[0088] Compound 2R (5 mmol) and 1,4-dimethylpyridinium iodide (117 mg, 5 mmol) were dissolved in 10 mL of anhydrous ethanol. Piperidine (49 μL, 0.5 mmol) was added dropwise to the mixture and stirred at 80 °C for 8 h. After the reaction was completed, the reactants were cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in a small amount of anhydrous ethanol and recrystallized by adding ether dropwise to obtain red solid NQPI1, 2, 3, 5 and 6 (yields were 71%, 63%, 60%, 69%, 72%, respectively).

[0089] NQPI1: 1H NMR(600MHz,DMSO-d6)δ8.98(d,J=6.5Hz,2H),8.65(d,J=8.5Hz,1H),8.46(d,J=6.9Hz,2H),8.39(d,J=2.5Hz,1H),8.32(dd,J=11.9,8.3Hz,3H),8.26(d,J=16.2Hz,1H),8.13-8.04(m,3H),7.56(dd,J=8.2,0.9Hz,2H),7.49(d,J=15.3Hz,2H),7.41-7.32(m,2H),4.32(s,3H). 13 C NMR(151MHz,DMSO-d6)δ154.43,151.81,146.70,145.62,140.30,139.76,137.81,135.97,131.56,129.53,129.10,128.94,127.02,124.99,124.91,123.42,122.13,121.13,121.08,110.17,47.72.MS(ESI):C 29 H 22 N3 + [M] + ,calcd412.18,found 412.1833.

[0090] NQPI2: 1 H NMR(600MHz,DMSO-d6)δ8.91(d,J=7.2Hz,2H),8.36(d,J=7.1Hz,2H),8.16-8.08(m,2H),7.92-7.83(m,2H),7.77(d,J=8.6Hz,1H),7.31(d,J=6.5Hz,1H),7.16(d,J=9.0Hz,4H),7.03(s,1H),6.99(d,J=9.1Hz,4H),4.28(s,3H),3.78(s,6H). 13 C NMR(151MHz,DMSO-d6)δ157.02,152.33,150.73,147.96,145.70,143.75,140.69,139.72,135.42,130.44,129.67,127.98,126.78,124.81,124.59,122.04,115.70,111.89,55.79,55.41,47.53.MS(ESI):C 31 H 28 N3O2 + [M] +,calcd 474.22,found 474.2166.

[0091] NQPI3: 1 H NMR(600MHz,Chloroform-d)δ9.19(d,J=5.9Hz,2H),8.30(dd,J=13.9,7.6Hz,3H),8.24-8.12(m,3H),8.03(d,J=8.6Hz,1H),7.75-7.63(m,1H),7.63-7.46(m,1H),7.32(d,J=7.7Hz,2H),7.16(t,J=7.7Hz,2H),7.09(t,J=7.5Hz,2H),6.92(d,J=8.1Hz,2H),4.68(s,3H). 13 C NMR(151MHz,Chloroform-d)δ167.71,153.31,145.92,140.55,139.01,137.85,137.71,132.24,129.88,129.64,126.99,126.63,126.33,124.37,123.99,123.88,121.38,120.76,120.61,119.93,116.16,109.69.MS(ESI):C 29 H 22 N3S + [M] + ,calcd444.15,found 444.1509.

[0092] NQPI5: 1 H NMR(600MHz,DMSO-d6)δ8.88(d,J=6.7Hz,2H),8.34(d,J=6.8Hz,2H),8.15(d,J=8.6Hz,1H),8.09(d,J=16.2Hz,1H),7.88-7.82(m,2H),7.78(d,J=8.6Hz,1H),7.10(dd,J=9.1,2.5Hz,1H),6.67(d,J=2.5Hz,1H),4.27(s,3H),4.01(t,J=7.3Hz,4H),2.43-2.36(m,2H). 13C NMR(151MHz,DMSO-d6)δ152.49,150.60,149.20,145.64,142.56,140.97,134.49,130.57,129 .93,126.02,124.46,121.96,119.13,103.29,52.29,47.47,40.53,16.59.MS(ESI):C20H20N3+

[0093] [M]+,calcd 302.17,found 302.1663.

[0094] NQPI6: 1 H NMR (600MHz, DMSO-d6) δ8.87(d,J=6.8Hz,2H),8.33(d,J=6.8Hz,2H),8.13(d,J=8.6Hz,1H),8.08(d,J=16.2Hz,1H),7.88-7.79(m,2H ),7.76(d,J=8.6Hz,1H),7.33(dd,J=9.3,2.6Hz,1H),6.77(d,J=2.5Hz,1H),4.27(s,3H),3.41(d,J=13.0Hz,4H),2.06-1.99(m,4H). 13 C NMR(151MHz,DMSO-d6)δ152.54,148.44,146.79,145.54,141.90,141.09,134.23,130.60,130 .40,125.46,124.33,121.92,120.56,103.61,48.01,47.44,40.52,25.54.MS(ESI):C21H22N3+

[0095] [M]+,calcd 316.18,found 316.1805.

[0096] Example 4

[0097]

[0098] A method for preparing a probe PAP, the preparation process of which is as follows

[0099] Isophorone (3.8 g, 27.6 mmol) and malononitrile (1.82 g, 27.6 mmol) were dissolved in anhydrous ethanol (150 mL). Piperidine (23 mg, 0.276 mmol) was added dropwise to the mixture, and the solution was stirred at 60 ° C for 12 h and monitored by TLC thin layer chromatography. After the reaction mixture was cooled to room temperature, the reaction solution was slowly poured into water (200 mL) and the precipitated solid was filtered out. The residue was dissolved in anhydrous ethanol at 60 ° C to obtain a saturated crude product ethanol solution, and petroleum ether was added dropwise until a solid precipitate was stably precipitated in the solution. The solution was cooled to room temperature and filtered to collect the filter residue to obtain 2-(3,5,5-trimethylcyclohexyl-2-enyl) malononitrile, which was a brown solid compound a with a yield of 80%.

[0100] Under argon protection, compound a (1.38 g, 10 mmol) and p-bromobenzaldehyde (1.85 g, 10 mmol) were dissolved in anhydrous acetonitrile (100 mL), and piperidine (8.3 mg, 1 mmol) was added to the mixture. The reaction was stirred at 80 ° C for 6 h and monitored by TLC thin layer chromatography. After the reaction was completed, the reaction solution was diluted with 100 mL of ethyl acetate at room temperature and washed with water and saturated sodium chloride, respectively. Finally, the crude product was dried over anhydrous sodium sulfate and purified by column chromatography (petroleum ether: ethyl acetate = 2: 1, V / V) to obtain a yellow powder b with a yield of 50%.

[0101] Compound b (1.0 mmol), 4-(4-methyl-1-piperazinyl)phenylboronic acid (1.3 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.1 mmol) were placed in a flask and stirred under argon protection. 30 mL of 1,4-dioxane and 10 mL of 2M sodium carbonate aqueous solution were added to the flask, heated to 110°C and stirred under reflux for 12 h. After cooling to room temperature, the aqueous phase was removed. The separated organic phase was washed with water and saturated brine, and then dried over anhydrous magnesium sulfate. After leaching out the magnesium sulfate, the organic phase was concentrated. The crude product was purified by elution with silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v) to obtain red powder PAP-1 with a yield of 30%. 1 H NMR (400MHz, CDCl3) δ7.63-7.56(m,6H),7.11-6.98(m,4H),6.87(s,1H),3.37(t,J=11 .6Hz,4H),2.72(t,J=7.6Hz,4H),2.63(s,2H),2.51(s,2H),2.46(s,3H),1.11(s,6H). 13C NMR (101MHz, CDCl3) δ154.04,150.94,136.90,133.68,128.49,128.12,127. 62,126.76,123.36,115.90,113.64,55.02,48.60,46.18,43.02.MS(ESI):C 30 H 32 N4[M+H] + ,calcd449.27,found 449.264.

[0102] Compound b (1.0 mmol), 4-(diphenylamino)phenylboronic acid (1.3 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.1 mmol) were placed in a flask and stirred under an argon atmosphere. 30 mL of 1,4-dioxane and 10 mL of 2M aqueous sodium carbonate solution were added to the flask, heated to 110 ° C and stirred under reflux for 12 h. After cooling to room temperature, the aqueous phase was removed. The separated organic phase was washed with water and saturated brine, and then dried over anhydrous magnesium sulfate. After leaching out the magnesium sulfate, the organic phase was concentrated. The crude product was purified by elution with silica gel column chromatography (dichloromethane: methanol = 50: 1, v / v) to obtain a brown solid PAP-2 with a yield of 20%. 1 H NMR (400MHz, CDCl3) δ7.66-7.51 (m, 7H), 7.32 (D, J = 7.9Hz, 3H), 7.19 -7.16(m,6H),7.11-7.07(m,4H),6.89(s,1H),2.64(s,2H),2.53(s,2H),1.13(s,6H). 13 C NMR (101MHz, CDCl3) δ169.27,153.97,147.80,147.46,141.92,136.78,133.47,129.3 7,128.73,128.14,127.59,126.98,124.68,123.48,123.26,112.84,43.03.MS(ESI):C 37 H 31 N3[M+H] + ,calcd 518.26,found518.264.

[0103] Example 5

[0104] Synthesis and preliminary evaluation of the probe

[0105] The final structures of the probes NPPI, NQPI1-6 and PAP1-2 prepared in Examples 1-4 were determined by 1 H and 13To demonstrate whether the probe can be used for myelin imaging, the effectiveness of NQPI for myelin imaging was investigated on sagittal brain slice samples from six-week-old C57BL / 6J mice.

[0106] To prepare tissue sections, mice were deeply anesthetized with isoflurane and killed by cervical dislocation. The brains were removed, embedded in OCT at the optimal cutting temperature, and sliced ​​using a cryomicrotome (Leica CM1950). Each brain slice was approximately 25 μm thick and adhered to an adhesive glass slide. After incubation with different probes (10 μM, DMSO) for 30 min and rinsed three times with PBS, the tissue sections were postfixed in paraformaldehyde (PFA) for 15 min, incubated with DAPI for 15 min, and rinsed three times with PBS before observation using a confocal fluorescence microscope (Leica TCS SP8 MP). DAPI channel, excitation wavelength: 405nm, emission wavelength: 410-500nm; PAP-1 channel, excitation wavelength: 561nm, emission wavelength: 590-690nm; PAP-2 channel, excitation wavelength: 561nm, emission wavelength: 590-690nm; NPPI channel, excitation wavelength: 488nm, emission wavelength: 500-690nm; NQPI1 channel, excitation wavelength: 405nm, emission wavelength: 610-700nm; NQPI2 channel, excitation wavelength: 561nm, emission wavelength: 610-700nm; NQPI3 channel, excitation wavelength: 405nm, emission wavelength: 610-700nm; NQPI4 channel, excitation wavelength: 476nm, emission wavelength: 500-580nm; NQPI5 channel, excitation wavelength: 488nm, emission wavelength: 510-610nm. NQPI6 channel, excitation wavelength: 488nm, emission wavelength: 530-565nm. Images were edited using ImageJ. Figure 1 AC shows that compared with PAP-1 and PAP-2, the pyridinium salt-containing probe NPPI showed strong fluorescence signals along the myelinated nerve fibers of the striatum (STR), corpus callosum (CC), and cerebellum (CB) in mouse brain sections. Given that the content of myelin in white matter is higher than that in gray matter, the fluorescence intensity ratio (FIR) was used as a parameter to evaluate the specificity of the probe binding to myelin in the brain and spinal cord. Figure 1 The results showed that the fluorescence intensity ratio of NPPI was significantly higher than that of PAP-1 and PAP-2, indicating that the NPPI fluorophore containing a pyridinium salt terminal group has a stronger binding affinity to myelin.

[0107] The design of NPPI mimics the amphiphilic structure of phospholipids in the membrane. The hydrophilic and positively charged pyridinium salt molecules face the surface of the lipid bilayer and interact with the negatively charged phospholipid phosphate groups through strong electrostatic interactions, thereby enhancing the plasma membrane targeting of NPPI. By imitating the NPPI structure, positively charged pyridinium salt molecules are introduced as hydrophilic groups in the NQPI series of myelin probes. In addition, a quinoline ring with good fluorescence properties is added to the lipophilic structure of the probe in the present invention as a fluorophore. In order to optimize these dyes for the complex brain environment, various cyclic aliphatic amines are also introduced as electron donors to replace N, N′-dimethylamine, thereby adjusting the properties of the probe, including fat solubility and fluorescence properties. Through the comparison of brain slice fluorescence imaging, it was found that NQPI1-6 all have a certain labeling ability for the myelin area in the brain slice, proving that the six probes constructed by the present invention all have the function of detecting and imaging myelin in brain tissue, among which NQPI6 with tetrahydropyrrole as an electron donor has good selectivity and a very high signal-to-noise ratio ( Figure 1 ).

[0108] Example 6

[0109] Given the importance of cytotoxicity assessment in biological applications of fluorescent probes, a standard MTT assay was performed before fluorescence imaging to assess the cytotoxicity of the probes in SH-SY5Y cells. SH-SY5Y cells were plated at 7x10 3 Cells were cultured in 96-well plates at a density of 100 μM and incubated in a 37°C, 5% CO2 incubator for 12 h to allow the cells to reach 80% confluency. Then, cells were treated with different concentrations of NQPI6 (0.01, 0.1, 0.5, 1, 2.5, 5, 10, 20, 50 μM) and cultured for another 24 h. The readings were then taken on an Infinite M200 Pro microplate reader (Tecan, Switzerland) at a wavelength of 570 nm (reference wavelength 630 nm). Figure 2 As shown, after being cultured with 10 μM NQPI6 for 24 h, the viability of SH-SY5Y cells still remained above 75%, indicating that the probe has low cytotoxicity and biocompatibility.

[0110] Example 7

[0111] Cell membrane cholesterol imaging

[0112] The probe NQPI6 was used to detect the changes in cholesterol content in living cell membranes. SH-SY5Y cells were plated at 7x10 5The cells were cultured in 12-well plates at a density of 100 μg / cm2 and cultured in a 37°C, 5% CO2 incubator for 12 h. The consumption and supplementation of cholesterol were performed using the literature method (Two-Photon Fluorescent Turn-On Probe for Lipid Rafts in Live Cell and Tissue. Journal of the American Chemical Society, 2008. 130(13): p. 4246-4247.). The cholesterol depletion method was to incubate the living cells with 10 mM methyl-β-cyclodextrin (M-β-CD) at 37°C for 30 min, rinse three times with PBS, and then incubate with NQPI6 (10 μM) in fresh culture medium for 30 min, and rinse three times with PBS to obtain a cholesterol depletion group. Cholesterol-depleted cells were incubated with 50 μM cholesterol in the presence of a cholesterol / M-β-CD (1 / 10) mixture at 37°C for 1 h, refilled with depleted cells, rinsed three times with PBS, and then incubated with NQPI6 (10 μM) in fresh medium for 30 min, rinsed three times with PBS, to obtain the cholesterol-supplemented group. Microscopic imaging was performed using a confocal fluorescence microscope (Leica TCS SP8 MP) with an excitation wavelength of 488 nm and a collection wavelength range of 530-565 nm.

[0113] like Figure 3 As shown, after M-β-CD-mediated cholesterol depletion, the green fluorescence of the probe became almost undetectable. However, after co-incubation with cholesterol solution, cellular cholesterol was replenished and the probe fluorescence was significantly enhanced, with the fluorescence signal mainly localized to the cell membrane. This phenomenon can be attributed to the increased cholesterol levels in the plasma membrane, which may reduce the cellular permeability of NQPI6 and thus enhance its localization in the plasma membrane. These results highlight the effectiveness of NQPI6 in detecting and observing different cholesterol levels in the plasma membrane of living cells, indicating its ability to label myelin, a cholesterol-rich membrane structure.

[0114] Example 8

[0115] Myelin colocalization imaging

[0116] To further validate the ability of NQPI6 to map the myelin sheath of the central nervous system (CNS), NQPI6 staining and immunohistochemistry of myelin components Plp1 or Mbp were performed in normal control mice and Akt1 / Akt2 / Akt3 triple conditional knockout (Akt cTKO) mice. Akt dysfunction is associated with white matter diseases, including corpus callosum dysgenesis. Oligodendrocyte lineage cell-specific Akt1 / Akt2 / Akt3 triple conditional knockout mice exhibited white matter developmental defects, loss of mature oligodendrocytes, loss of myelination, and unchanged apoptotic cell death in the CNS (Akt Regulates Sox10 Expression to Control Oligodendrocyte Differentiation via PhosphorylatingFoxO1. J Neurosci, 2021. 41(39): p.8163-8180.).

[0117] Whole brains of postnatal day 14 mice were dissected and fixed in 4% paraformaldehyde (PFA) at 4°C overnight. The tissues were dehydrated and embedded in paraffin. 10 μm sections were made using a microtome. Then, the sections were deparaffinized and rehydrated. Subsequently, the sections were subjected to antigen retrieval treatment with boiling sodium citrate buffer for 20 min. The sections were then incubated with NQPI6 (10 μM) for 30 min at room temperature (RT) and rinsed with 1x PBS. After blocking with 5% BSA for 30 min at room temperature (RT), the sections were stained with primary antibodies overnight at 4°C and then incubated with secondary antibodies for 30 min at room temperature (RT). Cell nuclei were stained with DAPI (1 mg / ml). Images were analyzed using a fluorescence microscope system (BX53, Olympus). The antibodies used were as follows: Mbp (Catalog # MAB386, Millipore, 1:100), Plp1 (Catalog # MAB388, Millipore, 1:1000), Alexa 488AffiniPure Donkey Anti-Mouse IgG(Catalog#715-545-150,JacksonImmunoResearch Labs,1:500)and Alexa 488AffiniPure Goat Anti-Rat IgG (Catalog #112-545-003, Jackson ImmunoResearch Labs, 1:500).

[0118] The co-staining results showed that NQPI6 fluorescence was detected in the Plp1+ myelin sheaths of the cerebellar white matter (CB) and corpus callosum (CC) of control mice; however, no obvious signal was detected in the cortical gray matter ( Figure 4 A and 4B). In contrast, no fluorescence signals of NQPI6 or Plp1 were observed in the CB, CC, or cortex of AktcTKO mice. Consistent with control mice, NQPI6 fluorescence was also detected in the Mbp+ myelin sheaths of the CB and CC white matter, but no obvious fluorescence signals were detected in the cortical gray matter ( Figure 4 C and 4D). Akt cTKO mice also had no fluorescent signals of NQPI6 or Mbp. The colocalization results showed that myelin in the white matter could be specifically stained by NQPI6.

[0119] Example 9

[0120] Mouse sciatic nerve imaging

[0121] Sciatic nerve fibers in the PNS are wrapped by Schwann cells. Therefore, this multi-layered, membrane-rich myelin sheath is a logical target for NQPI6. To test this possibility, sciatic nerve fibers from mice were stained with NQPI6.

[0122] Six-week-old C57BL / 6J mice were anesthetized with isoflurane and cervical dislocation was performed under deep anesthesia. After shaving the mouse hip, a 3 mm incision was made on the skin above the mouse trochanter, and the conjunctival tissue was cut on the right side of the hip. A small gap was opened from the conjunctival tissue. The muscle fascia was pushed away with fine forceps so that a small part of the nerve could be cut. The tissue was embedded by OCT at the optimal cutting temperature and sliced ​​using a cryomicrotome (Leica CM1950). Each slice was about 10 μm thick and adhered to an adhesive glass slide. After incubation with the probe NQPI6 (10 μM) for 30 minutes and rinsed three times with PBS, the 10 μm sciatic nerve slices were post-fixed in paraformaldehyde (PFA) for 15 minutes, incubated with DAPI for 15 minutes and rinsed three times with PBS, and observed using a confocal fluorescence microscope (Zeiss LSM 880). For the DAPI channel, excitation: 405 nm, emission: 410-500 nm. The excitation wavelength of the probe channel is 488nm, emission: 530-565nm. Use ImageJ to edit the image. Adjacent slices of the same tissue were stained with LFB (Luxol Fast Blue) myelin by Sevier Biotechnology Co., Ltd. and photographed (Pannoramic MIDI). LFB is a copper-phthalocyanine dye, generally used for staining the brain, spinal cord, and peripheral nerves, and the myelin sheath of the nerve appears bright blue. Figure 5 As shown, ( Figure 5 B) Fluorescence imaging area of ​​NQPI6 in sciatic nerve slices and ( Figure 5A) The bright blue area stained by LFB is consistent, indicating that NQPI6 can label sciatic nerve fibers.

[0123] Example 10

[0124] Mouse spinal cord myelin imaging

[0125] Six-week-old C57BL / 6J mice were deeply anesthetized by intraperitoneal injection of chlorpromazine (15 mg / kg). The chest and abdomen were sprayed with 75% ethanol, and then the abdominal cavity was cut open longitudinally along the midline. The chest cavity was opened to expose the heart, and the heart was grasped with forceps, and a 21-gauge needle was inserted into the left ventricle. An incision was made in the right atrium, and the heart was punctured with frozen Ca-free 2+ and Mg 2+ ACSF (containing 95% O2 / 5% CO2) was slowly perfused into mice through the left ventricle. After perfusion, the mice were decapitated, the spine was removed and quickly immersed in normal ice-cold ACSF. A small incision was made on each side of one end of the cervical vertebra, the ventral vertebral body was removed, the dorsal processes on both sides were cut, the spinal canal was opened, the nerves were cut and the spinal cord was exposed. The spinal cord was removed and immersed in frozen normal ACSF saturated with 95% O2 / 5% CO2, and the remaining spinal nerve roots close to the spinal cord were cut. At the optimal cutting temperature, the tissue was embedded in OCT and sliced ​​using a cryomicrotome (Leica CM1950). Each slice was approximately 10 μm thick and adhered to an adhesive glass slide. After incubation with the probe NQPI6 (10 μM) for 30 minutes and rinsing three times with PBS, 10 μm mouse spinal cord sections were post-fixed in paraformaldehyde (PFA) for 15 minutes, incubated with DAPI for 15 minutes and rinsed three times with PBS, and then observed using a confocal fluorescence microscope (Leica TCS SP8 MP). For the DAPI channel, excitation: 405 nm, emission: 410-500 nm. The excitation wavelength of the probe channel was 488 nm, emission: 530-565 nm. Images were edited using ImageJ. Figure 6 As shown, after incubation of spinal cord slices with NQPI6, obvious fluorescence appeared in the white matter area, indicating that NQPI6 specifically stained the myelin sheath in the white matter of the spinal cord.

[0126] Embodiment 11

[0127] Clinical tissue imaging

[0128] Clinical human left occipital lobe slices were obtained from Liaocheng People's Hospital (2024277). The slides were inserted into a slide rack and placed in a 60°C oven for 1.5 h. The slices were deparaffinized twice with xylene, each for ten minutes. The slices were rehydrated with decreasing concentrations of ethanol, anhydrous ethanol twice, 5 min each; 95% ethanol for 5 min; 70% ethanol for 5 min; 50% ethanol for 5 min. After a brief rinse with distilled water, the slices were incubated with the probe NQPI6 (10 μM) for 30 minutes and rinsed three times with PBS before observation using a confocal fluorescence microscope (Leica TCS SP8 MP). The excitation wavelength of the probe channel was 488 nm, and the emission: 530-565 nm. Images were edited using ImageJ. Adjacent slices of the same tissue were stained for myelin sheath with LFB by Sevier Biotechnology Co., Ltd. and photographed (Pannoramic MIDI). Figure 7 As shown, ( Figure 7 B) Fluorescence imaging area of ​​NQPI6 in sagittal slices of left occipital lobe of clinical human subjects and ( Figure 7 A) LFB staining is consistent with bright blue areas, indicating that NQPI6 can image myelin in clinical human left occipital lobe sagittal sections.

[0129] Example 12

[0130] 3D rendering of myelin sheath in brain tissue

[0131] Compared with single-photon fluorescent probes, two-photon fluorescent probes have obvious advantages such as lower excitation energy, less photodamage and deeper tissue penetration, and are gaining more and more attention in biomedical research. To further evaluate the superiority of NQPI6 in myelin fiber fluorescence imaging, two-photon imaging of NQPI6-stained mouse brain slices was performed.

[0132] Six-week-old C57BL / 6J mice were anesthetized with isoflurane and the cervical vertebrae were dislocated under deep anesthesia. The whole brain of the mouse was extracted using a scalpel, forceps, and fine scissors. The mouse brain was placed in a container containing an appropriate amount of liquid nitrogen for 10-15 seconds. The tissue turned white and was taken out. The mouse brain was wrapped with aluminum foil, labeled, and placed in a -80℃ refrigerator until ready for sectioning. At the optimal cutting temperature, the tissue was embedded in OCT and sectioned using a cryomicrotome (Leica CM1950). Each slice was about 100μm thick and adhered to an adhesive glass slide. After incubation with the probe NQPI6 (10μM) for 30 minutes and rinsed three times with PBS, 100μm mouse whole brain slices were post-fixed in paraformaldehyde (PFA) for 15 minutes. The NQPI6-incubated brain tissue slices were observed and imaged at different depths using a two-photon confocal fluorescence microscope (Leica TCS SP8MP), scanning every 5μm, with a total depth of 80μm. The excitation wavelength of the probe channel was 920 nm, emission: 530-565 nm. The three-dimensional construction was performed using Leica TCS SP8 MP software.

[0133] Under 920nm excitation, NQPI6-stained mouse brain slices showed strong fluorescence signals in the CB, STR, and CC regions of sagittal brain slices. NQPI6 clearly marked and located myelinated axonal fibers in the white matter ( Figure 8 ).like Fig. 9 As shown in the 3D reconstruction images, the probe can distinguish single nerve bundles with high signal-to-noise ratio in the caudate nucleus thalamus (CPu) at a depth of 80μm. These results confirm that NQPI6 is a reliable myelin probe that can label myelin fibers in different regions, providing a new tool and strategy for high signal-to-noise ratio 3D deep imaging of brain myelin.

[0134] Embodiment 13

[0135] NQPI6 responses to Aβ fibrils in vitro

[0136] Given that one potential application of highly environmentally sensitive probes is to develop them as tools for detecting amyloid plaques (AD-specific biomarkers), we further explored whether the probe NQPI6 has the potential to become a probe for dual detection of myelin and amyloid plaques.

[0137] Aβ 1-42The polymer was prepared using a reported method (In Vivo Brain Imaging of Amyloid-βAggregates in Alzheimer's Disease with a Near-Infrared Fluorescent Probe. ACSSensors, 2021.6(3): p.863-870. 3D dynamic tracking Aβplaques in live brains using vinyl-bridged dyes with two-photon excitation / NIR emission and large Stokes shifts. Biosensors and Bioelectronics, 2023.238: p.115563.). 1-42 The lyophilized powder (P9001, Bio-Tech) was dissolved in 44 μL pre-cooled hexafluoroisopropanol (HFIP) to prepare Aβ 1-42 After sealing with a film, the mixture was shaken and incubated at room temperature for 1 h, and then the film was removed and HFIP / Aβ 1-42 Leave at room temperature overnight (12h). After HFIP evaporates, a transparent Aβ 1-42 Then, Aβ 1-42 The peptide film was redissolved in 8.8 μL DMSO to obtain a 5 mM concentration of Aβ 1-42 Peptide stock solution. The stock solution was diluted with pre-cooled PBS (10 mM, containing 1 mM EDTA) solution and then centrifuged at 16,000 × g for 10 min to obtain 50 μM Aβ 1-42 monomers and stored at 193K. 1-42 The monomers were incubated at 310K for different time periods (0-72h) to prepare Aβ with different aggregation states. 1-42 Peptide.

[0138] In vitro, it was observed that the probe NQPI6 (10 μM) was incubated with Aβ in PBS for 72 h. 1-42 After incubation with aggregates, the fluorescence intensity at 640 nm increased in a concentration-dependent manner ( Fig.10 AB). Kd value is as high as 1.266μM ( Fig.10 C), showing the relationship between 1-42 In addition, to evaluate whether NQPI6 would be affected by other interfering species in the organism, the probe was incubated with different metal ions (K + 、Zn 2+ , Cu2+ Mg 2+ , Ca 2+ 、Na + , Fe 2+ , Fe 3+ , 50 μM), amino acids (Lys, His, Val, Ala, Cys, Ile, 50 μM), GSH (50 mM) and representative proteins (BSA, HSA, 50 μg / mL) ( Fig.10 D), and found that these effects were almost negligible. These results indicate that NQPI6 is an ideal molecular tool for the specific detection of Aβ fibrils.

[0139] Embodiment 14

[0140] Molecular docking studies

[0141] In order to study the binding mode of NQPI6 to Aβ aggregates, Aβ 1-42The cryo-EM structure of the fibril (PDB ID: 5OQV) was used for molecular docking studies. The crystal structure of Aβ protein used for docking studies has been deposited in the RCSB Protein Data Bank (RCSB PDB ID: 5OQV). The covalent docking of NQPI6 with the Aβ protein binding pocket was performed using Schrodinger softwaresuite 2015 (Docking covalent inhibitors: a parameter free approach to pose prediction and scoring. J Chem Inf Model, 2014. 54(7): p. 1932-40.). All parameter settings for the docking study were default settings. In addition, the binding free energy between the docking pose and the protein receptor was calculated by Schrodinger package Prime3.9 using the MM-GBSA (Molecular mechanics with generalized Born and surface area solvation) model (A hierarchical approach to all-atom protein loop prediction. Proteins, 2004. 55 (2): p. 351-67. On the role of the crystal environment in determining protein side-chain conformations. J Mol Biol, 2002. 320 (3): p. 597-608.). The first binding site determined includes a tunnel along the fiber axis, which is composed of the side chains of Val36, Asp1, and Ile41. It was observed that NQPI6 binds to Aβ along the 1-42 The fiber is inserted into the tunnel to form the fiber axis, and the docking score is -7.250 kcal / mol ( Fig.11 A). Similarly, a second binding site was identified as a tunnel along the fiber axis consisting of the side chains of Phe19, Asn27, Gly29, and Ile31. NQPI6 binds along Aβ 1-42 The fiber is inserted into the tunnel and the fiber axis is formed. The docking score is -10.346 kcal / mol ( Fig.11 B). The third binding site is located in a groove along the fiber axis on the exposed surfaces of adjacent Phe20 and Glu22. NQPI6 can attach to the groove formed by the exposed surfaces of adjacent amino acid residues along the fiber axis, which results in a docking score of -4.773 kcal / mol ( Fig.11C). The last binding site is located in a groove along the fiber axis on the exposed surface adjacent to Lys16 and His14. NQPI6 can attach along the fiber axis in the groove formed by the exposed surfaces of adjacent amino acid residues, which results in a docking score of -2.763 kcal / mol (11D). It is noteworthy that the intermolecular forces between the probe and the tunnel of the protein fiber axis provide higher binding affinity and more stable planar structure compared with the probe attached to the protein surface.

[0142] Embodiment 15

[0143] Co-localization imaging of Aβ plaques

[0144] Given that NQPI6 can inhibit Aβ in vitro 1-42 Aggregates reacted well, and their imaging ability in brain tissue sections of 5x FAD model mice was further studied to explore its potential for simultaneous imaging of Aβ plaques and myelin. Transgenic mouse (5xFAD, 6 months old, male) brains were dissected and fixed in 4% paraformaldehyde (PFA) at 4°C overnight. The tissue was dehydrated and embedded in paraffin. 10μm sections were made using a microtome. Then, the sections were deparaffinized and rehydrated. Subsequently, the sections were subjected to antigen retrieval treatment with boiling sodium citrate buffer for 20min. The sections were then incubated with NQPI6 (10μM) for 30min at room temperature (RT) and rinsed with 1x PBS. After blocking with 5% BSA at room temperature (RT) for 30min, the sections were stained with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature (RT) for 30min. Cell nuclei were stained with DAPI (1mg / ml). Images were analyzed using a fluorescence microscope system (BX53, Olympus). The antibodies used were as follows: β-Amyloid (Catalog #8243, Cell Signaling Technology, 1:500), Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG (Cat #111-545-003, Jackson ImmunoResearch Labs, 1:500).

[0145] like Fig.12 As shown, brain sections of 5xFAD mice showed obvious red fluorescent signals after incubation with NQPI6. Co-localization studies of amyloid plaque-specific antibodies and NQPI6 confirmed that NQPI6 could specifically stain Aβ plaques in brain sections of 5xFAD model mice.

[0146] Example 16

[0147] 5x Simultaneous imaging of Aβ plaques and myelin pathology in FAD mouse brain sections

[0148] Myelin structure changes with age, but it is more obvious in AD. Dense plaque cores often have the greatest deleterious effects on surrounding nerve fibers, and focal demyelination can be observed in human cases. Given that the probe NQPI6 has good dual labeling ability for myelin and Aβ plaques, this probe was used to study the pathological changes associated with Aβ plaques in different brain regions of AD mice, including changes in myelin number and morphology, and further investigate the correlation between myelin and Aβ plaques during the pathogenesis of AD. Sagittal sections of 5x FAD mice and age-matched WT mice were prepared. Transgenic mice (5x FAD, 1, 3, and 6 months, male) and age-matched wild-type mice (WT, 1, 3, and 6 months, male) were anesthetized with isoflurane and killed by cervical dislocation under deep anesthesia. The whole brain was dissected and fixed in 4% paraformaldehyde (PFA) at 4°C overnight. The tissue was dehydrated and embedded in paraffin. 10μm sections were made using a microtome. Then, these sections were deparaffinized and rehydrated. The slices were incubated with NQPI6 (10 μM) for 30 min at room temperature (RT) and rinsed with 1x PBS. Detailed fluorescence imaging and analysis of different brain regions of these brain slices were performed using a confocal fluorescence microscope (Leica TCS SP8MP). DAPI channel: Ex, 405 nm; Em, 420-480 nm. NQPI6 channel 1: Ex, 488 nm; Em, 530-565 nm. NQPI6 channel 2: Ex, 561 nm; Em, 690-730 nm.

[0149] By comparing the imaging results ( Fig.13), and found that the fluorescence signals of NQPI6-stained 5xFAD mice in the cerebral cortex, hippocampus, and corpus callosum were different from those of WT mice of the same age. Aβ deposition seemed to induce focal demyelination, with the maximum demyelination occurring in the core of the plaque, and no myelin fibers passing through the amyloid plaque. Demyelination decreased with increasing distance from the plaque core. Given that myelin plays a crucial role in regulating the speed and integrity of axonal transmission, myelin lesions are likely to significantly contribute to the systemic cognitive decline characteristic of AD. To further verify whether myelin degeneration is a significant feature of AD, the number and morphological changes of myelin sheaths in different brain regions of 5xFAD mice were studied, and age-matched WT mice were used as controls. Compared with WT mice, the brain tissue structure and material composition of AD model mice in the cerebral cortex, hippocampus, and corpus callosum were significantly changed. Among them, the myelin sheath of the hippocampus is severely affected in AD and morphologically atrophied, indicating that in the pathological process of AD, there is widespread myelin loss in brain regions such as the hippocampus that are critical for memory and other cognitive functions. In addition, enhancing myelin renewal can alleviate cognitive impairment associated with attention deficit disorder. These results indicate that demyelination is closely related to the development of cognitive dysfunction in 5x FAD model mice.

[0150] Embodiment 17

[0151] Immunofluorescence imaging of 5xFAD brain tissue sections

[0152] Brains of transgenic mice (5xFAD, 6 months old, male) and age-matched wild-type mice (WT, 6 months old, male) were dissected and fixed in 4% paraformaldehyde (PFA) overnight at 4°C. Tissues were dehydrated and embedded in paraffin. 10 μm sections were made using a microtome. Then, the sections were deparaffinized and rehydrated. Subsequently, the sections were subjected to antigen retrieval treatment with boiling sodium citrate buffer for 20 min. After blocking in 5% BSA for 30 min, the sections were stained with primary antibodies (Mbp and β-Amyloid) at 4°C overnight, followed by incubation with secondary antibodies (Alexa Fluor 594AffiniPure Donkey Anti-Rabbit IgG and Alexa Fluor 488AffiniPure Goat Anti-Rat IgG) at RT for 30 min. Nuclei were counterstained with DAPI (1 mg / ml). Images were analyzed using a fluorescence microscope system (BX53, Olympus). The antibodies used were as follows: Mbp (Catalog # MAB386, Millipore, 1:100), β-Amyloid (Catalog # 8243, Cell Signaling Technology, 1:500), Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (Cat # 711-585-152, Jackson ImmunoResearch Labs, 1:500) and Alexa Fluor 488 AffiniPure Goat Anti-Rat IgG (Catalog # 112-545-003, Jackson ImmunoResearch Labs, 1:500).

[0153] like Fig.14 As shown in the figure, the same phenomenon as that in the NQPI6-incubated brain slices was observed in the immunostained brain slices incubated with MBP and Aβ antibodies. There was no myelin sheath around the plaques, and the demyelination phenomenon in the cerebral cortex and hippocampus of 5x FAD mice was more obvious than that in the corpus callosum. This shows that the probe NQPI6 can accurately image the myelin sheath and Aβ plaques in the brain of AD mice, and compared with antibody incubation, the probe NQPI6 incubation is more convenient.

[0154] In summary, the probes prepared by the present invention, such as NQPI6, are proven to be a powerful tool that can simultaneously detect Aβ plaques and myelin in the brain through dual channels, and is more accurate and convenient. This ability is very valuable for clarifying the intricate relationship between demyelination and Aβ plaque formation, and is of great significance for future research on the prevention, diagnosis and treatment of attention deficit disorder.

Claims

1. A myelin-responsive amphiphilic fluorescent probe having a pyridinium salt structure, characterized in that: The structure of the probe is shown in any of the following: The R is selected from any one of the following:

2. A method for preparing the myelin-responsive amphiphilic fluorescent probe NQPI4 having a pyridinium salt structure according to claim 1, characterized in that: The steps include: N,N-dimethyl-p-toluidine and (E)-2-butenal were added to HCl solution, stirred at room temperature, and an organic solvent was added, heated and stirred, and after the reaction, extraction, washing, drying, and concentration under reduced pressure were performed to obtain a crude product, and the crude product was purified to obtain a yellow-brown solid compound 1; SeO2 was dissolved in 1,4-dioxane / H2O, heated and stirred, then compound 1 was added, heated and stirred, after the reaction was completed, the reactant was cooled to room temperature, filtered and concentrated under reduced pressure to obtain a crude product, and the crude product was purified by chromatography to obtain yellow solid compound 2; Compound 2 and 1,4-dimethylpyridinium iodide were dissolved in an organic solvent, piperidine was added dropwise to the mixture, and heated and stirred. After the reaction was completed, the reactants were cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The crude product was dissolved and recrystallized to obtain a red solid NQPI4; The reaction formula is as follows:

3. A method for preparing the myelin-responsive amphiphilic fluorescent probe having a pyridinium salt structure according to claim 1, characterized in that: The steps include: Cesium carbonate, 2,2'-bis(diphenylphosphine)-1,1'-binaphthyl and palladium (II) acetate are dissolved in an organic solvent, and then 6-bromo-2-methylquinoline and different secondary amines are added, and refluxed to react. After the reaction, the solution is cooled to room temperature, and the residue obtained by extraction and concentration under reduced pressure is purified to obtain the desired compound 1R, wherein the secondary amine required for preparing NQPI1 is carbazole; the secondary amine required for preparing NQPI2 is 4,4'-dimethoxydiphenylamine; The secondary amine required for the preparation of NQPI3 is phenothiazine; the secondary amine required for the preparation of NQPI5 is azocyclobutane; the secondary amine required for the preparation of NQPI6 is tetrahydropyrrole; SeO2 is dissolved in 1,4-dioxane / H2O, heated and stirred, and then compound 1R is added, and the mixture is heated and stirred. After the reaction is completed, the reactant is cooled to room temperature and filtered. The filtrate is concentrated under reduced pressure to obtain a crude product, and the crude product is purified to obtain the corresponding aldehyde 2R; Compound 2R and 1,4-dimethylpyridinium iodide were dissolved in anhydrous ethanol, piperidine was added dropwise to the mixture, and the mixture was heated and stirred. After the reaction was completed, the reactants were cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The crude product was dissolved and recrystallized to obtain red solid NQPI1, 2, 3, 5 and 6; The reaction formula is as follows:

4. Use of the myelin-responsive amphiphilic fluorescent probe having a pyridinium salt structure according to claim 1 in the preparation of a drug or reagent for detecting myelin.

5. The use according to claim 4, characterized in that: The myelin sheath is preferably central nervous system and peripheral nervous system myelin sheath.

6. The use according to claim 4, characterized in that The myelin sheaths are myelin sheaths in different brain regions of brain slices, myelin sheaths in spinal cord slices, myelin sheaths in sciatic nerve slices and myelin sheaths in right occipital lobe slices.

7. The use according to claim 4, characterized in that The probe detects myelin for analyzing myelin density and mapping the trajectory of myelinated nerve fiber bundles.

8. Use of the myelin-responsive amphiphilic fluorescent probe with a pyridinium salt structure according to claim 1 in the preparation of a drug or reagent for simultaneous detection and imaging of myelin and Aβ aggregates in AD brains.

9. The use according to claim 8, characterized in that: The amphiphilic fluorescent probe is used in preparing drugs or reagents for imaging myelin integrity and Aβ aggregation in brain tissue.

10. The use according to claim 8, characterized in that: The amphiphilic fluorescent probe is used in preparing drugs or reagents for three-dimensional mapping and imaging of brain tissue myelin sheath and marking Aβ plaques in brain slices.

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