Near-infrared organic amine BODIPY fluorescent probe capable of recognizing A beta fibers with high sensitivity as well as preparation method and application of near-infrared organic amine BODIPY fluorescent probe

Abstract

The invention belongs to the technical field of organic synthesis, and particularly discloses a near-infrared organic amine BODIPY fluorescent probe capable of recognizing A beta fibers with high sensitivity and a preparation method and application thereof.The near-infrared organic amine BODIPY fluorescent probe is synthesized by enabling para-organic amine substituted benzaldehyde and BODIPY fluorescent dye to be subjected to a Knoevenagel condensation reaction; according to the present invention, the synthesized organic amine BODIPY compound can be combined with diphenylalanine dipeptide so as to achieve the selective recognition of the Abeta fiber in the hippocampus, the organic amine is combined with the BODIPY fluorescent dye, such that the optical property of the BODIPY can be effectively regulated due to the strong electron supply ability and the low steric hindrance so as to achieve the selective recognition of the Abeta fiber in the hippocampus; and the sensitivity and the performance in AD application are improved. The enhanced signal response is helpful for realizing the rapid identification of the A beta fiber, and provides a potential strategy for the prevention and treatment of AD (Alzheimer's disease).

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and relates to the preparation of a fluorescent probe for AD diagnosis, and specifically to a near-infrared organic amine BODIPY fluorescent probe for highly sensitively identifying Aβ fibers, and a preparation method and application thereof. Background Art

[0002] Alzheimer's Disease (AD) is a common neurodegenerative disease, with main symptoms including memory loss, cognitive decline, and abnormal behavior and emotions. At present, about 50 million people in the world are suffering from AD, most of whom are elderly. It is predicted that by 2050, the number of AD patients worldwide will surge to 152 million. With the intensification of global aging and the pursuit of healthy quality of life, the demand for AD treatment continues to grow. According to the "World Alzheimer's Report" released by AD International, the global AD treatment market size is expected to increase from approximately US$180 billion in 2020 to approximately US$310 billion in 2027, with a compound annual growth rate of 5.8%. As one of the world's largest AD treatment markets, China's market size will continue to expand.

[0003] Studies have shown that in the pathological process of Alzheimer's disease (AD), excessive aggregation of Aβ (amyloid β protein) peptides forms fibrillar structures. The deposition of these structures in the brain leads to neuronal damage and death, which in turn causes cognitive dysfunction. However, in the process of Aβ folding and aggregation, phenylalanine dipeptide (FF) plays a key role. As the core motif of Aβ, FF can self-assemble into fibers and accelerate the occurrence of AD. If FF can be detected quickly and effectively in the human body, Aβ aggregates can be identified, thereby diagnosing AD. That is, Aβ peptide is one of the main targets for early diagnosis and intervention treatment of AD.

[0004] The development of BODIPY-type fluorescent probes can achieve specific identification of Aβ fibers. However, the existing BODIPY-type fluorescent probes have the problem of low detection sensitivity. Therefore, the development of a BODIPY-type fluorescent probe with high stability, high selectivity and high sensitivity will make an important contribution to enriching the types of BODIPY-type fluorescent probes and promoting the early diagnosis and intervention treatment of AD. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers. The present invention combines organic amine with fluoroboron dipyrrole fluorescent dye, which can specifically identify Aβ fibers and significantly improve the detection sensitivity, which has important research value for the fluorescence detection of AD. The present invention also provides a preparation method of the near-infrared organic amine BODIPY fluorescent probe.

[0006] The present invention is achieved through the following technical solutions:

[0007] A near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers, the structural formula is shown below:

[0008]

[0009] Wherein, R is selected from dimethylamine or diethylamine.

[0010] The present invention introduces dimethylamino or diethylamine into the BODIPY molecule, and both dimethylamino and diethylamine improve the optical properties of BODIPY through electron supply capacity and stereo effect. Both dimethylamino and diethylamine have strong electron supply capacity, and can increase the electron density of BODIPY through resonance effect, so that the absorption and emission wavelengths are red-shifted, and this red-shift phenomenon is beneficial to improving the sensitivity of BODIPY in the near-infrared region.

[0011] A further improvement of the present invention is:

[0012] A method for preparing a near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers comprises the following steps:

[0013] (1) reacting 4-(dimethylamino)benzaldehyde with 2,4-dimethylpyrrole to generate a compound BDP-N;

[0014] (2) reacting the compound BDP-N with benzaldehyde substituted with an organic amine at the para position to generate the target compound;

[0015] The reaction route is as follows:

[0016]

[0017] Wherein, R is selected from dimethylamine or diethylamine.

[0018] A further improvement of the present invention is:

[0019] The process of step (1) is as follows: 4-(dimethylamino)benzaldehyde and 2,4-dimethylpyrrole are dissolved in dichloromethane, trifluoroacetic acid is added dropwise, 2,3-dimethyl-5,6-dicyanobenzoquinone is used as a catalyst, and boron trifluoride ether is used as a complexing agent to react to obtain the fluoroboron dipyrrole fluorescent dye BDP-N.

[0020] Furthermore, the molar ratio of 4-(dimethylamino)benzaldehyde to 2,4-dimethylpyrrole is 1:2 to 2.5.

[0021] Furthermore, the process of step (2) is as follows: the compound BDP-N, the benzaldehyde substituted with the para-position organic amine, and the catalyst toluenesulfonamide are dissolved in a mixed solution of toluene and piperidine, placed in a round-bottom flask equipped with a Dean-Stark apparatus, heated to reflux until all the solvent is collected by the Dean-Stark apparatus, and then toluene and piperidine are added to the reaction medium, and the process is repeated at least once. After the reaction is complete, the target compound is purified.

[0022] Furthermore, the molar ratio of the compound BDP-N, the benzaldehyde substituted with the para-position organic amine and the catalyst toluenesulfonamide is 1:1-3:0.01-0.03.

[0023] Furthermore, the reaction temperature of step (2) is 110°C-160°C.

[0024] A further improvement of the present invention is:

[0025] Application of a near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers in the preparation of Alzheimer's disease diagnostic reagents.

[0026] The beneficial effects of the present invention are:

[0027] The present invention utilizes fluoroboron dipyrrole (BODIPY) fluorescent dye and 4-(dimethylamino)benzaldehyde and 4-(diethylamine)benzaldehyde to synthesize a novel compound. BODIPY compound can bind to diphenylalanine dipeptide in Aβ protein, and through fluorescence imaging technology, their distribution and aggregation in the brain can be observed, thereby revealing the basic morphology of diphenylalanine dipeptide fibers, which is helpful for the identification of Aβ fibers. Organic amines, such as dimethylamino and diethylamine, are important bioactive molecules. When considering improving the sensitivity of BODIPY dye, it is key to select appropriate amino substituents. Both dimethylamino and diethylamine enhance the optical properties of BODIPY through electron supply capacity and stereo effect. Both dimethylamino and diethylamine have strong electron supply capacity, can increase the electron density of BODIPY through resonance effect, and make the absorption and emission wavelengths red-shifted. This red-shift phenomenon is conducive to improving the sensitivity of BODIPY in the near-infrared region.

[0028] The present invention uses a probe to test the sensitivity of Aβ fibers and obtains that the detection limits of BDP, BDP-3N-2 and BDP-3N-1 are 1350pg / ml, 183pg / ml and 157pg / ml respectively. Due to the role of the amine (dimethylamino, diethylamine) substituents in BDP-3N-2 and BDP-3N-1, their detection limits are low and they are easier to detect, thereby improving the sensitivity. In addition, the dimethylamino group has a strong electron supply capacity and lower steric hindrance, so the sensitivity is stronger than that of BDP-3N-2.

[0029] The present invention combines organic amines with fluoroboron dipyrrole fluorescent dyes, which can more effectively regulate the optical properties of BODIPY due to their strong electron supply capacity and low steric hindrance, and improve its sensitivity and performance in AD applications. This enhanced signal response helps to achieve rapid identification of Aβ fibers and provides a potential strategy for the prevention and treatment of AD. In addition, the preparation process of BODIPY compounds is mild and simple, which shows their great potential and application value in the biomedical field, especially in the early diagnosis of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the nuclear magnetic hydrogen spectrum of the near-infrared organic amine BODIPY fluorescent probe for identifying Aβ fibers obtained by the present invention.

[0031] Figure 2 This is the mass spectrum of the near-infrared organic amine BODIPY fluorescent probe for identifying Aβ fibers obtained in the present invention.

[0032] Figure 3 This is a UV-fluorescence spectrum characterization diagram of the near-infrared BODIPY fluorescent probe BDP-3N-1 for identifying Aβ fibers obtained in the present invention.

[0033] Figure 4 This is a UV-fluorescence spectrum characterization diagram of the near-infrared BODIPY fluorescent probe BDP-3N-2 for identifying Aβ fibers obtained in the present invention.

[0034] Figure 5 The BODIPY compounds obtained in the present invention are presented by fluorescent staining for the recognition of Aβ fibers in the mouse hippocampus.

[0035] Figure 6 The sensitivity detection limit of the near-infrared organic amine BODIPY fluorescent probe for identifying Aβ fibers obtained by the present invention is measured. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with specific embodiments.

[0037] Example 1: Synthesis of fluoroboron dipyrrole fluorescent dye BDP-N

[0038] The reaction equation is as follows:

[0039]

[0040] The preparation method of the near-infrared organic amine BODIPY fluorescent probe intermediate used in the following examples is as follows: weigh 212 mg (2.0 mmol) of benzaldehyde, dissolve it in 200 mL of freshly distilled dichloromethane, inject 412 mg (4.4 mmol) of 2,4-dimethylpyrrole with a syringe, quickly add 2 drops of trifluoroacetic acid, quickly stir magnetically, avoid light, stir overnight at room temperature, and protect with nitrogen. Then add 454 mg (2 mmol) of 2,3-dimethyl-5,6-dicyanobenzoquinone under stirring. Continue stirring and add 3 mL of freshly distilled triethylamine, and stir for 10 minutes. Slowly add 3 mL of boron trifluoride ether in an ice bath. After 10 hours, TLC is tracked until the raw material reacts completely, column chromatography, and the solvent is removed by reduced pressure distillation to obtain a green fluorescent fluoroboron dipyrrole fluorescent dye.

[0041] Example 2: Synthesis of target compound BDP-3N-1

[0042] The reaction equation is as follows:

[0043]

[0044] Fluoroboron dipyrrole fluorescent dye (0.54 mmol), 4-(dimethylamino)benzaldehyde (1.18 mmol), and p-toluenesulfonamide (0.01 mmol) were dissolved in a mixed solution of toluene (25 mL) and piperidine (1 mL), placed in a round-bottom flask equipped with a Dean-Stark apparatus, and heated under reflux at 142°C until all the solvent was collected by the Dean-Stark apparatus. Toluene (25 mL) and piperidine (1 mL) were added to the reaction medium, and heating under reflux at 142°C was continued until all the solvent was collected by the Dean-Stark apparatus. The process of adding toluene (25 mL) and piperidine (1 mL) and heating under reflux was repeated 4 times. This process was tracked by TLC. After the raw material reaction was complete, column chromatography was performed and the solvent was removed by reduced pressure distillation to obtain a dark green solid product BDP-3N-1. The compound was characterized by nuclear magnetic resonance. 1 HNMR spectrum, mass spectrum MS EI + To characterize, 1H NMR (300MHz, DMSO-D6): δ=7.59(d,J=8.8Hz,4H), 7.16(d,J=8.8Hz,2H), 6.95-6.99(m,2 H), 6.75-6.79(m,7H), 6.01(d,2H), 5.67(s,H), 3.02(s,6H), 2.29(s,H), 1.95(s,H); MS EI + :629.6.

[0045] Example 3: Synthesis of target compound BDP-3N-2

[0046] The reaction equation is as follows:

[0047]

[0048] Fluoroboron dipyrrole fluorescent dye (0.54 mmol), 4-(diethylamine)benzaldehyde (1.62 mmol), and p-toluenesulfonamide (0.01 mmol) were dissolved in a mixed solution of toluene (25 mL) and piperidine (1 mL), placed in a round-bottom flask equipped with a Dean-Stark apparatus, and heated under reflux at 142°C until all the solvent was collected by the Dean-Stark apparatus. Toluene (25 mL) and piperidine (1 mL) were then added to the reaction medium, and the heating under reflux at 142°C was continued until all the solvent was collected by the Dean-Stark apparatus. The addition of toluene (25 mL) and piperidine (1 mL) and the heating under reflux were repeated twice. The process was tracked by TLC. After the raw material reacted completely, column chromatography was performed and the solvent was removed by reduced pressure distillation to obtain a black solid product. The compound was characterized by nuclear magnetic resonance. 1 HNMR spectrum, mass spectrum MS EI + To characterize, 1 H NMR (300MHz, DMSO-D6): δ = 7.59 (d, J = 8.8Hz, 4H), 7.16 (d, J = 8.8Hz, 2H), 6.77-6.99 (m, 9H) , 6.01(d,2H), 5.67(s,H), 3.4(m,4H), 3.02(s,2H), 2.29(s,H), 1.95(s,H), 1.12(m,4H); MS EI + :685.7.

[0049] Example 4: Analysis of the recognition of Aβ fibers by compounds BDP-3N-1 and BDP-3N-2

[0050] Diphenylalanine dipeptide (3 mg) was dissolved in 45 μl hexafluoroisopropanol, and then 105 μl of water was added by injection. After ultrasonication at room temperature for 5 minutes, diphenylalanine dipeptide self-assembled into nanofibers. Hexafluoroisopropanol was dialyzed with water to obtain diphenylalanine dipeptide nanofibers stored in water for later use. Compound BDP-3N-1 or BDP-3N-2 (1 mg) was dissolved in 45 μl hexafluoroisopropanol and added to the diphenylalanine dipeptide nanofiber aqueous solution by injection. After 2 hours, fluorescently labeled diphenylalanine dipeptide nanofibers were obtained to achieve the effect on Aβ fibers.

[0051] Example 5: Detection and analysis of AD mouse brain tissue sections using compounds BDP-3N-1 and BDP-3N-2:

[0052] The brain tissue of adult Alzheimer's disease model mice APPswe / PS1dE9 was used for histological analysis. Part of the hippocampus tissue was embedded in paraffin and sliced ​​10 μm thick. The slices were stained with 50% ethanol compound BDP-3N-1 or BDP-3N-2 solution (100uM) for 2 hours, washed with 50% ethanol for 20 minutes, and washed and covered with fluorescent preservation reagent for histological evaluation under a confocal laser microscope at 640nm. The results are shown in Figure 5 As shown, it was found that compound BDP-3N-1 or BDP-3N-2 had a special response to Aβ fibers, which indicates that compound BDP-3N-1 or BDP-3N-2 has a certain recognition effect on Aβ fibers and has potential application value in the early diagnosis of Alzheimer's disease.

[0053] Figure 6 The detection limit results of the compounds BDP-3N-1 and BDP-3N-2 synthesized by the present invention show that the detection limits of BDP-3N-1 and BDP-3N-2 are very low; due to the role of the amine (dimethylamino, diethylamine) substituents in BDP-3N-2 and BDP-3N-1, their detection limits are low and they are easier to detect, thereby improving the sensitivity; and the dimethylamino group has a strong electron donation capacity and lower steric hindrance, so the sensitivity is stronger than that of BDP-3N-2.

[0054] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers, characterized in that: The structural formula is as follows: Wherein, R is selected from dimethylamine or diethylamine.

2. The method for preparing a near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers according to claim 1, characterized in that: The following steps are involved: (1) reacting 4-(dimethylamino)benzaldehyde with 2,4-dimethylpyrrole to generate a compound BDP-N; (2) reacting the compound BDP-N with benzaldehyde substituted with an organic amine at the para position to generate the target compound; The reaction route is as follows: Wherein, R is selected from dimethylamine or diethylamine.

3. The method for preparing a near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers according to claim 2, characterized in that: The process of step (1) is as follows: 4-(dimethylamino)benzaldehyde and 2,4-dimethylpyrrole are dissolved in dichloromethane, trifluoroacetic acid is added dropwise, 2,3-dimethyl-5,6-dicyanobenzoquinone is used as a catalyst, and boron trifluoride ether is used as a complexing agent to react to obtain the fluoroboron dipyrrole fluorescent dye BDP-N.

4. A method for preparing a near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers according to claim 2 or 3, characterized in that: The molar ratio of the 4-(dimethylamino)benzaldehyde to 2,4-dimethylpyrrole is 1:2 to 2.

5.

5. The method for preparing a near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers according to claim 2, characterized in that: The process of step (2) is as follows: the compound BDP-N, the benzaldehyde substituted with the para-position organic amine, and the catalyst toluenesulfonamide are dissolved in a mixed solution of toluene and piperidine, placed in a round-bottom flask equipped with a Dean-Stark apparatus, heated to reflux until all the solvent is collected by the Dean-Stark apparatus, and then toluene and piperidine are added to the reaction medium, and the process is repeated at least once. After the reaction is complete, the target compound is purified.

6. The method for preparing a near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers according to claim 5, characterized in that: The molar ratio of the compound BDP-N, the benzaldehyde substituted by the para-position organic amine and the catalyst toluenesulfonamide is 1:1-3:0.01-0.

03.

7. The method for preparing a near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers according to claim 2 or 5, characterized in that: The reaction temperature of step (2) is 110°C-160°C.

8. Use of the near-infrared organic amine BODIPY fluorescent probe for highly sensitive identification of Aβ fibers as claimed in claim 1 in the preparation of diagnostic reagents for Alzheimer's disease.

Images