CN116003448B - BODIPY near infrared fluorescent probe containing indoline polyethylenically recognized Abeta fiber and preparation method thereof - Google Patents

Abstract

The invention discloses a BODIPY near infrared fluorescent probe (TF-BODIPY) containing indoline polyethylenic acid for identifying Abeta fibers and a preparation method thereof. The compound is synthesized by adopting a Noruger condensation reaction of a fluoboric dipyrrole fluorescent dye and an aldehyde compound. Its maximum absorption and maximum emission wavelength in chloroform is in the near infrared region. Alzheimer's disease biomarkers are plaques formed in the brain of patients, which are mainly precipitated after aggregation of beta-amyloid (Abeta) fibers. The TF-BODIPY has a hydrophilic group and a hydrophobic group distributed at two ends, the distance between the two ends is exactly the same as the length of the HHQKLVFF amino acid fragment in the Abeta fiber, HHQK is a hydrophilic end, LVFF is a hydrophobic end, and the hydrophilic/hydrophobic structure of the TF-BODIPY is similar to that of the TF-BODIPY, so that the TF-BODIPY interacts with the amino acid fragment HHHQKLVFF of the Abeta fiber. The fluorescent probe designed from the angles of molecular structure and interval can target and specifically identify Abeta fiber, thereby achieving the purpose of diagnosing Alzheimer's disease.

Description

A BODIPY near-infrared fluorescent probe containing indoline polyene bonds for recognizing Aβ fibers and its preparation method

Technical Field

The invention belongs to the technical field of organic synthesis, relates to the preparation of fluorescent probes, and specifically relates to a BODIPY near-infrared fluorescent probe containing indoline polyene bonds for identifying Aβ fibers and a preparation method thereof.

Background technique

With the intensification of global aging, the elderly population is gradually increasing, and Alzheimer's disease (AD), as a neurodegenerative disease, often occurs in middle-aged and elderly people. There is a new case of AD every 3 seconds in the world. There are about 10 million AD patients in China, which is one of the countries with the largest number of AD patients in the world. There are currently two methods for diagnosing AD. One is to extract the patient's cerebrospinal fluid for testing through lumbar puncture; the other is the PET method that uses radioactive tracers to measure Aβ protein. Fluorescent probe technology is widely used in biological detection and can label biological macromolecules such as proteins containing specific groups. Fluorescent probes have particularly strong designability, and their own structural design is closely related to polymers.

Over the past few decades, various fluorescent probes have been developed for in vitro Aβ fluorescence imaging techniques and near-infrared (NIR) fluorescent probes for the detection of Aβ. However, these fluorescent probes still have some disadvantages, such as the ability to detect high background noise signals. Therefore, the study of new fluorescent probes with high sensitivity and high selectivity is of great significance for the diagnosis of AD.

Summary of the invention

In view of the shortcomings of the prior art, the purpose of the present invention is to provide a BODIPY near-infrared fluorescent probe containing indolinium polyene bonds for identifying Aβ fibers. The fluorescent probe has the advantages of near-infrared wavelength, high sensitivity and high selectivity for identifying Aβ fibers, a biomarker of late AD. Another purpose of the present invention is to provide a method for preparing the fluorescent probe.

The present invention is achieved through the following technical solutions:

A BODIPY near-infrared fluorescent probe containing indoline polyene bonds for recognizing Aβ fibers has a molecular structure as shown below:

A further improvement of the present invention is:

A method for preparing a BODIPY near-infrared fluorescent probe containing indoline polyene bonds for recognizing Aβ fibers comprises the following steps:

(1) using dimethylpyrrole and 6-(diethylamino)pyridine-3-carboxaldehyde as raw materials, dichloromethane as solvent, 2,3-dimethyl-5,6-dicyanobenzoquinone as catalyst, and boron trifluoride etherate as complexing agent to prepare BODIPY compound;

(2) using BODIPY and 2-furancarboxaldehyde as raw materials, toluene and piperidine as a mixed solvent, and reacting in the presence of p-toluenesulfonamide as a catalyst to prepare an intermediate compound O-BODIPY;

(3) Using O-BODIPY and 2-methylindoline as raw materials, tetrahydrofuran as solvent, and aluminum oxide as catalyst, the final product, near-infrared fluorescent probe TF-BODIPY, was prepared by reaction;

The reaction route is shown below:

A further improvement of the present invention is:

The specific process of step (1) is as follows: dimethylpyrrole and 6-(diethylamino)pyridine-3-carboxaldehyde are mixed and dissolved in dichloromethane, triethylamine is added, and the mixture is stirred at room temperature for 0.5 h to 6 h. Boron trifluoride ether is slowly added dropwise in an ice bath, and the mixture is stirred for 0.5 h to 6 h. 2,3-dimethyl-5,6- _{dicyanobenzoquinone} is added. The mixture is extracted with dichloromethane, dried over anhydrous _Na2SO4 , and the solvent is removed in vacuo at 25°C to 100°C. The mixture is separated and purified by a chromatography column to obtain a light yellow solid product, fluoroboron dipyrrole fluorescent dye BODIPY.

Furthermore, the molar ratio of dimethylpyrrole, 6-(diethylamino)pyridine-3-carboxaldehyde, triethylamine, boron trifluoride etherate, and 2,3-dimethyl-5,6-dicyanobenzoquinone is 1-3.3: 0.3-2.3: 0.3-2.3: 1-3: 0.03-0.23.

Furthermore, the specific process of reaction step (2) is as follows: BODIPY, 2-furancarboxaldehyde and p-toluenesulfonamide are dissolved in a mixed solution of toluene and piperidine, placed in a round-bottom flask equipped with a Dean-Stark apparatus, heated under reflux at 100° C. to 150° C. 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 TLC tracking until the reaction of the raw materials is complete, column chromatography is performed, and the solvent is removed by vacuum distillation to obtain a black solid product O-BODIPY.

Furthermore, the molar ratio of BODIPY, 2-furancarboxaldehyde and p-toluenesulfonamide is 0.1-1: 0.1-1.3: 0.01-0.13; and the volume ratio of toluene and piperidine is 1-3: 0.1-0.3.

Furthermore, the specific process of reaction step (3) is as follows: O-BODIPY is dissolved in tetrahydrofuran, placed in a round-bottom flask, 2-methylindoline is added, aluminum oxide is added, heated at 25°C to 100°C, the mixture is stirred for reaction until the raw material is consumed as observed by TLC, and then the product after the reaction is filtered and washed with ether to obtain TF-BODIPY. In this process, the introduced 2-methylindoline can bind to the hydrophobic segment of Aβ fiber to enhance fluorescence.

Furthermore, the molar ratio of O-BODIPY, 2-methylindoline and aluminum oxide is 1-1.5: 0.3-2.3: 0.03-0.23.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention is synthesized by using fluoroboron dipyrrole fluorescent dye and an aldehyde compound, and is used to detect Aβ fibers, a biomarker of late AD. This TF-BODIPY has a hydrophilic and a hydrophobic group, distributed at its two ends, and the distance between the two ends is exactly the same as the length of the HHQKLVFF amino acid fragment in the Aβ fiber. HHQK is the hydrophilic end and LVFF is the hydrophobic end. This is similar to the hydrophilic/hydrophobic structure of TF-BODIPY, so that TF-BODIPY interacts with the amino acid fragment HHQKLVFF of the Aβ fiber. This fluorescent probe designed from the perspective of molecular structure and spacing can target and specifically identify Aβ fibers to achieve the purpose of diagnosing Alzheimer's disease. At the same time, the preparation conditions of the compound are mild, the steps are simple, and it has good potential application value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a hydrogen NMR spectrum of TF-BODIPY obtained in Example 1 of the present invention;

FIG. 2 is a fluorescent staining presentation of AD mouse brain slices by TF-BODIPY obtained in Example 1 of the present invention.

Detailed ways

In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

The preparation method of the BODIPY fluorescent dye used in the following examples is as follows:

Weigh 356 mg (2.0 mmol) of 6-(diethylamino)pyridine-3-carboxaldehyde, dissolve it in 200 mL of freshly distilled dichloromethane, inject 412 mg (4.4 mmol) of 2,4-dimethylpyrrole and 3 ml of triethylamine with a syringe, stir rapidly with magnetic stirring, avoid light, stir at room temperature overnight, protect with nitrogen, slowly dropwise add 3 mL of boron trifluoride ether in an ice bath, stir for 10 minutes, then add 45.4 mg (0.2 mmol) of 2,3-dimethyl-5,6-dicyanobenzoquinone under stirring, track by TLC until the reaction of the raw material is complete, perform column chromatography, remove the solvent by reduced pressure distillation, extract with dichloromethane, dry over _{anhydrous Na2SO4} , remove the solvent in vacuo at 50°C, separate and purify by chromatography column to obtain fluoroboron dipyrrole fluorescent dye, i.e., BODIPY compound.

Example 1

(1) 214 mg (0.54 mmol) of BODIPY fluorescent dye, 52 mg (0.54 mmol) of 2-furancarboxaldehyde, and 1.7 mg (0.01 mmol) of p-toluenesulfonamide 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 mixture was heated under reflux at 142° C. 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 4 times. The process was tracked by TLC. After the reaction of the raw materials was complete, column chromatography was performed and the solvent was removed by vacuum distillation to obtain a solid product O-BODIPY.

(2) 194 mg (0.41 mmol) of O-BODIPY was dissolved in 2 mL of tetrahydrofuran, placed in a round-bottom flask, 54 mg (0.41 mmol) of 2-methylindoline and 15 mg (0.15 mmol) of aluminum oxide were added, and the mixture was heated at 90° C. and stirred until the raw material was consumed as observed by TLC. The product after the reaction was then filtered and rinsed with ether to obtain a black solid compound TF-BODIPY (211.5 mg, yield 85%). The compound was identified as the target product by nuclear magnetic 1H NMR spectrum.

Example 2

(1) 198 mg (0.5 mmol) of BODIPY fluorescent dye, 96 mg (1 mmol) of 2-furancarboxaldehyde, and 1.7 mg (0.01 mmol) of p-toluenesulfonamide 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 4 times. The process was tracked by TLC. After the reaction of the raw materials was complete, column chromatography was performed and the solvent was removed by vacuum distillation to obtain a solid product O-BODIPY.

(2) 194 mg (0.41 mmol) of O-BODIPY was dissolved in 4 mL of tetrahydrofuran, placed in a round-bottom flask, 108 mg (0.82 mmol) of 2-methylindoline and 15 mg (0.15 mmol) of aluminum oxide were added, heated at 90° C., and the mixture was stirred until the starting material was consumed as observed by TLC. The product after the reaction was then filtered and rinsed with ether to obtain a black solid compound TF-BODIPY (224 mg, yield 90%). The compound was identified as the target product by nuclear magnetic 1H NMR spectrum.

Example 3

(1) 198 mg (0.54 mmol) of BODIPY fluorescent dye, 78 mg (0.81 mmol) of 2-furancarboxaldehyde, and 1.7 mg (0.01 mmol) of p-toluenesulfonamide 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 mixture was heated under reflux at 142° C. 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 4 times. The process was tracked by TLC. After the reaction of the raw materials was complete, column chromatography was performed and the solvent was removed by distillation under reduced pressure to obtain a solid product O-BODIPY.

(2) 194 mg (0.41 mmol) of O-BODIPY was dissolved in 2 mL of tetrahydrofuran, placed in a round-bottom flask, 82 mg (0.62 mmol) of 2-methylindoline and 10 mg (0.01 mmol) of aluminum oxide were added, heated at 90° C., and the mixture was stirred until the starting material was consumed as observed by TLC. The product after the reaction was then filtered and rinsed with ether to obtain a black solid compound TF-BODIPY (228 mg, yield 92%). The compound was identified as the target product by nuclear magnetic 1H NMR spectrum.

Example 4: Application of the substance prepared in Example 1

TF-BODIPY detection and analysis of brain tissue slices of Alzheimer's mice:

Adult Alzheimer's disease model mouse APPswe/PS1dE9 brain tissue was used for histological analysis. Part of the hippocampus tissue was embedded in paraffin and sliced 10μm thick. The slices were stained with 50% ethanol TF-BODIPY solution (100uM) for 2 hours, washed with 50% ethanol for 20 minutes, and rinsed and covered with fluorescent preservation reagent (Merck Millipore) under a 640nm confocal laser microscope (Nikon A1R) for histological evaluation. It was found that TF-BODIPY had a special response to Aβ fibers, which indicates that TF-BODIPY has potential application value in the diagnosis of Alzheimer's disease.

The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims (8)

1. A BODIPY near-infrared fluorescent probe containing indolinium polyene bonds for recognizing Aβ fibers, characterized in that the fluorescent probe TF-BODIPY has the molecular structure shown below: 2. The method for preparing a BODIPY near-infrared fluorescent probe containing indoline polyene bonds for recognizing Aβ fibers according to claim 1, characterized in that it comprises the following steps: (1) using dimethylpyrrole and 6-(diethylamino)pyridine-3-carboxaldehyde as raw materials, dichloromethane as solvent, 2,3-dimethyl-5,6-dicyanobenzoquinone as catalyst, and boron trifluoride etherate as complexing agent to prepare BODIPY compound; (2) using BODIPY and 2-furancarboxaldehyde as raw materials, toluene and piperidine as a mixed solvent, and reacting in the presence of p-toluenesulfonamide as a catalyst to prepare an intermediate compound O-BODIPY; (3) Using O-BODIPY and 2-methylindoline as raw materials, tetrahydrofuran as solvent, and aluminum oxide as catalyst, the final product, near-infrared fluorescent probe TF-BODIPY, was prepared by reaction; The reaction route is shown below: 3. The method for preparing a BODIPY near-infrared fluorescent probe containing indolinium polyene bonds for recognizing Aβ fibers according to claim 2, characterized in that: the specific process of reaction step (1) is: dissolving dimethylpyrrole and 6-(diethylamino)pyridine-3-carboxaldehyde in dichloromethane, adding triethylamine, stirring at room temperature for 0.5h to 6h, slowly dropping boron trifluoride ether in an ice bath, stirring for 0.5h to 6h, adding 2,3-dimethyl-5,6-dicyanobenzoquinone, extracting with _{dichloromethane} , drying with anhydrous _Na2SO4 , removing the solvent in vacuo at 25°C to 100°C, separating and purifying with a chromatography column to obtain a light yellow solid product, fluoroboron dipyrrole fluorescent dye BODIPY. 4. The method for preparing a BODIPY near-infrared fluorescent probe containing indolinium polyene bonds for recognizing Aβ fibers according to claim 3, characterized in that the molar ratio of dimethylpyrrole, 6-(diethylamino)pyridine-3-carboxaldehyde, triethylamine, boron trifluoride etherate, and 2,3-dimethyl-5,6-dicyanobenzoquinone is 1-3.3:0.3-2.3:0.3-2.3:1-3:0.03-0.23. 5. The method for preparing a BODIPY near-infrared fluorescent probe containing indolinium polyene bonds for recognizing Aβ fibers according to claim 2, characterized in that: the specific process of reaction step (2) is: BODIPY, 2-furancarboxaldehyde, and p-toluenesulfonamide are dissolved in a mixed solution of toluene and piperidine, placed in a round-bottom flask equipped with a Dean-Stark device, heated under reflux at 100° C. to 150° C. until all the solvent is collected by the Dean-Stark device, then toluene and piperidine are added to the reaction medium, and the process is repeated at least once. After TLC tracking is performed until the raw materials react completely, column chromatography is performed, and the solvent is removed by reduced pressure distillation to obtain a black solid product O-BODIPY. 6. The method for preparing a BODIPY near-infrared fluorescent probe containing indolinium polyene bonds for recognizing Aβ fibers according to claim 5, characterized in that the molar ratio of BODIPY, 2-furancarboxaldehyde, and p-toluenesulfonamide is 0.1-1: 0.1-1.3: 0.01-0.13; and the volume ratio of toluene and piperidine is 1-3: 0.1-0.3. 7. The method for preparing a BODIPY near-infrared fluorescent probe containing indoline polyene bonds for recognizing Aβ fibers according to claim 2, characterized in that: the specific process of reaction step (3) is: dissolving O-BODIPY in tetrahydrofuran, placing it in a round-bottom flask, adding 2-methylindoline, adding aluminum oxide, heating at 25° C. to 100° C., reacting and stirring the mixture until the raw material is observed to be consumed by TLC, and then filtering the product after the reaction and washing it with ether to obtain TF-BODIPY. 8. The method for preparing a BODIPY near-infrared fluorescent probe containing indoline polyene bonds for recognizing Aβ fibers according to claim 7, characterized in that the molar ratio of O-BODIPY, 2-methylindoline and aluminum oxide is 1-1.5: 0.3-2.3: 0.03-0.23.