CN110229120B

CN110229120B - Long-wavelength fluorescent dye molecule and preparation method thereof

Info

Publication number: CN110229120B
Application number: CN201910523036.0A
Authority: CN
Inventors: 郑开波; 陈慧; 李梦; 周洋
Original assignee: China Three Gorges University CTGU
Current assignee: China Three Gorges University CTGU
Priority date: 2019-06-17
Filing date: 2019-06-17
Publication date: 2022-09-02
Anticipated expiration: 2039-06-17
Also published as: CN110229120A

Abstract

The invention discloses a long-wavelength fluorescent dye molecule, wherein the chemical formula of the fluorescent dye is C ₄₂ H ₂₉ NO ₂ S and its synthesis and biological application of fluorescent dye. The method for synthesizing the compound comprises the following steps: stirring 4- (1,2, 2-triphenylethylene) acetophenone and 4-benzothiazole-3-hydroxybenzaldehyde in an ethanol solution under an alkaline condition at room temperature for reacting for 2-16 hours, and separating and purifying after the reaction is finished to obtain the fluorescent dye molecule; the structure of the tetraphenylethylene selected by the invention has a larger conjugated system, a benzothiazole group introduced by utilizing an aldehyde-ketone condensation reaction has good light stability, and the compound has a chalcone structure to increase the conjugated degree; the compound has longer wavelength, small background interference, high fluorescence capable of entering cells and emitting strong signals, good cell membrane permeability and good light transmission.

Description

Long-wavelength fluorescent dye molecule and preparation method thereof

Technical Field

The invention belongs to the field of fluorescent dyes, and particularly relates to a novel organic fluorescent dye which is simple and easy to synthesize.

Background

The organic small molecule fluorescent dye is widely applied to aspects of biological imaging, biological molecule marking, environmental analysis, cell dyeing and clinical examination diagnosis, photochemical treatment, organic light emitting diodes and the like, and is an indispensable important tool in the fields of chemistry, biology, environmental science, medicine and material research.

Some traditional fluorescent dyes are easily influenced by temperature, solution pH value, concentration and the like, so that the fluorescent dyes are often limited in the process of carrying out fluorescent dyeing, and cannot meet the requirements of continuously-developed biological analysis and other application fields. In order to meet the requirement of the biological analysis application on the fluorescent dye, research and development of novel fluorescent materials with more excellent properties and fluorescence spectrum performance are the key and core of the development of the fluorescence analysis technology.

Disclosure of Invention

The invention aims to provide a novel organic fluorescent dye which is easy to synthesize and has fluorescence in solution.

The purpose of the invention is realized by the following technical scheme:

a novel fluorescent dye, the chemical structural formula of the dye is as follows:

the specific synthetic route of the fluorescent dye 1 is as follows:

the method comprises the following steps:

adding alkali into a reaction bottle at room temperature, and then adding a solvent to fully dissolve the alkali; adding 4- (1,2, 2-triphenylethylene) acetophenone into the solution, stirring to dissolve, adding 4-benzothiazole-3-hydroxybenzaldehyde, and stirring to react for 2-16 h at room temperature; adding water into the reaction solution, extracting with dichloromethane, drying with anhydrous sodium sulfate, spin-drying to obtain a crude product, and further separating and purifying by silica gel column chromatography to obtain yellow fluorescent dye 1;

wherein, the structural formulas of all the compounds are shown in a synthetic route; the molar ratio of the 4- (1,2, 2-triphenylethylene) acetophenone to the 4-benzothiazole-3-hydroxybenzaldehyde is 1 (0.5-4); the stationary phase used for the column chromatography is 100-200 mesh silica gel; the mobile phase of the column chromatography used for separating and purifying the compound 1 is a petroleum ether-dichloromethane mixed solution, and the volume ratio is (1-3): 1. The alcohol solution is absolute ethyl alcohol or a mixed solution of 10-12% NaOH solution and absolute ethyl alcohol in mass fraction, wherein the volume ratio of the 10-12% NaOH solution to the absolute ethyl alcohol is 1: 4.5-6.

Tetraphenylethylene (TPE) is a typical organic molecular chromophore with a large pi conjugated structure, and has been widely used in the related research fields of material chemistry, biochemistry and the like due to its characteristics of easy synthesis, convenience for functional group modification, good luminescence property and the like. Chalcones are α, β unsaturated carbonyl compounds, which are generally obtained by cross aldol condensation of an aromatic aldehyde and an aromatic ketone. The chalcone derivative has biological activities of oxidation resistance, depression resistance, cancer resistance, virus resistance and the like, can be used as a medicine, has a large conjugated structure and good optical properties, and can also be used as a functional material for organic nonlinear optics, optical storage, light sensitivity and the like. Chemical modification of the chalcone structure can be realized by changing aryl at two ends of an unsaturated bond, and the fluorescence quantum efficiency of a product and the wavelength of a compound can be greatly changed by changing the type of a substituent on the position of an aromatic ring.

The fluorescent dye emits light close to a red light area, can effectively avoid interference of self-absorption of cells and self-fluorescence, has stronger penetrating power of red light signals in organisms, has active site hydroxyl groups, can be simply modified into a fluorescent probe, and can be directly used for cell imaging dyeing and other fields.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of the fluorescent dye prepared in example 1 or example 2.

FIG. 2 shows the nuclear magnetic carbon spectrum of the fluorescent dye obtained in example 1 or example 2.

FIG. 3 is a graph showing fluorescence spectra of the fluorescent dye of example 3 in various solvents including dichloromethane, N-dimethylformamide, dimethylsulfoxide, acetonitrile, tetrahydrofuran, and toluene.

FIG. 4 is imaging of example 4 fluorescent dyes in B-16 cells: panel A shows the cellular imaging of fluorescence emission signals in the 530nm to 610nm range after dye solution treatment, and panel B shows the superposition of fluorescent imaging of cells treated with dye solution and bright field.

Detailed Description

Synthesis of dyes

The invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the scope of the examples.

Example 1: synthesis of fluorescent dye 1

Preparing 10% NaOH solution (EtOH: H) ₂ O, V is 5:1), stirring at room temperature to fully dissolve sodium hydroxide; 4- (1,2, 2-triphenylvinyl) acetophenone (74.7mg,0.2mmol) was added to the above 10% sodium hydroxide solution, and after stirring and dissolution, 4-benzothiazole-3-hydroxybenzaldehyde (50.8mg,0.2mmol) was added, and the reaction was continued at room temperature for 12 hours. After the reaction, 20mL of water was added to the reaction solution, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate and then spin-dried to obtain a crude product, which was then separated by silica gel column chromatography (petroleum ether: dichloromethane ═ 1: 1) to obtain a pure yellow product, dye 1, with a yield of 42.5%.

Example 2

Weighing 0.2g of sodium hydroxide, dissolving in 10mL of ethanol, and stirring at room temperature to fully dissolve the sodium hydroxide; 4- (1,2, 2-triphenylvinyl) acetophenone (74.9mg,0.2mmol) was added to the above ethanol solution of sodium hydroxide, and after stirring and dissolution, 4-benzothiazole-3-hydroxybenzaldehyde (51.2mg,0.2mmol) was added, and the reaction was stirred at room temperature for 16 hours. After the reaction, 20mL of water was added to the reaction solution, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate and then spin-dried to obtain a crude product, which was then separated by silica gel column chromatography (petroleum ether: dichloromethane ═ 1: 1) to obtain a pure yellow product, dye 1, with a yield of 54.1%.

Example 3: fluorescent Properties of Compounds

The fluorescent dye is accurately weighed, the compound is prepared into a fluorescent dye mother solution with the concentration of 500 mu M by using N, N-dimethylformamide, 30 mu L of the fluorescent dye mother solution is absorbed by a pipette and is respectively added into 3mL of different organic solvents (dichloromethane, N-dimethylformamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran and toluene), the fluorescent spectrum of the compound is tested after the compound is fully shaken up at room temperature, and the optical property data are shown in figure 3. The dye has good solvent denaturation and solvent compatibility.

Example 4:

dissolving a dye in DMF (dimethyl formamide) to prepare a fluorescent dye solution with the concentration of 500 mu M, culturing mouse B-16 melanoma cells by using the dye with the concentration of 5 mu M at 37 ℃ for 20 minutes, receiving a fluorescence emission signal in the range of 530 nm-610 nm by using a laser confocal scanning microscope with 405nm as an excitation wavelength, and observing that the signal is stronger in the cells, thereby indicating that the probe can be used for cell imaging.

Claims

1. Use of a long wavelength fluorescent dye molecule for the preparation of an organic fluorescent dye for B-16 melanoma cell imaging, characterized by: when the fluorescent dye is used in the process of B-16 melanoma cell imaging, 405nm is used as an excitation wavelength, a fluorescence emission signal is received in the range of 530 nm-610 nm, and the chemical formula of the fluorescent dye is C ₄₂ H ₂₉ NO ₂ S, the structural formula is as follows:

2. use according to claim 1, characterized in that the long wavelength fluorescent dye molecule preparation method comprises the following steps:

adding alkali into a reaction bottle at room temperature, adding absolute ethyl alcohol to fully dissolve the alkali, adding 4- (1,2, 2-triphenylethylene) acetophenone, stirring to fully dissolve the 4-benzothiazole-3-hydroxybenzaldehyde, stirring to react for 2-16 h, pouring the mixture into water gradually after the reaction is finished, extracting with dichloromethane, washing, drying, performing rotary evaporation concentration on an organic phase, and performing silica gel column chromatography separation to obtain a yellow fluorescent dye, wherein the reaction equation is as follows

3. The use according to claim 2, wherein the molar ratio of 4- (1,2, 2-tristyryl) acetophenone to 4-benzothiazole-3-hydroxybenzaldehyde is 1:0.5 to 4.

4. The use according to claim 2, wherein the temperature is 0 to 80 ℃; the alkali is any one of sodium hydroxide, potassium carbonate and piperidine.