阅读说明：本技术 一类基于香豆素骨架的近红外荧光染料及其合成方法 (Near-infrared fluorescent dye based on coumarin skeleton and synthetic method thereof ) 是由 李楠 赵娜 尹伟 李悦 于 2018-12-21 设计创作，主要内容包括：本发明公开了一类基于香豆素结构的近红外荧光染料及其合成方法,该荧光染料的结构通式为<Image he="308" wi="656" file="DDA0001914709740000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>式中R代表C<Sub>1</Sub>烷基、X<Sup>-</Sup>代表PF<Sub>6</Sub><Sup>-</Sup>,或者R代表C<Sub>2</Sub>～C<Sub>12</Sub>烷基中任意一种,X<Sup>-</Sup>代表Br<Sup>-</Sup>。本发明将4-(二乙氨基)香豆素通过Vilsmeier-Haack反应合成4-(二乙氨基)香豆素醛,再依次与氰基吡啶、不同碳链长度的卤代烷烃反应即得到系列荧光染料。本发明荧光染料通过对铵盐侧链的调控可以特异性的标记活细胞中的亚细胞器,而且R代表C<Sub>3</Sub>烷基的荧光染料可实现标记过程的快速染色以及免洗；荧光染料的最大发射波长处于红色发光区域,可显著消除细胞成像中自荧光现象的干扰；此外R代表C<Sub>9</Sub>、C<Sub>12</Sub>烷基的荧光染料具有产生单线态氧的性质,在细胞及细菌的光动力治疗中有一定的应用潜能。(The invention discloses a near-infrared fluorescent dye based on a coumarin structure and a synthesis method thereof, wherein the structural general formula of the fluorescent dye is shown in the specification Wherein R represents C 1 Alkyl radical, X ‑ Represents PF 6 ‑ Or R represents C 2 ～C 12 Any one of alkyl radicals, X ‑ Represents Br ‑ . The invention synthesizes 4- (bis-diethylamino) coumarin into 4- (bis-coumarin) through Vilsmeier-Haack reactionEthylamino) coumarin aldehyde, and then reacting with cyanopyridine and halogenated alkanes with different carbon chain lengths in sequence to obtain the series of fluorescent dyes. The fluorescent dye can specifically mark subcellular organelles in living cells by regulating and controlling ammonium salt side chains, and R represents C 3 The alkyl fluorescent dye can realize quick dyeing and no-washing in the marking process; the maximum emission wavelength of the fluorescent dye is in a red light emitting area, so that the interference of an autofluorescence phenomenon in cell imaging can be obviously eliminated; in addition R represents C 9 、C 12 The alkyl fluorescent dye has the property of generating singlet oxygen and has certain application potential in the photodynamic treatment of cells and bacteria.)

1. A near-infrared fluorescent dye based on a coumarin skeleton is characterized in that the structural general formula of the fluorescent material is as follows:

wherein R represents C₁Alkyl radical, X^-Represents PF₆ ^-Or R represents C₂～C₁₂Any one of alkyl radicals, X^-Represents Br^-。

2. A method of preparing a fluorescent dye according to claim 1, wherein:

(1) reacting phosphorus oxychloride and N, N-dimethylformamide at a molar ratio of 1: 1.1-1.3 at room temperature, adding an N, N-dimethylformamide solution of 4- (diethylamino) coumarin shown in formula I, reacting at 50-70 ℃ for 20-24 hours, and separating and purifying a product to obtain a compound shown in formula II;

(2) taking ethanol as a solvent, reacting the compound of the formula II, 4-acetonitrile pyridine hydrochloride and triethylamine at 40-50 ℃ for 10-12 hours, and separating and purifying a product to obtain a compound of the formula III;

(3) taking N, N-dicarboximide as a solvent, reacting the compound shown in the formula III with methyl iodide at 70-90 ℃ for 8-10 hours, separating and purifying a product, dissolving the product in acetonitrile, adding potassium hexafluorophosphate, reacting at room temperature for 10-12 hours, and filtering after the reaction is finished to obtain the compound with R as C₁Alkyl radical, X^-Is PF₆ ^-The fluorescent dye of (1);

or using N, N-dimethyl imide as solventReacting a compound shown in the formula III with halogenated alkane at the temperature of 70-90 ℃ for 8-10 hours, and separating and purifying a product to obtain a product with R as C₂～C₁₂Any one of alkyl, X^-Is Br^-The fluorescent dye of (1); wherein said haloalkane is C₂～C₁₂Any one of the above brominated alkanes.

3. The method for producing a fluorescent dye according to claim 2, characterized in that: in the step (1), the molar ratio of the compound shown in the formula I to phosphorus oxychloride is 1: 8-9.

4. The method for producing a fluorescent dye according to claim 2, characterized in that: in the step (2), the molar ratio of the compound of the formula II, 4-acetonitrile pyridine hydrochloride and triethylamine is 1: 2-3: 3-5.

5. The method for producing a fluorescent dye according to claim 2, characterized in that: in the step (3), the molar ratio of the compound shown in the formula III to methyl iodide and potassium hexafluorophosphate is 1: 8.5-12.

6. The method for producing a fluorescent dye according to claim 2, characterized in that: in the step (3), the molar ratio of the compound shown in the formula III to the halogenated alkane is 1: 8.5-12.

Technical Field

The invention belongs to the technical field of fluorescent dyes for biomedicine, and particularly relates to a fluorescent dye with near-infrared luminescence property and a preparation method of the fluorescent dye.

Background

The cytoplasmic membrane is the two-dimensional boundary between a living cell and its environment. Vital activities associated with cytoplasmic membranes including dynamic membrane remodeling, signal transduction, and nutrient transport are the focus of research by cell biologists. To study these cellular processes, it is important to develop subcellular fluorescence imaging probes that can recognize the cytoplasmic membrane and record the dynamic changes. To date, only a small number of commercial fluorescent dyes have been available to track cell membranes, such as DiO₃And DiI. However, most of the current commercial fluorescent dyes emit light in the visible region, producing background fluorescence, resulting in a low signal-to-noise ratio (S/N). The red luminescent fluorescent dye can not only overcome the defect, but also avoid the damage of the laser light source to the cell tissue. Therefore, it is important to design and synthesize a fluorescent dye having red luminescence property.

Mitochondria are "energy factories" of cells that use oxygen for oxidative phosphorylation to produce Adenosine Triphosphate (ATP) to provide energy to cells and organisms. At the same time, with the leakage of electrons in the respiratory chain, a variety of Reactive Oxygen Species (ROS) are rapidly produced within mitochondria. Mitochondrial ROS play an important role in maintaining redox balance and participating in regulation of cell proliferation, differentiation, apoptosis and other behaviors. When the ROS level exceeds the body's antioxidant defenses, disease can result. Therefore, the development of a method for accurately detecting the ROS in the mitochondria has great significance for deeply exploring the regulation and control of the cell function of the ROS and the occurrence and development of related diseases. Due to the characteristics of low concentration, short service life, high reactivity and the like of ROS, accurate detection of ROS is a great challenge in the fields of chemistry, biology and medicine. The fluorescence imaging technology has the remarkable advantages of high space-time resolution, good biocompatibility, high sensitivity and the like, and becomes a powerful tool for detecting ROS in cells and living bodies in real time.

Disclosure of Invention

The invention aims to provide a near-infrared fluorescent dye based on a coumarin skeleton and a preparation method of the fluorescent dye.

In view of the above object, the structural general formula of the fluorescent dye adopted by the invention is as follows:

wherein R represents C₁Alkyl radical, X^-Represents PF₆ ^-Or R represents C₂～C₁₂Any one of alkyl radicals, X^-Represents Br^-。

The preparation method of the near-infrared fluorescent dye based on the coumarin skeleton comprises the following steps:

1. reacting phosphorus oxychloride and N, N-dimethylformamide at a molar ratio of 1: 1.1-1.3 at room temperature, adding an N, N-dimethylformamide solution of 4- (diethylamino) coumarin shown in formula I, reacting at 50-70 ℃ for 20-24 hours, and separating and purifying a product to obtain a compound shown in formula II.

2. And (2) taking ethanol as a solvent, reacting the compound of the formula II, 4-acetonitrile pyridine hydrochloride and triethylamine at 40-50 ℃ for 10-12 hours, and separating and purifying a product to obtain the compound of the formula III.

3. Taking N, N-dicarboximide as a solvent, reacting the compound shown in the formula III with methyl iodide at 70-90 ℃ for 8-10 hours, separating and purifying a product, and dissolving the product in the solventAdding potassium hexafluorophosphate into acetonitrile, reacting for 10-12 hours at room temperature, and filtering after the reaction is finished to obtain R as C₁Alkyl radical, X^-Is PF₆ ^-The fluorescent dye of (1);

or taking N, N-dicarboximide as a solvent, reacting the compound shown in the formula III with halogenated alkane at 70-90 ℃ for 8-10 hours, and separating and purifying the product to obtain the product with R as C₂～C₁₂Any one of alkyl, X^-Is Br^-The fluorescent dye of (1); wherein said haloalkane is C₂～C₁₂Any one of the above brominated alkanes.

In the step 1, the molar ratio of the compound of the formula I to the phosphorus oxychloride is preferably 1: 8-9.

In the step 2, the compound of the formula II, the 4-acetonitrile pyridine hydrochloride and the triethylamine are preferably in a molar ratio of 1: 2-3: 3-5.

In the step 3, the molar ratio of the compound of the formula III to methyl iodide and potassium hexafluorophosphate is preferably 1: 8.5-12, and the molar ratio of the compound of the formula III to haloalkane is preferably 1: 8.5-12.

The invention has the following beneficial effects:

1. the method utilizes a simple framework of coumarin, firstly takes N, N-diethyl as a power supply group and cyanopyridine as an electron-withdrawing group to form an intramolecular electron-withdrawing-electron-effect system, and then obtains a target compound through modification of different alkyl side chains. The compounds have red light emission property, can obviously avoid interference of autofluorescence in a fluorescence imaging process, and can carry out positioning labeling on different subcellular structures through side chain regulation.

2. In the structural formula of the fluorescent dye, R represents C₉And C₁₂The alkyl group has the characteristic of generating singlet oxygen, and can effectively generate the singlet oxygen, thereby being applied to photodynamic therapy. By utilizing the characteristics, the bacteriostatic and bactericidal experiment can be carried out.

Drawings

FIG. 1 is a graph showing the UV absorption spectra of the fluorescent dyes prepared in examples 1 to 5 in DMSO.

FIG. 2 is a fluorescence spectrum of the fluorescent dye prepared in examples 1 to 5 in DMSO.

FIG. 3 is a photograph showing a cell image of the fluorescent dye prepared in example 1.

FIG. 4 is a photograph of a cell image of the fluorescent dye prepared in example 2.

FIG. 5 is a photograph of a cell image of the fluorescent dye prepared in example 3.

FIG. 6 is a photograph of a cell image of the fluorescent dye prepared in example 4.

FIG. 7 is a photograph showing a cell image of the fluorescent dye prepared in example 5.

FIG. 8 is a photograph of a cell wash-free image of the fluorescent dye prepared in example 2.

FIG. 9 is a graph of cellular images of fluorescent dye-labeled cell membranes prepared in example 2 over time.

FIG. 10 is a graph showing the UV-VIS absorption spectrum of the fluorescent dye prepared in example 1 during irradiation with white light in DI water.

FIG. 11 is a graph showing the UV-VIS absorption spectrum of the fluorescent dye prepared in example 2 during irradiation with white light in DI water.

FIG. 12 is a graph showing the UV-VIS absorption spectrum of the fluorescent dye prepared in example 3 during irradiation with white light in DI water.

FIG. 13 is a graph showing the UV-VIS absorption spectrum of the fluorescent dye prepared in example 4 during irradiation with white light in DI water.

FIG. 14 is a graph showing the UV-VIS absorption spectrum of the fluorescent dye prepared in example 5 during irradiation with white light in DI water.

FIG. 15 is a graph of the relative intensity of the absorbance at 378nm during white light irradiation in deionized water for the fluorescent dyes prepared in examples 1-5 and in the absence of the fluorescent dye.

FIG. 16 is a graph showing the bactericidal effect of the fluorescent dye prepared in example 5 on E.coli under white light irradiation.

FIG. 17 is a graph showing the bactericidal effect of the fluorescent dye prepared in example 5 on Staphylococcus aureus under white light irradiation.

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.