阅读说明：本技术 一种检测溶酶体极性变化的荧光探针及其制备方法和应用 (Fluorescent probe for detecting polarity change of lysosome and preparation method and application thereof ) 是由 强涛涛 王宝帅 梁天宇 胡伟 于 2021-06-21 设计创作，主要内容包括：本发明公开了一种检测溶酶体极性变化的荧光探针及其制备方法和应用,属于分析化学技术领域。所述检测溶酶体极性变化的荧光探针的结构式为：所述制备方法先通过4-溴-1,8-萘二甲酸酐与N-(2-氨基乙基)吗啉的取代反应制得Nap-Br,再继续与4-乙炔基苯胺进行缩合反应制得。所述检测溶酶体极性变化的荧光探针能够检测溶酶体极性改变,具有良好的光学稳定性以及对极性的特异性响应；能够有效应用于检测溶酶体极性变化,同时解决了特定细胞器靶向性差和生物样品损伤大的问题。(The invention discloses a fluorescent probe for detecting polarity change of lysosome, and a preparation method and application thereof, and belongs to the technical field of analytical chemistry. The structural formula of the fluorescent probe for detecting the polarity change of the lysosome is as follows:)

1. A fluorescent probe for detecting polarity change of lysosome is characterized in that the structural formula is as follows:

2. the method of claim 1, comprising the steps of:

(1) uniformly dispersing 4-bromo-1, 8-naphthalic anhydride and N- (2-aminoethyl) morpholine in a solvent A for substitution reaction, and after the substitution reaction is finished, separating and purifying the obtained substitution product mixture to obtain Nap-Br;

(2) mixing CuI and Pd (PPh)₃)₂Cl₂Uniformly dispersing Nap-Br and 4-ethynylaniline obtained in the step (1) in a solvent B, and taking Pd (PPh) in a CuI environment₃)₂Cl₂And carrying out condensation reaction for a catalyst, and after the condensation reaction is finished, separating and purifying the obtained condensation product mixture to obtain the fluorescence probe for detecting polarity change of lysosomes.

3. The method according to claim 2, wherein the molar ratio of 4-bromo-1, 8-naphthalic anhydride to N- (2-aminoethyl) morpholine in step (1) is 1.0: 1.0-1.5.

4. The preparation method according to claim 2, wherein in the step (2), the molar ratio of Nap-Br to 4-ethynylaniline is 1.0: 1.0-1.5.

5. The method according to claim 2, wherein in step (2), CuI and Pd (PPh)₃)₂Cl₂The mol ratio of the Nap-Br to the Nap-Br obtained in the step (1) is 0.003:0.4: 1.0.

6. The method according to claim 2, wherein in the step (1), the temperature of the substitution reaction is 40 ℃;

in the step (2), the temperature of the condensation reaction is 25-30 ℃.

7. The preparation method according to claim 2, wherein in the step (1), the time of the substitution reaction is 1-5 h;

in the step (2), the condensation reaction time is 10-24 h.

8. The method according to claim 2, wherein in the step (1), the solvent A is ethanol;

in the step (2), a mixed solution of tetrahydrofuran and triethylamine is used as a solvent B; wherein the volume ratio of the tetrahydrofuran to the triethylamine is 1-3: 1.

9. The use of the fluorescent probe for detecting polarity changes of lysosomes according to claim 1 or prepared by the preparation method of any one of claims 2 to 8 in the detection of polarity changes of lysosomes.

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a fluorescent probe for detecting polarity change of lysosomes, and a preparation method and application thereof.

Background

Lysosome belongs to one of cellular microenvironment and plays a very important role in pathological research, and lysosome is a recycling and processing center of intracellular substances and plays an important role in aspects of autophagy, metabolism, energy circulation, cell membrane repair and the like. The polarity change of lysosomes often influences the progress of various metabolism in cells. Lysosomes have a major role in the regulation of biological physiological processes, and abnormalities in polarity are associated with many diseases. Therefore, the detection of the change of polarity in the lysosome is of great significance for the study of autophagy and the pathological analysis of organisms.

The fluorescent probe technology has the advantages of real-time monitoring, high efficiency, accuracy, high sensitivity and the like, so that the fluorescent probe technology becomes an important detection means for detecting the micro environment of biomolecules and organisms. Compared with a single photon probe, the two-photon probe has unique advantages in the aspects of high tissue penetration depth, small biological sample damage and the like, and is applied to biological imaging. Therefore, the development of a novel two-photon fluorescent probe has important significance for detecting the polarity of lysosomes.

The polar probe at the present stage cannot position important organelles generated by autophagy due to lack of targeting property and two-photon characteristic, and is often uniformly diffused in the organelles after entering a cell body, so that the polar probe is lack of targeting property. Meanwhile, the single photon probe at the present stage has the characteristic of short excitation wavelength, so that the damage to the sample is large, and the long-time observation of the biological sample cannot be realized, so that the relationship between autophagy and inflammation cannot be further researched.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a fluorescent probe for detecting polarity change of lysosomes, a preparation method and application thereof, solves the problems of poor targeting property of a specific organelle and large damage of a biological sample, and provides a reasonable tool for exploring the occurrence of intracellular inflammation and autophagy.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a fluorescent probe for detecting polarity change of lysosome, which has the following structural formula:

the invention discloses a preparation method of the fluorescence probe for detecting polarity change of lysosomes, which comprises the following steps:

(1) uniformly dispersing 4-bromo-1, 8-naphthalic anhydride and N- (2-aminoethyl) morpholine in a solvent A for substitution reaction, and after the substitution reaction is finished, separating and purifying the obtained substitution product mixture to obtain Nap-Br;

(2) mixing CuI and Pd (PPh)₃)₂Cl₂Uniformly dispersing Nap-Br and 4-ethynylaniline obtained in the step (1) in a solvent B, and taking Pd (PPh) in a CuI environment₃)₂Cl₂And carrying out condensation reaction for a catalyst, and after the condensation reaction is finished, separating and purifying the obtained condensation product mixture to obtain the fluorescence probe for detecting polarity change of lysosomes.

Preferably, in the step (1), the molar ratio of the 4-bromo-1, 8-naphthalic anhydride to the N- (2-aminoethyl) morpholine is 1.0: 1.0-1.5.

Further preferably, the molar ratio of 4-bromo-1, 8-naphthalic anhydride to N- (2-aminoethyl) morpholine is 1.0: 1.3.

Preferably, in the step (2), the molar ratio of Nap-Br to 4-ethynylaniline is 1.0: 1.0-1.5.

Further preferably, the mole ratio of Nap-Br to 4-ethynylaniline is 1.0: 1.1.

Preferably, in step (2), CuI, Pd (PPh)₃)₂Cl₂The mol ratio of the Nap-Br to the Nap-Br obtained in the step (1) is 0.003:0.4: 1.0.

Preferably, in step (1), the temperature of the substitution reaction is 40 ℃.

Preferably, in the step (2), the temperature of the condensation reaction is 25-30 ℃.

Preferably, in the step (1), the time of the substitution reaction is 1-5 h.

Preferably, in the step (2), the condensation reaction time is 10-24 h.

Further preferably, the time of the substitution reaction is 2 h.

Further preferably, the time of the condensation reaction is 12 h.

Preferably, in step (1), the solvent a is ethanol.

Preferably, in the step (2), a mixed solution of tetrahydrofuran and triethylamine is used as the solvent B; wherein the volume ratio of the tetrahydrofuran to the triethylamine is 1-3: 1.

Further preferably, the mixing volume ratio of tetrahydrofuran and triethylamine is 2: 1.

The invention discloses an application of the fluorescence probe for detecting polarity change of lysosomes or the fluorescence probe for detecting polarity change of lysosomes prepared by the preparation method in detecting polarity change of lysosomes.

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

the invention discloses a fluorescence probe for detecting polarity change of lysosomes, wherein in the structure of the fluorescence probe for detecting polarity change of the lysosomes, naphthalimide is a two-photon fluorophore, a morpholine group is a lysosome targeting group, and 4-ethynylaniline is an optical adjusting handle. Probe molecule Nap-NH₂As a D-pi-A compound, the compound has stronger polar response effect. For probe molecules Nap-NH₂In other words, due to the existence of the 4-ethynylaniline structure, the probe has a polar response property. Therefore, the fluorescent probe for detecting the polarity change of the lysosome can effectively solve the problems of poor targeting property and large damage to biological samples.

The invention discloses a preparation method of the fluorescence probe for detecting the polarity change of the lysosome, which can carry out substitution reaction on the oxygen element of 4-bromo-1, 8-naphthalic anhydride through the high activity of the amino group in N- (2-aminoethyl) morpholine, and can quickly and efficiently combine a two-photon naphthalimide fluorophore with a lysosome targeting group morpholine. The optical adjusting handle ethynylaniline can be rapidly connected to the molecule through electrophilic reaction of the acetylene group in the 4-ethynylaniline to halogen. Therefore, the preparation method has the advantages of simple and convenient process and high yield.

The invention discloses the application of the fluorescent probe for detecting polarity change of lysosome in detecting the polarity of the lysosomeIn the case of the variant, the polarity of lysosomes is reduced during autophagy. The fluorescent probe molecule Nap-NH for detecting polarity change of lysosome in the invention₂In other words, since the 4-ethynylaniline structure enables the probe to have a polarity response property, the fluorescence intensity of the probe gradually increases with the decrease of the polarity of the solvent. The invention utilizes the alkalescence property of morpholine groups to target acidic organelle lysosomes. Meanwhile, the polar response is realized by using the two-photon property of naphthalimide as a two-photon fluorophore and 4-aminoacetylene as an optical adjusting handle. Therefore, the fluorescent probe for detecting polarity change of the lysosome can describe the polarity change of the lysosome through the change of fluorescence intensity, and further explore the relation between autophagy and inflammation.

In conclusion, the invention discloses a fluorescent probe for detecting polarity change of lysosomes, which can detect polarity change of the lysosomes and has good optical stability and specific response to polarity; realizes the detection of the polarity change in the living body and the detection of the relationship between the autophagy and the inflammation. Meanwhile, the invention provides a preparation method of the probe, which has the advantages of simple steps, convenience in optimization and high yield. Can be applied to the aspect of researching the relation between the cell autophagy and the inflammation of a living body.

Drawings

FIG. 1 shows a fluorescent probe for detecting polarity changes of lysosomes according to the invention¹H NMR spectrum;

FIG. 2 shows the detection of polarity changes of lysosome with the fluorescent probe of the present invention¹³A C NMR spectrum;

FIG. 3 is an emission spectrum of the lysosomal polarity change-detecting fluorescent probe of the present invention in solvents of different polarities;

FIG. 4 is a graph of fluorescence imaging of the lysosomal polarity alteration detection fluorescent probe of the present invention in PC12 cells for lysosomal polarity alteration; wherein, (a) is fluorescence confocal imaging of polar response in an inflammation model constructed by PC12 cells, and (b) is a histogram of relative fluorescence intensity under different conditions after the inflammation model induces autophagy.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The fluorescent probe for detecting the polarity change of the lysosome has lysosome positioning capacity and is named as Nap-NH₂The structural formula of the fluorescence probe for detecting the polarity change of the lysosome is shown as a formula (1):

the fluorescent probe for detecting the polarity change of the lysosome has good response and sensitivity to the polarity, and takes PC12 cells as a research object, namely a probe Nap-NH₂Can be applied to the process of cell lysosome autophagy to monitor the cell inflammation induced by autophagy inhibiting drugs in tanning residuesThe therapeutic effect in the process promotes the understanding of autophagy and inflammation. Meanwhile, the fluorescent probe for detecting polarity change of lysosome provided by the invention is simple to synthesize, convenient to separate and purify and high in yield. The following is a method for preparing the fluorescent probe for detecting polarity change of lysosome according to the present invention.

The preparation method of the fluorescence probe for detecting polarity change of the lysosome comprises the following steps:

(1) 4-bromo-1, 8-naphthalic anhydride (1) and N- (2-aminoethyl) morpholine (2) are subjected to substitution reaction in ethanol as a solvent A, and Nap-Br (3) is obtained through separation and purification, wherein the chemical reaction formula of the step is as follows:

(2) in a CuI environment, a mixed solution of tetrahydrofuran and triethylamine is used as a solvent B (wherein the volume ratio of the mixture of the tetrahydrofuran and the triethylamine is 1: 1-3), and Pd (PPh) is added to the mixed solution of Nap-Br (3) and 4-ethynylaniline (4)₃)₂Cl₂Carrying out condensation reaction for a catalyst, and separating and purifying to obtain a final product Nap-NH₂(5) Namely the fluorescent probe for detecting the polarity change of the lysosome. The chemical reaction formula of this step is as follows.

In the step (1), the molar ratio of the 4-bromo-1, 8-naphthalic anhydride to the N- (2-aminoethyl) morpholine is 1.0: 1.0-1.5, preferably 1.0: 1.3. In the step (1), the temperature of the substitution reaction was 40 ℃. In the step (1), the time of the substitution reaction is 1-5 h; preferably, the reaction time is 2 h.

In the step (2), the molar ratio of Nap-Br to 4-ethynylaniline is 1.0: 1.0-1, and preferably is 1.0: 1.1. In the step (2), the condensation reaction temperature is 25-30 ℃, and preferably, the reaction temperature is 25 ℃. In the step (2), the condensation reaction time is 10-24 h; preferably, the reaction time is 12 h.

In the step (2), CuI and Pd (PPh)₃)₂Cl₂The mol ratio of the Nap-Br to the Nap-Br obtained in the step (1) is 0.003:0.4: 1.0.

In the step (1), the separation and purification step comprises: and cooling the reacted system to room temperature, performing suction filtration, and performing vacuum drying.

In the step (2), the separation and purification step is as follows: and (3) carrying out column chromatography separation to obtain a purified product, wherein mobile phases separated by the column chromatography separation comprise dichloromethane and petroleum ether with the volume ratio of 10: 1.

In the step (2), the reaction is carried out in an inert gas protective atmosphere.

In the step (2), the mixing volume ratio of tetrahydrofuran and triethylamine is preferably 2: 1.

The mechanism of the fluorescent probe for detecting the polarity change of the lysosome is as follows: in the structure of the fluorescence probe for detecting the polarity change of the lysosome, naphthalimide is a two-photon fluorophore, a morpholine group is a lysosome targeting group, and 4-ethynylaniline is an optical adjusting handle. Probe molecule Nap-NH₂As a D-pi-A compound, the compound has stronger polar response effect. In the process of autophagy, lysosome endocytosis causes the polarity of the lysosome membrane to gradually increase, and thus the polarity of lysosome becomes smaller. For probe molecules Nap-NH₂In other words, due to the existence of the 4-ethynylaniline structure, the probe has a polarity response property, and the fluorescence intensity of the probe gradually increases along with the decrease of the polarity of the solvent. Therefore, the fluorescent probe for detecting polarity change of the lysosome can describe the polarity change of the lysosome through the change of fluorescence intensity, and further explore the relation between autophagy and inflammation. Therefore, the fluorescent probe for detecting polarity change of lysosome can be applied to detect polarity of lysosome of cells, and the fluorescent probe for detecting polarity change of lysosome is applied to change of polarity of lysosome caused by cell autophagy so as to research the relationship between the cell autophagy and inflammation.

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples.

Example 1

(1) 277mg of 4-bromo-1, 8-naphthalic anhydride (1mmol) and 169mg of N- (2-aminoethyl) morpholine (1.3mmol) were weighed into a 2ml ethanol round-bottomed flask and heated under reflux at 40 ℃ for 2h for substitution. And after the substitution reaction is finished, cooling to room temperature, carrying out suction filtration and vacuum drying on the substitution product mixture to obtain Nap-Br.

(2) 3.89g of the compound Nap-Br (10mmol), 36.79mg of Pd (PPh)₃)₂Cl₂(0.03mmol) and 7.62mg of CuI (4mmol) were charged into a round-bottomed flask, dissolved in a mixed solution of 50ml of tetrahydrofuran and 50ml of triethylamine, and then purged with nitrogen for 15 minutes to obtain a reaction system. 1.18g of 4-ethynylaniline (10.10mmol) was further dissolved in 50ml of tetrahydrofuran, and the solution was added to the above reaction system at 25 ℃ in a CuI atmosphere using Pd (PPh)₃)₂Cl₂A condensation reaction is carried out for the catalyst. And (2) refluxing and stirring the mixture for 12h, finishing the condensation reaction to obtain a condensation product mixture, performing rotary evaporation to remove various solvents in the condensation product mixture to obtain a reaction system concentrated solution, and purifying the obtained reaction system concentrated solution by using column chromatography, wherein mobile phases separated by the column chromatography are dichloromethane and petroleum ether with the volume ratio of 10: 1. Removing the mobile phase solvent from the purified product by a rotary evaporation method, and finally drying in vacuum to obtain the fluorescent probe Nap-NH for detecting the polarity change of the lysosome₂. The yield in this example was 95%.

The fluorescent probe for detecting polarity change of lysosome prepared in the example¹H NMR spectrum as shown in FIG. 1, of the Probe¹³C NMR spectrum is shown in FIG. 2:

¹HNMR(400MHz,DMSO-d6)δ8.66(d,J＝8.3Hz,1H),8.47(d,J＝7.1Hz,1H),8.34(d,J＝7.6Hz,1H),8.01–7.81(m,2H),7.43(d,J＝8.2Hz,2H),6.63(d,J＝8.3Hz,2H),5.86(s,2H),4.14(t,J＝6.5Hz,2H),3.53(s,4H),2.59–2.51(m,2H),2.45(s,4H).

¹³CNMR(101MHz,DMSO)δ163.62,163.31,151.19,133.97,132.57,131.59,130.95,130.65,129.98,128.29,128.24,127.85,122.84,120.86,114.11,107.27,102.84,84.93,66.69,55.98,53.87,37.32.HRMS m/z calc.for C₂₆H₂₃N₃O₃:426.18122；found:426.18127[M+H]⁺.

example 2

(1) 277mg of 4-bromo-1, 8-naphthalic anhydride (1mmol) and 130mg of N- (2-aminoethyl) morpholine (1.0mmol) were weighed into a 2ml ethanol round-bottomed flask and heated under reflux at 40 ℃ for 1h for substitution. And after the substitution reaction is finished, cooling to room temperature, carrying out suction filtration and vacuum drying on the substitution product mixture to obtain Nap-Br.

(2) 3.89g of the compound Nap-Br (10mmol), 36.79mg of Pd (PPh)₃)₂Cl₂(0.03mmol) and 7.62mg of CuI (4mmol) were charged into a round-bottomed flask, dissolved in a mixed solution of 100ml of tetrahydrofuran and 50ml of triethylamine, and then purged with nitrogen for 15 minutes to obtain a reaction system. 1.61g of 4-ethynylaniline (10.50mmol) was further dissolved in 50ml of tetrahydrofuran and added to the above reaction system at 30 ℃ in a CuI atmosphere as Pd (PPh)₃)₂Cl₂A condensation reaction is carried out for the catalyst. And (2) refluxing and stirring the mixture for 24h, finishing the condensation reaction to obtain a condensation product mixture, performing rotary evaporation to remove various solvents in the condensation product mixture to obtain a reaction system concentrated solution, and purifying the obtained reaction system concentrated solution by using column chromatography, wherein mobile phases separated by the column chromatography are dichloromethane and petroleum ether with the volume ratio of 10: 1. Removing the mobile phase solvent from the purified product by a rotary evaporation method, and finally drying in vacuum to obtain the fluorescent probe Nap-NH for detecting the polarity change of the lysosome₂。

The yield of this example was 70%.

Example 3

(1) 277mg of 4-bromo-1, 8-naphthalic anhydride (1mmol) and 195mg of N- (2-aminoethyl) morpholine (1.5mmol) were weighed into a 2ml ethanol round-bottomed flask and heated under reflux at 40 ℃ for 3 hours for substitution. And after the substitution reaction is finished, cooling to room temperature, carrying out suction filtration and vacuum drying on the substitution product mixture to obtain Nap-Br.

(2) 3.89g of the compound Nap-Br (10mmol), 36.79mg of Pd (PPh)₃)₂Cl₂(0.03mmol), 7.62mg of CuI (4mmol) were charged into a round-bottomed flask, and dissolved with a mixed solution of 80ml of tetrahydrofuran and 50ml of triethylamineThen, nitrogen gas was blown for 15 minutes to obtain a reaction system. 1.07g of 4-ethynylaniline (10.00mmol) was further dissolved in 50ml of tetrahydrofuran, and the resulting solution was added to the above reaction system at 27 ℃ in the presence of Pd (PPh) in a CuI atmosphere₃)₂Cl₂A condensation reaction is carried out for the catalyst. And (2) refluxing and stirring the mixture for 18h, finishing the condensation reaction to obtain a condensation product mixture, performing rotary evaporation to remove various solvents in the condensation product mixture to obtain a reaction system concentrated solution, and purifying the obtained reaction system concentrated solution by using column chromatography, wherein mobile phases separated by the column chromatography are dichloromethane and petroleum ether with the volume ratio of 10: 1. Removing the mobile phase solvent from the purified product by a rotary evaporation method, and finally drying in vacuum to obtain the fluorescent probe Nap-NH for detecting the polarity change of the lysosome₂。

The yield in this example was 80%.

Example 4

(1) 277mg of 4-bromo-1, 8-naphthalic anhydride (1mmol) and 143mg of N- (2-aminoethyl) morpholine (1.1mmol) were weighed out and introduced into a 2ml round-bottomed flask with ethanol, and the mixture was heated under reflux at 40 ℃ for 5 hours to effect substitution reaction. And after the substitution reaction is finished, cooling to room temperature, carrying out suction filtration and vacuum drying on the substitution product mixture to obtain Nap-Br.

(2) 3.89g of the compound Nap-Br (10mmol), 36.79mg of Pd (PPh)₃)₂Cl₂(0.03mmol) and 7.62mg of CuI (4mmol) were charged into a round-bottomed flask, dissolved in a mixed solution of 35ml of tetrahydrofuran and 50ml of triethylamine, and then purged with nitrogen for 15 minutes to obtain a reaction system. 1.39g of 4-ethynylaniline (10.30mmol) was further dissolved in 50ml of tetrahydrofuran and added to the above reaction system at 28 ℃ in a CuI atmosphere as Pd (PPh)₃)₂Cl₂A condensation reaction is carried out for the catalyst. And (2) refluxing and stirring the mixture for 10 hours, finishing the condensation reaction to obtain a condensation product mixture, performing rotary evaporation to remove various solvents in the condensation product mixture to obtain a reaction system concentrated solution, and purifying the obtained reaction system concentrated solution by using column chromatography, wherein mobile phases separated by the column chromatography are dichloromethane and petroleum ether with the volume ratio of 10: 1. Removing the mobile phase solvent from the purified product by rotary evaporation, and vacuum drying to obtain the final productThe fluorescent probe Nap-NH for detecting polarity change of lysosome₂。

The yield in this example was 65%.

Example 5

(1) 277mg of 4-bromo-1, 8-naphthalic anhydride (1mmol) and 182mg of N- (2-aminoethyl) morpholine (1.4mmol) were weighed into a 2ml ethanol round-bottomed flask and heated under reflux at 40 ℃ for 3.5h for substitution. And after the substitution reaction is finished, cooling to room temperature, carrying out suction filtration and vacuum drying on the substitution product mixture to obtain Nap-Br.

(2) 3.89g of the compound Nap-Br (10mmol), 36.79mg of Pd (PPh)₃)₂Cl₂(0.03mmol) and 7.62mg of CuI (4mmol) were charged into a round-bottomed flask, dissolved in a mixed solution of 25ml of tetrahydrofuran and 50ml of triethylamine, and purged with nitrogen for 15 minutes to obtain a reaction system. 1.29g of 4-ethynylaniline (10.20mmol) was further dissolved in 25ml of tetrahydrofuran, and the resulting solution was added to the above reaction system at 26 ℃ in a CuI atmosphere using Pd (PPh)₃)₂Cl₂A condensation reaction is carried out for the catalyst. And refluxing and stirring the mixture for 15h, finishing the condensation reaction to obtain a condensation product mixture, performing rotary evaporation to remove various solvents in the condensation product mixture to obtain a reaction system concentrated solution, and purifying the obtained reaction system concentrated solution by using column chromatography, wherein mobile phases separated by the column chromatography are dichloromethane and petroleum ether with the volume ratio of 10: 1. Removing the mobile phase solvent from the purified product by a rotary evaporation method, and finally drying in vacuum to obtain the fluorescent probe Nap-NH for detecting the polarity change of the lysosome₂。

The yield in this example was 60%.

Example 6

The emission spectrum of the fluorescence probe for detecting the polarity change of the lysosome in solvents with different polarities

A fluorescent probe Nap-NH for detecting polarity changes of lysosomes with the concentration of 1mM obtained in example 1 is configured₂The test mother liquor of dimethyl sulfoxide (DMSO) is ready for use. In the test solution, organic solvents with different polarities are respectively taken, including Dioxane, dichromethane and dimethyl formamideDimethylformamide), dimethyl sulfoxide (dimethyl sulfoxide), Toluene (Toluene), Methanol (Methanol), Chloroform (Chloroform), Tetrahydrofuran (Tetrahydrofuran) and glycerol (glycerol) are added, a probe mother solution (the final concentration is 10 mu M) is added, fluorescence scanning is carried out (the excitation wavelength is 415nm, the detection wavelength is 475-800nm), and the relative fluorescence intensity in each system is measured, as shown in FIG. 3. As can be seen from FIG. 3, the fluorescent probe Nap-NH for detecting polarity change of lysosome as the polarity of solvent is decreased₂Gradually increases in fluorescence intensity.

Example 7

Application of the fluorescence probe for detecting polarity change of lysosome in detection of polarity change of lysosome caused by cell autophagy

A fluorescent probe Nap-NH for detecting polarity changes of lysosomes with the concentration of 1mM obtained in example 1 is configured₂The test mother liquor of dimethyl sulfoxide (DMSO) is ready for use.

PC12 cells in DMEM (Dulbecco's medium) were supplemented with 10% (v/v) fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin, and at 37 ℃ with 5/95(v/v) CO₂Cultivation in a humid atmosphere in air. The day before imaging, cells were detached and suspended in culture medium by treatment with 0.2% (w/v) trypsin-EDTA solution. The cell suspension was then transferred to a confocal culture dish for adherent growth. For imaging, PC12 cells with 80% confluence were collected by scraping and transferred to confocal dishes for adherent growth. Two-photon excited fluorescence images were obtained by excitation at 810nm with a multiphoton laser scanning confocal microscope, as shown in fig. 4. As is clear from FIG. 4(a), as shown by a 1-a 3, b 1-b 3, and c 1-c 3, no significant change in fluorescence was observed in the images of the cells incubated at 25 ℃ and 4 ℃ as compared with the cells incubated at 37 ℃ under 810nm two-photon excitation. Meanwhile, the relative strength of PC12 in d 1-d 3 after treatment with dexamethasone also showed almost no change in cell viscosity. For example, the graphs of e 1-e 3 and f 1-f 3 show lower fluorescence intensity signals of PC12 cells after adding the autophagy inducer rapamycin and cell starvation treatment than the control group. Meanwhile, as in g 1-g 3, significant observations were observed in cells treated with the autophagy inhibitor 3-MA (3-methyladenine)And the change in fluorescence intensity is mainly due to the appearance of autophago-lysosomes, resulting in increased lysosome polarity and decreased fluorescence intensity. Furthermore, the addition of the inflammation inducer LPS (bacterial lipopolysaccharide) to the cells, as in h 1-h 3, resulted in a lower fluorescence intensity signal than the control. E.g., i 1-i 3, the enhancement of fluorescence of cells cultured in LPS +3-MA resembles that of FIG. 4 (b). Indicating that the fluorescence change of the probe is associated with inflammation-induced autophagy. In conclusion, the designed molecule can be completely used as a lysosome targeting and polarity response probe, and the relation between inflammation and autophagy is revealed.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.