Preparation and application of fluorescent probe for specifically analyzing hydrogen sulfide in Golgi apparatus
阅读说明：本技术 一种特异性分析高尔基体中硫化氢的荧光探针制备与应用 (Preparation and application of fluorescent probe for specifically analyzing hydrogen sulfide in Golgi apparatus ) 是由 祝汉闯 柳彩云 梁长旭 张涵铭 贾盼 李子璐 朱宝存 于 2019-12-16 设计创作，主要内容包括：本发明涉及一种特异性分析高尔基体中硫化氢的荧光探针制备与应用,具体地,本发明的探针是一类萘酰亚胺类化合物的荧光探针,可作为硫化氢荧光探针,尤其是其能够靶向定位高尔基体,用于测量、检测或筛选高尔基体中的硫化氢。这类探针可实现如下技术效果中的至少一个：选择性高,能够测量、检测或筛选高尔基体中的硫化氢；抗干扰能力强,能够防止生命体内的其他物质对应该探针检测的干扰；灵敏度高,适合于生命体内微量硫化氢的检测；合成简单,性质稳定,适用于商业推广使用。(The invention relates to preparation and application of a fluorescent probe for specifically analyzing hydrogen sulfide in a Golgi apparatus, in particular to a fluorescent probe for naphthalimide compounds, which can be used as a hydrogen sulfide fluorescent probe, especially can be used for targeting positioning of the Golgi apparatus and is used for measuring, detecting or screening the hydrogen sulfide in the Golgi apparatus. Such probes can achieve at least one of the following technical effects: the selectivity is high, and hydrogen sulfide in a Golgi apparatus can be measured, detected or screened; the anti-interference capability is strong, and the interference of other substances in the life body corresponding to the detection of the probe can be prevented; the sensitivity is high, and the method is suitable for detecting trace hydrogen sulfide in a living body; simple synthesis and stable property, and is suitable for commercial popularization and use.)
1. A compound having the structure:
wherein: r1、R2、R3、R4、R5、R6、R7、R8And R9Is hydrogenAtom, straight chain or branched chain alkyl, straight chain or branched chain alkoxy, sulfonic group, ester group and carboxyl; r1、R2、R3、R4、R5、R6、R7、R8And R9May be the same or different.
2. A compound according to claim 1, characterized in that R1、R2、R3、R4、R5、R6、R7、R8And R9Are all hydrogen atoms.
3. A process for the preparation of a compound according to claim 1 or 2, comprising the steps of: dissolving a compound shown in a formula (III) and sodium azide in dimethyl sulfoxide (DMSO), heating and stirring until the reaction is finished, extracting and filtering reaction liquid to obtain a crude product, and separating and purifying to obtain a pure compound shown in a formula (I), wherein the reaction formula is as follows:
wherein: r1、R2、R3、R4、R5、R6、R7、R8And R9Is hydrogen atom, straight chain or branched chain alkyl, straight chain or branched chain alkoxy, sulfonic group, ester group, carboxyl; r1、R2、R3、R4、R5、R6、R7、R8And R9May be the same or different.
4. The method of claim 3, comprising the steps of:
r is to be1、R2、R3、R4、R5、R6、R7、R8And R9Dissolving a compound of formula (III) and sodium azide, both hydrogen atoms, in dimethyl sulfoxide (DMSO) in a molar ratio of 1:1 to 1:5, and addingStirring under heat for 6-48 hours. And (3) extracting and filtering the reaction liquid to obtain a crude product, and purifying and separating the crude product by using a mixed solvent of dichloromethane and petroleum ether as an eluent through a chromatographic column to obtain the pure compound shown in the formula (I).
5. A fluorescent probe composition for measuring, detecting or screening hydrogen sulfide, comprising the compound of any one of claims 1-2.
6. The fluorescent probe composition of claim 5, wherein the compound is:
7. the fluorescent probe composition of claim 5 or 6, wherein the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.
8. A method for detecting the presence of or determining the amount of hydrogen sulfide in a sample, comprising:
a) contacting a compound of any one of claims 1-2 with a sample to form a fluorescent compound;
b) determining the fluorescent properties of the fluorescent compound.
9. Use of a compound according to any one of claims 1-2 in fluorescence imaging of cells.
10. Use of a compound according to any one of claims 1-2 for the targeted localization of golgi to measure, detect or screen for hydrogen sulfide.
Technical Field
The invention belongs to the field of fluorescent probes, and particularly relates to a fluorescent probe of a naphthalimide compound and application thereof in a method for measuring, detecting or screening hydrogen sulfide in Golgi apparatus and live cell fluorescence imaging; the invention also provides a method for preparing the fluorescent probe.
Background
The hydrogen sulfide is colorless gas with irritation and asphyxiation, the low-concentration contact only has the local irritation of respiratory tract and eyes, and the general action is obvious when the hydrogen sulfide is high in concentration, and the hydrogen sulfide is manifested as central nervous system symptom and asphyxiation symptom.
In view of this, it is extremely important and interesting to develop analytical methods that allow the effective detection of fluctuations in the content of hydrogen sulfide, in particular in certain organelles. Analytical methods reported today for detecting hydrogen sulfide include mercury methods, detector tube methods, methylene blue colorimetric methods, and the like. Fluorescent probes have been the focus of attention among these numerous detection methods due to their unique advantages. However, the fluorescent probes reported so far still have some problems, including poor selectivity, slow response speed, complex synthesis, and inability to detect and analyze hydrogen sulfide in specific organelles. Due to other components in vivo such as alanine (Ala), phenylalanine (Phe), methionine (Met), glycine (Gly), glutamic acid (Glu), arginine (Arg), lysine (Lys), tryptophan (Trp), and sulfate radical (SO)4 2-) Bisulfite (HSO)3 -) Chloride ion (Cl)-) Carbonate (CO)3 2-) Bicarbonate radical (HCO)3 -) Nitrate radical (NO)3 -) Sulfite (SO)3 2-) Hydrogen peroxide (H)2O2) Hypochlorite (ClO)-) And the like, which potentially interfere with the detection of hydrogen sulfide, and therefore, a fluorescent probe capable of measuring, detecting or screening hydrogen sulfide in the golgi, which is rapid, highly selective, highly sensitive, and simple in synthesis, has become a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the present invention aims to provide a class of fluorescent probes for analyzing hydrogen sulfide in golgi with high selectivity, and their preparation methods and uses, and the fluorescent probes have the characteristics of simple synthesis, good selectivity, high sensitivity, and capability of measuring, detecting or screening hydrogen sulfide in golgi.
Specifically, the invention provides a compound having a structure represented by formula (I):
R1、R2、R3、R4、R5、R6、R7、R8and R9Is hydrogen atom, straight chain or branched chain alkyl, straight chain or branched chain alkoxy, sulfonic group, ester group, carboxyl; r1、R2、R3、R4、R5、R6、R7、R8And R9May be the same or different.
In some embodiments of the invention, the compound of the invention is R1、R2、R3、R4、R5、R6、R7、R8And R9A compound of formula (I) each being a hydrogen atom, having the formula:
the invention also provides a preparation method of the compound shown in the formula (I) or the formula (II), which comprises the following steps: dissolving a compound shown in a formula (III) and sodium azide in dimethyl sulfoxide (DMSO), heating and stirring until the reaction is finished, extracting and filtering reaction liquid to obtain a crude product, and separating and purifying to obtain a pure compound shown in a formula (I), wherein the reaction formula is as follows:
wherein: r1、R2、R3、R4、R5、R6、R7、R8And R9Is hydrogen atom, straight chain or branched chain alkyl, straight chain or branched chain alkoxy, sulfonic group, ester group, carboxyl; r1、R2、R3、R4、R5、R6、R7、R8And R9May be the same or different.
In some embodiments of the invention, the reaction time is from 6 to 48 hours.
In some embodiments of the invention, the molar ratio of the compound of formula (III) to sodium azide is from 1:1 to 10: 1.
In some embodiments of the invention, the heating reaction temperature is from 80 to 100 ℃.
In some embodiments of the invention, the separation and purification method is chromatography column separation.
In some embodiments of the invention, the eluent used for the chromatographic column separation is a mixed solvent of dichloromethane and petroleum ether.
In some embodiments of the invention, R is1、R2、R3、R4、R5、R6、R7、R8And R9The compound of formula (III) which is a hydrogen atom and sodium azide are dissolved in dimethyl sulfoxide (DMSO) at a molar ratio of 1:1 to 1:5, and then heated and stirred for 10 to 16 hours.And (3) extracting and filtering the reaction liquid to obtain a crude product, and purifying and separating the crude product by using a mixed solvent (the volume ratio is 1:1-1:3) of dichloromethane and petroleum ether as an eluent through a chromatographic column to obtain the pure compound shown in the formula (I).
The invention also provides a fluorescent probe composition for measuring, detecting or screening hydrogen sulfide, which comprises the compound of formula (I) of the invention.
In some embodiments of the invention, the fluorescent probe composition has the following structure:
in some embodiments of the invention, the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.
The invention also provides a method of detecting the presence of hydrogen sulfide in a sample or determining the amount of hydrogen sulfide in a sample, comprising:
a) contacting the compound of formula (I) or formula (ii) with a sample to form a fluorescent compound;
b) determining the fluorescent properties of the fluorescent compound.
In some embodiments of the invention, the sample is a chemical sample or a biological sample.
In some embodiments of the invention, the sample is a biological sample comprising water, blood, microorganisms, or animal cells or tissues.
The invention also provides a kit for detecting the presence of hydrogen sulfide in a sample or determining the amount of hydrogen sulfide in a sample, comprising the compound of formula (I) or formula (II).
The invention also provides application of the compound shown in the formula (I) or the formula (II) in cell fluorescence imaging.
The invention also provides application of the compound shown in the formula (I) or the formula (II) in measuring, detecting or screening hydrogen sulfide by the targeted positioning Golgi apparatus.
Compared with the prior art, the invention has the following remarkable advantages and effects: the selectivity is high, and hydrogen sulfide in a Golgi apparatus can be specifically identified; the anti-interference capability is strong, and the interference of other substances in the life body corresponding to the detection of the probe can be prevented; the sensitivity is high, and the method is suitable for detecting trace hydrogen sulfide in a living body; simple synthesis and stable property, and is suitable for commercial popularization and use.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1(a) fluorescence spectra before and after addition of probe (5. mu.M) to hydrogen sulfide (0-100. mu.M);
FIG. 1(b) probe (5. mu.M) was used to quantify the working curves for different concentrations of hydrogen sulfide (0-30. mu.M);
FIG. 2 influence of substances commonly found in human body on fluorescence intensity of probe (5. mu.M). The bar graph represents the fluorescence intensity values of the probes at 550nm in the presence of different analytes;
FIG. 3 probes and different commercial organelle dyes were incubated on cells and images were taken using a confocal microscope.
Detailed Description
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 should be understood that the described embodiments are only a part of the embodiments of the present invention, and should not be used to limit the scope of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, belong to the scope of the present invention.