阅读说明：本技术 一种pH敏感型探针分子及其应用 (pH sensitive probe molecule and application thereof ) 是由 关锋 周小满 宋志辉 张建健 李想 于 2019-11-19 设计创作，主要内容包括：本发明公开了一种pH敏感型探针分子及其应用,属于生物医药技术领域。本发明提供的基于N-羟基琥珀酰亚胺(NHS)与氨基共价结合激发荧光基团进而标记细胞外囊泡的检测方法,该探针化合物具有标记条件温和、产物稳定、无需额外的化学修饰、操作简单、灵敏度高等特点,在pH 2～6的环境下有强荧光,解决了胞外囊泡在体内体外研究中准确定位的问题,对于胞外囊泡的体内示踪有突出优势。(The invention discloses a pH sensitive probe molecule and application thereof, belonging to the technical field of biological medicines. According to the detection method for labeling the extracellular vesicles based on the covalent binding of N-hydroxysuccinimide (NHS) and amino to excite the fluorescent group, the probe compound has the characteristics of mild labeling condition, stable product, no need of additional chemical modification, simplicity in operation, high sensitivity and the like, has strong fluorescence in an environment with pH of 2-6, solves the problem of accurate positioning of the extracellular vesicles in-vivo and in-vitro research, and has outstanding advantages for in-vivo tracing of the extracellular vesicles.)

1. A compound having a structural formula shown in formula 1:

wherein X includes, but is not limited to, halogen, perchlorate, BF₄(ii) a Y includes but is not limited to C (CH)₃)₂Or S; r₁Including but not limited to- (CH)₂)₂COOH，-(CH₂)₅COOH；R₂Is an electron donating group; the electron donating group includes, but is not limited to, phenyl, C1 to C4 alkyl, methoxyphenyl, or tolyl.

2. The compound of claim 1, wherein the compound is

3. A composition comprising a compound of claim 1 or 2.

4. The composition of claim 3, wherein the composition is a protein, microorganism or exosome carrying the compound of claim 1 or 2.

5. A pharmaceutical composition comprising a compound of claim 1 or 2.

6. A process for the preparation of a compound according to claim 1 or 2, comprising the steps of: (1) mixing 1,1, 2-trimethyl-1H-benzindole and 3-bromopropionic acid in toluene according to the ratio of 1: 1-1.5, and reacting for 2-5H at 90-120 ℃; (2) mixing the compound prepared in the step (1) and 4-diphenylaminobenzaldehyde in an alcohol according to a ratio of 1: 0.8-1.2, and refluxing for 8-12 h at 60-80 ℃ under the protection of nitrogen; the alcohol includes, but is not limited to, ethanol, methanol, isopropanol.

7. Use of a compound according to claim 1 or 2 for microbial, protein labelling or exosome tracing.

8. An exosome-labeling method comprising mixing a compound according to claim 1 or 2 with an exosome or an exosome-containing cell to label the exosome.

9. The method according to claim 8, wherein the compound is used in an amount of 50 to 100. mu.M/1.0X 10⁴～1.0×10⁷(ii) individual cells; the labeling was performed in a dark environment.

10. Use of a compound according to claim 1 or 2, or an exosome labelled with a compound according to claim 1 or 2, in the preparation of a tracer drug.

Technical Field

The invention relates to a pH sensitive probe molecule and application thereof, belonging to the technical field of biological medicines.

Background

Extracellular vesicles (extracellular vesicles) are a subcellular component released by cells spontaneously or under certain conditions and are closed spherical vesicles surrounded by lipid bilayers. Vesicles can be classified according to the biosynthetic or release pathways of extracellular vesicles: exosomes (exosomes) have a diameter of 30-150nm, originating from the endocytic pathway; microparticles/microvesicles (microviscles) with a diameter of about 100-1000nm, released directly from the plasma membrane; apoptotic bodies (bleb) are about 50nm-2 μm in diameter and arise from apoptosis; tumor vesicles (tumor vesicles) about 1-10 μm in diameter, produced by release from tumor cells; and various EV subgroups. In recent years, extracellular vesicles such as exosomes have been studied, and this study is also exemplified. Usually, these extracellular vesicles carry lipid substances such as cholesterol, sphingomyelin, phosphatidylserine, ganglioside and the like, are rich in bioactive substances such as various proteins and RNA and the like, play an important role in cell signal communication, and participate in cell survival and apoptosis, angiogenesis, thrombosis, inflammatory immune response and the like. In addition, the extracellular vesicles are also used as markers for diagnosing diseases and evaluating prognosis; as a drug or drug carrier, plays an important role in the maintenance of physiological states and the progression of diseases. Therefore, in vitro labeling and in vivo tracing of isolated exosomes is very important.

There are many methods for labeling extracellular vesicles, including lipophilic dyes (e.g., PKH-67/PKH-26, Dil/DiD/DiO/DiR), membrane-permeable compounds (e.g., CFSE/CFDA/Calcein-AM), and fluorescent labeling methods based on thiol groups on the surface of extracellular vesicles. However, these dye labeling methods have some disadvantages, one is that the dye is mostly embedded into the membrane bilayer of the extracellular vesicle in a non-covalent manner, which results in that the dye can form dye aggregates or lumps in an aqueous solution similar to exosomes, and may bring misleading information to researchers in exosome uptake experiments; secondly, after the dye is marked, the marked object is structurally modified, the physical property of the marked object is changed, and the functional property of the marked object can be influenced; thirdly, most dyes have weak fluorescence signals in an acid environment and cannot be well labeled in a buffer solution with low pH value.

Disclosure of Invention

The invention designs and synthesizes a pH sensitive probe molecule, and has the characteristics of mild labeling condition, stable product, no need of additional chemical modification, simple operation, high sensitivity and the like.

The first object of the present invention is to provide a compound represented by formula 1:

wherein X includes, but is not limited to, halogen, perchlorate, BF₄(ii) a Y includes but is not limited to C (CH)₃)₂Or S; r₁Including but not limited to- (CH)₂)₂COOH，-(CH₂)₅COOH；R₂Are electron donating groups.

In one embodiment, X is Cl, Br, ClO₄Or BF₄(ii) a Y is C (CH)₃)₂Or S; r₁Is- (CH)₂)₂COOH or- (CH)₂)₅COOH；R₂Is phenyl, C1 to C4 alkyl, methoxyphenyl or tolyl.

In one embodiment, the compound is

It is a second object of the invention to provide compositions containing said compounds.

In one embodiment, the composition includes, but is not limited to, a protein carrying the compound.

In one embodiment, the composition includes, but is not limited to, a microorganism carrying the compound.

In one embodiment, the composition includes, but is not limited to, exosomes carrying the compound.

In one embodiment, the composition comprises a medicament comprising the exosome.

A third object of the present invention is to provide a process for preparing the compound, comprising the steps of:

(1) mixing 1,1, 2-trimethyl-1H-benzindole and 3-bromopropionic acid in toluene according to the ratio of 1: 1-1.5, and reacting for 2-5H at 90-120 ℃; (2) mixing the compound prepared in the step (1) and 4-diphenylaminobenzaldehyde in an alcohol according to a ratio of 1: 0.8-1.2, and refluxing for 8-12 h at 60-80 ℃ under the protection of nitrogen; the alcohol includes, but is not limited to, ethanol, methanol, isopropanol.

The fourth purpose of the invention is to provide the application of the compound in the aspect of exosome tracing.

The fifth purpose of the invention is to provide an exosome-labeling method, wherein the compound is mixed with an exosome to label the exosome.

In one embodiment, the compound is used in an amount of 50 to 100. mu.M/1.0X 10⁴～1.0×10⁷And (4) cells.

In one embodiment, the labeling is performed in a light-protected environment.

In one embodiment, the source of the exosomes includes, but is not limited to, bladder cancer cells.

The invention also claims the application of the compound or the exosome marked by the compound in the preparation of tracer drugs.

The invention also claims the application of the compound in microorganism or target protein labeling.

Has the advantages that: according to the detection method based on the covalent combination of N-hydroxysuccinimide (NHS) and amino to excite the fluorescent group so as to label the extracellular vesicles, the probe compound has strong fluorescence in the environment of pH 2-4, the problem of accurate positioning of the extracellular vesicles in vivo and in vitro research is solved, and the detection method has a prominent advantage on in vivo tracking of the extracellular vesicles.

Drawings

FIG. 1 is a scheme of synthesis of probe compounds;

FIG. 2 shows a mass spectrum (A) and a nuclear magnetic spectrum (B) of a probe compound;

FIG. 3 is a schematic of a probe compound labeling exosomes;

FIG. 4 is a graph of the fluorescence spectrum of a probe compound in the presence or absence of exosomes;

FIG. 5 is a graph of flow screening for optimal use concentrations of probe compounds;

FIG. 6 is a graph showing particle size analysis of probe compound-labeled exosomes;

FIG. 7 is an IC50 test graph of probe compounds for cytotoxicity;

FIG. 8 is a graph of fluorescence spectra of probe compounds at different pH conditions;

FIG. 9 is a plot of the maximum excitation and emission wavelengths of the probe compounds;

FIG. 10 is a graph of the cell uptake of flow cytometry detection probe-labeled exosomes;

FIG. 11 is a diagram of the endocytosis of living cells of exosomes labeled with confocal laser scanning detection probes.

EXAMPLE 1 preparation of Probe Compounds

The synthesis of the dye is shown in figure 1:

(1) synthesis of Compound 2: the compound 1,1, 2-trimethyl-1H-benzindole (3.13g,15.0mmol) and 3-bromopropionic acid (2.43g,16.0mmol) are mixed in 5.0mL of toluene, the mixture is refluxed at 100 ℃ for 3H under the protection of nitrogen, after TLC monitoring reaction is completed, the reaction solution is gradually cooled to room temperature, the reaction solution is dropwise added into an ether (150mL) solvent, a large amount of powder particles are separated out, the powder is collected by filtration and washed by cooled ether (50mL), and crimson solid powder, namely compound 2(4.11g, yield 76%) is obtained. HRMS (ESI, m/z) Calcd. for [ C₁₈H₂₀BrNO₂-Br^-],282.1489；Found,282.1613.

(2) Synthesis of Compound 3: mixing compound 2(722.0mg,2.0mmol) and 4-diphenylaminobenzaldehyde (546.0mg,2.0mmol) in 5.0mL ethanol, refluxing at 80 deg.C for 9h under nitrogen protection, after TLC monitoring reaction, gradually cooling the reaction solution to room temperature, adding dropwise into diethyl ether (150mL) solvent to precipitate a large amount of powder particles, filtering, washing, and passing through silica gel Column (CH)₂Cl₂MeOH ═ 40:1) yielded the target product, compound 3(1.07g, 87% yield). HRMS (ESI, m/z) Calcd. for [ C₃₇H₃₃BrN₂O₂-Br^-],537.2537；Found,537.2604.

(3) The mass spectrum and nuclear magnetic spectrum of the compound are shown in figure 2.

(4) The principle of compound labeling exosomes is shown in figure 3. EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) activates carboxyl on the probe compound to promote the carboxyl and NHS (N-hydroxysuccinimide) to form an intermediate compound containing N-hydroxysuccinimide ester, and the N-hydroxysuccinimide ester is irreversibly combined with amino specificity of protein on the membrane in a covalent mode to form amido bond, so that fluorescent groups are excited to carry out chromogenic reaction.