阅读说明：本技术 苯并吲哚类化合物与制备方法及其检测半胱氨酸中的应用 (Benzoindole compound, preparation method and application thereof in detection of cysteine ) 是由 唐波 王慧 张晓婷 董明燕 王洪统 于 2020-11-13 设计创作，主要内容包括：本发明公开了苯并吲哚类化合物与制备方法及其检测半胱氨酸中的应用,其化学结构如下式所示：该化合物可以作为检测线粒体内半胱氨酸的荧光探针,具有线粒体靶向能力,且能够实现双光子荧光成像灵敏检测线粒体内半胱氨酸变化。(The invention discloses a benzindole compound, a preparation method and application thereof in detecting cysteine, wherein the chemical structure of the benzindole compound is shown as the following formula: the compound can be used as a fluorescent probe for detecting cysteine in mitochondria, has the mitochondria targeting capability and can realize sensitive detection of cysteine change in mitochondria by two-photon fluorescence imaging.)

1. A benzindole compound is characterized in that the chemical structure is shown as the following formula:

2. a process for preparing the benzoindole compound as claimed in claim 1, wherein 1,1, 2-trimethyl-1H-benzo [ e ] indole and iodoethane are quaternized to obtain compound 1, p-hydroxybenzaldehyde and methyl group at position 2 of compound 1 are condensed to obtain compound 2, and acryloyl chloride and compound 2 are alcoholyzed to obtain the benzoindole compound.

3. The method for preparing benzindole compounds according to claim 2, wherein the temperature of the quaternization reaction is 80-130 ℃;

or the time of the quaternization reaction is 18-48 h.

4. The process for preparing benzindoles according to claim 2, wherein the catalyst in the condensation reaction is a mixture of piperidine and acetic acid.

5. The process for preparing benzindole compounds according to claim 2, wherein compound 2 and acryloyl chloride are subjected to alcoholysis under basic conditions and in an inert atmosphere.

6. The process for preparing benzindole compounds according to claim 5, wherein the compound 2 and triethylamine are added into the solvent, then acryloyl chloride is added dropwise, the mixture is mixed uniformly at-35 to-25 ℃, and then the temperature is raised to room temperature for reaction.

7. The process for producing benzindole compounds according to claim 6, wherein the dropping time of acryloyl chloride is 10 to 30 min.

8. The use of the benzindole compound of claim 1 for the detection of cysteine.

9. The application of the benzindole compound of claim 1 in preparing a two-photon fluorescent probe for detecting mitochondrial cysteine.

10. A method for detecting cysteine in mitochondria, which is characterized in that cells are put into a solution containing the benzindole compound of claim 1 for incubation, and the incubated cells are subjected to ultraviolet absorption detection, fluorescence detection or confocal imaging detection.

Technical Field

The invention relates to the technical field of fluorescent probes, in particular to a benzindole compound, a preparation method and application thereof in cysteine detection.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Mitochondria are a common organelle in cells, are sites for eukaryotic organisms to carry out oxidative metabolism, and are sites for eventual oxidation of sugars, fats and amino acids to release energy. The common pathways responsible for the final oxidation by mitochondria are the tricarboxylic acid cycle and oxidative phosphorylation, corresponding to the second and third stages of aerobic respiration, respectively. Mitochondria are the most sensitive organelles of a cell to various oxidative damages, and when a cell is damaged, the size, number, and structure of mitochondria are changed.

Cysteine can be involved in a variety of biological processes, such as cell growth, and the like. Cysteine in mitochondria is sensitive to mitochondrial oxidative stress and needs to be monitored in real time.

Disclosure of Invention

The invention aims to provide a benzindole compound, a preparation method and application thereof in cysteine detection, wherein the benzindole compound can be used as a fluorescent probe to detect cysteine with lower dose, and has the advantages of high sensitivity, good selectivity and simple and convenient synthesis.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in one aspect, a benzindole compound has a chemical structure shown as the following formula:

on the other hand, the preparation method of the benzindole compound comprises the steps of carrying out quaternization reaction on 1,1, 2-trimethyl-1H-benzo [ e ] indole and iodoethane to generate a compound 1, carrying out condensation reaction of methyl and aldehyde group on p-hydroxybenzaldehyde and 2-methyl of the compound 1 to generate a compound 2, and carrying out alcoholysis reaction on acryloyl chloride and the compound 2 to obtain the benzindole compound.

In a third aspect, the application of the benzindole compound in cysteine detection is provided.

In a fourth aspect, the benzindole compound is applied to preparation of a two-photon fluorescent probe for detecting mitochondrial cysteine.

In a fifth aspect, a method for detecting cysteine in mitochondria, comprising the steps of placing cells in a solution containing the benzindole compound for incubation, and performing ultraviolet absorption detection, fluorescence detection or confocal imaging detection on the incubated cells.

The invention has the beneficial effects that:

1. the invention provides a benzindole compound which can be used as a fluorescent probe for mitochondrial targeting detection of cysteine and has a sensitive cysteine detection effect.

2. The emitted light of the benzindole compound provided by the invention is located in an orange region, has low phototoxicity on tissues, and is suitable for living body imaging and the like.

3. The benzindole compound provided by the invention has good biocompatibility and small damage to cells and living bodies.

4. The benzindole compound provided by the invention has a simple synthetic route, and the raw materials are cheap and easily available, so that the benzindole compound can be applied to market production.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph of absorption and fluorescence spectra of In-CYS prepared In example 1 before and after reaction with cysteine, wherein the abscissa is wavelength (nm), the left ordinate is UV absorption intensity, and the right ordinate is fluorescence intensity emitted by In-CYS at 520 nm;

FIG. 2 is a graph of In-CYS response curves of a nano fluorescent probe prepared In example 1 according to the present invention with different concentrations of cysteine, wherein the abscissa is the wavelength (nm) and the ordinate is the fluorescent intensity, and the fluorescent intensity of the probe gradually increases with the increase of the concentration;

FIG. 3 is a linear relationship diagram of In-CYS with cysteine concentration increase of the nano fluorescent probe prepared In example 1 of the present invention, the abscissa is cysteine concentration, the ordinate is fluorescence intensity of the nano fluorescent probe, and the fluorescence intensity of the probe tends to increase linearly with cysteine concentration increase;

FIG. 4 is a graph showing the response of In-CYS prepared In the fluorescent nanoprobe of example 1 of the present invention to other related intracellular components, and it can be seen from the graph that In-CYS has a significant increase In fluorescence intensity after incubation with cysteine, which is 17 times that of the original In-CYS, and has almost no response to other analytes, which indicates that In-CYS has good selectivity to cysteine.

FIG. 5 is a diagram showing the fluorescence imaging of the nano fluorescent probe In-CYS prepared In example 1 after co-staining with a commercial dye for mitochondrial organelles In HL-7702 cells (normal human liver cells);

FIG. 6 is a diagram of the cellular fluorescence imaging of the In-CYS nano-fluorescence probe prepared In example 1 under the irradiation of a two-photon confocal fluorescence microscope In HL-7702 cells (normal human hepatocytes);

FIG. 7 is a living imaging of In-CYS prepared by the fluorescence nanoprobe of example 1 In arthritis mice, wherein the right leg of the mice is a model of arthritis injected by lambda-carrageenan, the left leg is a control group injected by normal saline, and the fluorescence intensity of the left leg is significantly higher than that of the right leg.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The invention aims to provide a benzindole compound, a preparation method and application thereof in cysteine detection.

In a typical embodiment of the present invention, a benzindole compound is provided, which has a chemical structure as shown in the following formula:

in another embodiment of the present invention, a preparation method of the above-mentioned benzindole compound is provided, wherein 1,1, 2-trimethyl-1H-benzo [ e ] indole and iodoethane are subjected to a quaternization reaction to form a compound 1, p-hydroxybenzaldehyde and a methyl group at position 2 of the compound 1 are subjected to a condensation reaction of a methyl group and an aldehyde group to form a compound 2, and acryloyl chloride and the compound 2 are subjected to an alcoholysis reaction to obtain the benzindole compound.

1,1, 2-trimethyl-1H-benzo [ e ]]The chemical structural formula of the indole is

The structural formula of the iodoethane is

The structural formula of the parahydroxybenzaldehyde is

The acryloyl chloride has the structure of

The synthetic route is as follows:

in some embodiments, the temperature of the quaternization reaction is 80-130 ℃, and the effect is better when the temperature is 80-100 ℃.

In some embodiments, the quaternization reaction time is 18-48 h, and the effect is better when the reaction time is 48 h.

In some embodiments, the solvents for the quaternization reaction are acetonitrile and toluene. Among them, acetonitrile is less toxic. In the reaction, the adding ratio of 1,1, 2-trimethyl-1H-benzo [ e ] indole to acetonitrile is 1-2: 20, mol: and L.

In some embodiments, the catalyst in the condensation reaction is a mixture of piperidine and acetic acid. Wherein, when the molar ratio of the piperidine to the acetic acid is 1: 0.9-1.1, the reaction effect is better.

In some embodiments, compound 2 and acryloyl chloride are subjected to an alcoholysis reaction under basic conditions and an inert atmosphere. When triethylamine is used for providing an alkaline environment, the reaction effect is better.

In the series of embodiments, the compound 2 and triethylamine are added into a solvent, then acryloyl chloride is added dropwise, the mixture is uniformly mixed at-35 to-25 ℃, and then the temperature is raised to room temperature for reaction. The room temperature in the invention refers to the temperature of indoor environment, and is generally 15-30 ℃.

In the series of embodiments, the dropping time of the acryloyl chloride is 10-30 min. The reaction effect is better after the dropwise addition is finished in the time.

In a third embodiment of the invention, the application of the benzindole compound in cysteine detection is provided. Preferably, the use is for the diagnosis and treatment of non-diseases.

The fourth embodiment of the invention provides an application of the benzindole compound in preparing a two-photon fluorescent probe for detecting mitochondrial cysteine.

In a fifth embodiment of the present invention, a method for detecting cysteine in mitochondria is provided, wherein cells are incubated in a solution containing the above-mentioned benzindole compound, and the incubated cells are subjected to ultraviolet absorption detection, fluorescence detection or confocal imaging detection. Preferably, the method for detecting cysteine in mitochondria is aimed at diagnosis and treatment of non-diseases.

When the benzindole compound provided by the invention meets cysteine, the acrylate and the cysteine in the compound are subjected to addition cyclization and decyclization to form hydroxyl. Hydroxyl destroys the PET effect of the probe, so that the emission of the reacted probe is increased at 575nm, and the probe is positively charged and the mitochondrial inner membrane is negatively charged, so that mitochondria can be targeted.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1:

synthesis of fluorescent probe:

1,1, 2-trimethyl-1H-benzo [ e ] indole (0.5g, 2.39mmol) was weighed out and dissolved in 40mL of acetonitrile under stirring, ethyl iodide (250. mu.L, 3.63mmol) was added thereto under nitrogen protection, and the mixture was stirred under reflux at 80 ℃ for 48 hours. After the reaction is finished, most of the solvent is spun dry and precipitated in ether, and after suction filtration and drying, a silver gray solid is obtained and is a compound 1 which is directly used for the next reaction.

Under the protection of nitrogen, compound 1(0.0476g, 0.2mmol) and p-hydroxybenzaldehyde (0.0293g, 0.24mmol) were charged into a 50mL two-necked flask, 10mL of absolute ethanol was further added thereto to dissolve the mixture, 0.01mL of piperidine and 0.01mL of acetic acid were then added thereto, and the mixture was stirred and refluxed at 80 ℃ for 6 hours. Cooling, distilling under reduced pressure to remove most of solvent, and purifying by silica gel column chromatography with the ratio of developing agent being dichloromethane: methanol 30: 1, obtaining a rosy compound 2 for the next reaction.

Compound 2(0.0926g, 0.27mmol), triethylamine (0.0546g, 0.54mmol) and 5mL of tetrahydrofuran were added to a 25mL two-necked flask under nitrogen, acryloyl chloride (40.6. mu.L, 0.5mmol) was added dropwise to the flask, and the mixture was stirred at-30 ℃ for 3 hours, after which it was transferred to room temperature and stirred for another 3 hours. After the reaction is finished, removing the solvent by reduced pressure distillation, and purifying by a silica gel column chromatography method, wherein the ratio of the developing agent is dichloromethane: first of allAlcohol 30: 1, a golden yellow solid was obtained, denoted as In-CYS. (¹H NMR(400MHz,CDCl₃)δ＝8.35(t,3H),8.22(d,J＝8.57,1H),8.14(d,J＝8.94,1H),8.07(d,J＝7.56,1H),7.89(d,J＝15.50,1H),7.82-7.84(m,2H),7.68(t,1H),7.35(d,J＝7.56,2H),6.63(d,J＝17.51,1H),6.32(q,1H),6.07(d,J＝10.84,1H),5.18(q,2H),2.12(s,6H),1.68(q,1H).¹³C NMR(101MHz,CdCl₃)δ＝182.18,163.79,154.88,152.50,133.54,131.97,130.41,127.74,127.35,122.80,112.45,112.32,77.37,77.05,76.73,54.20,44.91,29.71,26.82,14.96.HRMS(ESI)m/z:[M]⁺calculated for C₂₇H₂₆NO²⁺,396.1964found 396.1904)。

In-CYS effect test:

in general, the dye molecule may be dissolved in a water-soluble organic solvent such as physiological saline, PBS buffer, or Hepes buffer, and then the test may be performed by adding an appropriate buffer. This example explores the absorption and fluorescence spectral response of the probe In-CYS as a function of cysteine concentration In PBS buffer solutions of different concentrations of cysteine (1% DMSO, pH 7.4), respectively, and was used for live cell and inflammatory mouse imaging experiments. The living cell staining method is to incubate the cultured cells in a culture solution containing probe molecules, remove the incubation solution after incubation for a certain time, and perform a confocal imaging experiment. The mouse staining method is to inject the probe into the normal part and inflammation part of mouse in situ, and after some time, to take the living fluorescence imaging of the mouse injected probe.

And (3) performing ultraviolet absorption, fluorescence emission and reversible selection experiments on the probe In-CYS In PBS (phosphate buffer solution) solutions with different concentrations of cysteine:

the UV absorption and fluorescence response properties of In-CYS probe In PBS solutions (20. mu.M) of different concentrations of cysteine were investigated. As the concentration of cysteine is increased (2-100 mu M), the absorption spectrum of the probe In-CYS (20 mu M) shows a change with high sensitivity. As shown in FIG. 1, the maximum absorbance red of the probe shifted to around 520nm with increasing cysteine concentration. FIG. 2 shows the fluorescence spectral response of the probe In-CYS (20. mu.M) at different concentrations of cysteine. Under the excitation of 520nm light, the fluorescence intensity of the probe at 575nm is gradually enhanced along with the continuous increase of the concentration of cysteine, which indicates that the probe has high sensitivity fluorescence response to cysteine.

FIG. 3 is a line graph showing the fluorescence response at 575nm collected under excitation at 520nm after cysteine of 0-10 μ M added with 20 μ M In-CYS probe at different concentrations and incubated at 37 ℃ for 10 minutes. The result shows that In-CYS always shows sensitive fluorescence response when the concentration of cysteine is 0-10 mu M, and R of In-CYS²The value was 0.9945.

FIG. 4 is a graph showing the response of In-CYS to other relevant components In a cell, and the fluorescence response of In-CYS (20. mu.M) to 25 different analytes including: glycine, lysine, serine, threonine, proline, arginine, glutamine, isoleucine, aspartic acid, valine, phenylalanine, glutamic acid, methionine, aspartic acid, hydrogen peroxide, ClO^-、Cu²⁺、Fe²⁺、Ca²⁺、Na⁺、K⁺、Zn²⁺Homocysteine, glutathione and cysteine.

Detection Limit experiment of In-CYS:

the detection limit calculation formula of the probe In-CYS is as follows: LOD is 3S₀and/K. Wherein LOD is the detection limit, S₀The standard deviation of fluorescence intensity measured for the probes In-CYS for a plurality of times (more than 13 times), and K is the slope of the linear relation between the fluorescence intensity of the probes In-CYS and the cysteine concentration. Calculated S₀3.65, K121.75, and the detection limit of probe CCYS was calculated to be 0.09. mu.M.

Mitochondrial targeting experiments for In-CYS:

human normal hepatocyte HL-7702 was cultured in DMEM medium containing polyclonal antibody and fetal bovine serum albumin. After incubating the cells with 50 μ M probes with commercial dyes (mitochondria, lysosomes) that localize different subcellular organelles, respectively, for 15min, the co-localization imaging experiments were performed using confocal laser microscopy. Co-localized cell imaging experiments As shown In FIG. 5, the In-CYS probe of FIG. 5 only has a good overlap with the fluorescence of the mitochondrial commercial localization dye.

Two-photon fluorescence imaging experiment of In-CYS:

human normal hepatocyte HL-7702 was cultured in DMEM medium containing polyclonal antibody and fetal bovine serum albumin. After incubating the cells with 50 μ M probe and cells for 15min, a two-photon confocal fluorescence microscope was used for the localization imaging experiment. The positioning cell imaging experiment is shown In figure 6, and figure 6 shows that the In-CYS can well show the positioning effect with mitochondria under the excitation of 800 nm.

In situ in vivo imaging experiments with probes against normal and inflammatory sites in a mouse model of inflammation:

mice were injected with normal saline at the left leg joint site as a control, and lambda-carrageenan (5mg/mL, 100 μ L) at the same position on the right side to induce the generation of arthritis inflammation. After 48 hours, the mice were anesthetized with chloral hydrate and the experiment was performed. In vivo fluorescence imaging was performed by injecting probe In-CYS (50. mu.M, 50. mu.L) at the site injected with lambda-carrageenan (right side) and physiological saline (left side) and detecting with a 520nm laser as shown In FIG. 7. As a result, the fluorescence intensity of the mouse lambda-carrageenan stimulation side is slightly higher than that of the side injected with physiological saline In a 570nm window under 520nm laser, which shows that the probe In-CYS can be used for detecting the content of cysteine In arthritis.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.