CN114957082B

CN114957082B - Lysosome targeting fluorescent probe and preparation method and application thereof

Info

Publication number: CN114957082B
Application number: CN202210711226.7A
Authority: CN
Inventors: 谈雪良; 吴运阁; 钱近春; 郑慧敏; 夏继波
Original assignee: Suzhou Youyi Landi Biotechnology Co ltd
Current assignee: Suzhou Youyi Landi Biotechnology Co ltd
Priority date: 2022-06-22
Filing date: 2022-06-22
Publication date: 2024-03-26
Anticipated expiration: 2042-06-22
Also published as: CN114957082A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a lysosome targeting fluorescent probe, a preparation method and application thereof, which are acid organelle dyes in living cells. Most lysosome probes currently marketed have emission peaks in the blue and yellow light ranges, while fluorescent antibodies or other functional fluorescent dyes usually appear in this band, usually the intracellular autofluorescence is in the blue-green region, if the fluorescent indicators are in the ultraviolet region at or near this band, which will overlap each other and affect imaging.

Description

Lysosome targeting fluorescent probe and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a lysosome targeting fluorescent probe, a preparation method and application thereof, which are acid organelle dyes in living cells.

Background

Lysosomes, which are an organelle in eukaryotic cells, contain various hydrolases, which not only break down exogenous macromolecular substances, but also digest the local cytoplasm or organelle of the cell itself. In senescent cells, the programmed disrupted lysosomes release hydrolytic enzymes, allowing the cells to be digested and die.

Lysosomes have low pH values and enzymes within lysosomes mostly only function in such acidic environments. Lysosomes are involved in various vital activities such as metabolism of substances in cells, circulation in living bodies, apoptosis, etc. Lysosomes are highly dynamic in nature, and changes in number and morphology are directly indicative of the state of vital activity in which the cell is located, and lysosomal abnormalities often cause a variety of diseases, such as gout, lysosomal storage disorders, silicosis, and the like. Therefore, the research and analysis of the strong lysosomes can understand the molecular mechanism of the lysosomes participating in biological activities, and has important guiding effect on the treatment of related diseases. In order to achieve the above functions, the developed probe is required to have high specificity to lysosomes and little interference to physiological functions.

Commercial lysosome probes realize lysosome localization analysis by labeling lysosome membranes, such as classical LysoTracker series, specifically labeling acidic organelles at millimolar concentrations, and realizing the function of lysosome localization in fluorescence imaging. However, most lysosome probes currently marketed have emission peaks in the blue and yellow light ranges, and fluorescent antibodies or other functional fluorescent dyes are usually present in this wavelength band, which brings about great difficulty to the experimenters who need to dye lysosomes in multicolor imaging in the selection of the wavelength band of the dye. In general, the autofluorescence in the cell is in the blue-green region, and if the fluorescence indicators are in the ultraviolet region at or near the wavelength band, the fluorescence indicators overlap with each other, so that imaging is affected, and in order to solve the dilemma, a new luminous lysosome dye needs to be developed, so that the requirement of multichannel fluorescence imaging can be met. For the above reasons, the development of lysosomal dyes in the red spectral region is a desirable choice, but no such probes have been reported in the prior art.

Disclosure of Invention

The invention relates to a red fluorescent dye, which has excitation and emission wavelengths of 630-640/655-665nm, can freely penetrate cell membranes, mark living cells, and can specifically mark and track acidic organelles in the living cells at a low nanomolar concentration. Another feature of the invention is that one-step staining can be achieved without the need for secondary antibody detection as with other probes such as Dinitrobenzene (DNP), and specific labeling of acid cell organelle lysosomes within living cells, and detection of cells at nanomolar levels is achieved while rapid introduction. Meanwhile, compared with other lysosome dyes, the dye has more obvious dyeing effect, is not easy to quench and has better optical stability, and can be kept in cells for a long time. The excitation emission wavelength of the invention is closer to the near infrared spectrum, and the invention can also meet the requirement of multicolor dyeing.

The invention adopts the following technical scheme:

a lysosomal targeting fluorescent probe having the following chemical formula:

the invention discloses a preparation method of the lysosome targeting fluorescent probe, which comprises the steps of reacting an indole compound with a hydroxycyclobutene compound to obtain the lysosome targeting fluorescent probe; the chemical structural formula of the indole compound is one or two of the following structural formulas:

、/>、/>

wherein the substituent is consistent with the lysosome targeting fluorescent probe.

In the present invention, the chemical structural formula of the hydroxycyclobutene compound is as follows:

or->

In the present invention, R ₁ 、R ₂ Independently selected from hydrogen, alkyl, ether, and the like; r is R ₃ 、R ₄ Independently selected from hydrogen, alkyl, alkylsulfonic acid ions, and the like.

In the above technical scheme, the alkyl is straight-chain alkyl or cycloalkyl, and the alkylThe number of carbon atoms is 1 to 20, preferably 2 to 15, more preferably R ₁ 、R ₂ The number of carbon atoms of the alkyl group in the formula (I) is 2-6, R ₃ 、R ₄ The number of carbon atoms of the alkyl group is 2 to 12.

In the above embodiments, the alkyl group in the ether group has 2 to 6 carbon atoms, preferably 2 to 5 carbon atoms, such as methyl ether group, ethyl ether group, methyl ethyl ether group, etc.

In the above technical scheme, the alkylsulfonic acid is (CH ₂ )nSO ₃ H, n is 1 to 10, preferably 2 to 8, more preferably 3 to 5.

In the above technical scheme, the alkylsulfonic acid ion is (CH) ₂ )mSO ₃ ^- M is 1 to 10, preferably 2 to 8, more preferably 3 to 5.

In the above technical solution, the lysosome targeting fluorescent probe has balanced electrovalence, and when the lysosome targeting fluorescent probe has positive charge and does not contain alkylsulfonic acid ions, the lysosome targeting fluorescent probe has anion coordination, and the specific anion coordination is a conventional technology, such as halogen ions; if the inclusion of an alkylsulfonic acid ion causes the molecular body to carry a negative charge, it has a cationic coordination, and the specific cationic coordination is a conventional technique such as sodium ion.

In the above technical scheme, the temperature of the reaction of the indole compound and the hydroxycyclobutene compound is 120-200 ℃, preferably 150-180 ℃ for 2-6 hours, preferably 3-5 hours.

The invention discloses an application of the lysosome targeting fluorescent probe as a lysosome probe, or an application in preparing the lysosome probe, or an application in cell development, or an application in preparing a cell developer.

The invention discloses a cell developing method, which comprises the steps of co-culturing cells and the lysosome targeted fluorescent probe, and then developing to complete cell development; preferably, the co-incubation time is from 5 to 30 minutes, preferably from 10 to 25 minutes, and the development is carried out in a conventional manner; the concentration of lysosomal targeted fluorescent probes can be as low as nanomolar, even below 0.05 μm. By way of example, biological cells and the lysosome targeted fluorescent probe of the invention are co-cultured for 15 minutes, and can be developed by utilizing a laser confocal microscope or a common fluorescent microscope.

The invention develops a novel red-light lysosome dye, which has the advantages of stable targeting performance and high fluorescence intensity, and meets the requirement of diversified selection of clients. The excitation and emission wavelengths are 630-640/655-665nm, can freely penetrate cell membranes, label living cells, and can specifically label and track acidic cell organelles in the living cells at a low nanomolar concentration. Another feature of the invention is that one-step staining can be achieved without the need for secondary antibody detection as with other probes such as Dinitrobenzene (DNP), and specific labeling of acid cell organelle lysosomes within living cells, and detection of intracellular at nanomolar concentration levels is achieved while rapid introduction. Meanwhile, compared with other lysosome dyes, the dye has more obvious dyeing effect, is not easy to quench and has better optical stability, and can be kept in cells for a long time.

Drawings

FIG. 1 is a mass spectrum of Compound 1;

FIG. 2 is a mass spectrum of Compound 2;

FIG. 3 is a mass spectrum of Compound 3;

FIG. 4 is a mass spectrum of Compound 4;

FIG. 5 is a mass spectrum of Compound 5;

FIG. 6 is spectral data for Compound 1, excitation 634nm, emission 661nm;

FIG. 7 is a compound 1 cell visualization;

FIG. 8 is a compound 2 cell visualization;

FIG. 9 is a compound 3 cell visualization;

FIG. 10 is a co-staining pattern of Compound 1 and control;

FIG. 11 is a compound 3 cell visualization;

FIG. 12 is a photograph showing a conventional dye cell.

Detailed Description

The raw materials of the invention are conventional compounds, and the specific preparation operation and test are conventional techniques.

Example 1

500 9 g of 4-dipropylaminomethyl-aniline and 70 mL of concentrated hydrochloric acid are added into a mL reaction flask, stirred and cooled to-10 ℃, 6.1 g sodium nitrite solution dissolved in 30 mL water is dropwise added, the internal temperature is not higher than-5 ℃, stirring is carried out for 30 minutes after the completion of the dropwise addition, and 25 mL of concentrated hydrochloric acid (37 wt%) solution of 32.6 g stannous chloride dihydrate precooled to 5 ℃ is dropwise added. After the dripping is finished, the reaction solution is stirred for 1.5 hours at the temperature of minus 10 ℃, then the temperature is increased to 5 ℃ for reaction for 18 hours, after the reaction is finished, the solvent is removed by rotary evaporation, the isopropanol 120 ml is added, the stirring is carried out for 2 hours, the filtration is carried out, the filter cake is washed once by the isopropanol, the filtration is carried out, and the filter cake is dried in vacuum to obtain the product 11.2 g of the intermediate I.

11.2 g of intermediate I,11.3 g of methyl isopropyl ketone and 110 mL of acetic acid are added into a 250 mL reaction bottle, heated to 120 ℃, cooled to room temperature after the reaction is finished for 2 hours, solvent is removed by rotary evaporation, ethyl acetate 160 ml and pure water 160 ml are added, the mixture is filtered after stirring for 30 minutes, the filtrate is separated, the aqueous phase is extracted once by 120 ml of ethyl acetate, the organic phases are combined, saturated saline is washed once, dried by anhydrous sodium sulfate, filtered, the filtrate is rotary evaporated to dryness, silica gel is purified by column chromatography, eluent is dichloromethane to methanol=96:4 (mass ratio), rotary drying is collected, and 10.7g of intermediate II is obtained by vacuum drying.

250 10.7g intermediate II,2.2 g of 3, 4-dihydroxyl-3-cyclobutene-1, 2-dione, 55 mL toluene and 55 mL n-butanol are added into a mL reaction bottle, stirring is carried out, heating is carried out to 160 ℃ for 4 hours, after the reaction is finished, heating is stopped, cooling is carried out to room temperature, solid is separated out, filtering is carried out, filter cakes are washed by diethyl ether for 2 times, filter cakes are collected, and vacuum drying is carried out, thus obtaining the final product 6.1 g of compound 1, and the mass spectrum is shown in figure 1.

Example two

250 5g of 4-dimethoxy aminomethyl-aniline and 55. 55 mL of concentrated hydrochloric acid (37%) are added into a mL reaction flask, then a solution of 3.8. 3.8 g sodium nitrite dissolved in 20 mL water is dripped at-10 ℃ and the internal temperature is not higher than-5 ℃, stirring is carried out for 30 minutes after the dripping is finished, and then 18. 18 mL of concentrated hydrochloric acid (37 wt%) of 20.5. 20.5 g stannous chloride dihydrate precooled to 5 ℃ is dripped. After the dripping is finished, the reaction solution is stirred for 1.5 hours at the temperature of minus 10 ℃, then the temperature is increased to 5 ℃ for reaction for 18 hours, after the reaction is finished, the solvent is removed by rotary evaporation, the isopropanol 75 ml is added, the stirring is carried out for 2 hours, the filtration is carried out, the filter cake is washed once by the isopropanol, the filtration is carried out, and the filter cake is dried in vacuum to obtain the product 4.6 g of the intermediate I.

4.6 g of intermediate I,5.1 g of methyl isopropyl ketone and 50 mL of acetic acid are added into a 100 mL reaction bottle, the mixture is reacted for 2 hours at 120 ℃, the mixture is cooled to room temperature after the reaction is finished, the solvent is removed by rotary evaporation, ethyl acetate 80 ml and pure water 80 ml are added, the mixture is filtered after stirring for 30 minutes, the filtrate is separated, the aqueous phase is extracted once by 60ml of ethyl acetate, the organic phases are combined, saturated saline is washed once, anhydrous sodium sulfate is dried, the mixture is filtered, the filtrate is rotary evaporated to dryness, silica gel is purified by column chromatography, the eluent is methylene dichloride and methanol=95:5 (mass ratio), rotary drying is collected, and 4.1g of intermediate II is obtained by vacuum drying.

4.1g of intermediate II, 9.0 g of 1, 4-butanesultone are added into a 100 mL reaction bottle, then the reaction is carried out for 12 hours at 130 ℃, the reaction is cooled to room temperature after the completion of the reaction, ethyl acetate 60ml is added, the mixture is heated and refluxed, the mixture is cooled to room temperature after stirring for 1 hour, the mixture is filtered, the filter cake is added with 60ml of ethyl acetate again, the mixture is filtered after stirring for 2 hours, the filter cake is collected, and the filter cake is dried in vacuum to obtain 4.7 g of intermediate III.

100 4.7 g intermediate III,0.7 g of 3, 4-dihydroxyl-3-cyclobutene-1, 2-dione, 25 mL toluene and 25 mL n-butanol are added into a mL reaction bottle, then the reaction is carried out for 3 hours at 160 ℃, after the reaction is finished, heating is stopped, the reaction is cooled to room temperature, 0.6 mL sodium hydroxide aqueous solution (0.85 g/mL) is dropwise added, stirring is carried out for 30 minutes, solid is separated out, filtering is carried out, a filter cake is collected, silica gel is purified by passing through a column, eluting agent is dichloromethane to methanol=90 to 10 (mass ratio), spin-drying is collected, and 2.5g of a final product of the compound 2 is obtained through vacuum drying.

According to the first or second embodiment, the starting materials are changed to obtain the compounds of table 1, in particular, the conventional techniques; FIG. 2 is a mass spectrum of Compound 2; FIG. 3 is a mass spectrum of Compound 3; FIG. 4 is a mass spectrum of Compound 4; figure 5 is a mass spectrum of compound 5.

Example III

FIG. 6 is spectral data for Compound 1, excitation 634nm, emission 661nm, red dye.

1. Preparing a lysosome staining stock solution: taking a jar with a cover, adding 10 mg compound 1 powder, adding 20 mL DMSO solution, diluting with sterilized water until the OD is 65 (OD) _{Standard of} ) Obtaining the lysosome staining stock solution.

2. Lysosome dye solution staining cells

(1) Cell preparation: cells were plated on the day before the experiment, 1.5X10 cells were plated at 100. Mu.L per well of 96 well plate ⁴ Individual hela cells, i.e.at a cell concentration of 1.5X10 ⁵ Culturing in basic culture medium (DMEM high sugar liquid culture medium) at 37deg.C and 5% CO ₂ ）；

(2) Preparing a dyeing working solution: diluting the prepared lysosome staining stock solution with a basic culture medium according to the mass ratio of 1:20000 to obtain a staining working solution, and preheating at 37 ℃ for later use; at this time, the concentration of Compound 1 was 2.5X10 ^-5 mg/mL（0.04μmol/L）；

(3) Dyeing: removing the cell culture medium, cleaning twice by 1 XPBS, adding 100 mu L of staining working solution into each hole, and incubating for 15 minutes at 37 ℃ in a dark place; after completion, the dye solution was aspirated, washed three times with 1 XPBS, and finally 50. Mu.L of 1 XPBS was added to prevent the cells from drying out, and observed with a fluorescence microscope, see FIG. 7.

Example IV

1. Preparing a lysosome staining stock solution: taking a jar with a cover, adding 10 mg compound 2 powder, adding 20 mL DMSO solution, diluting with sterilized water until the OD is 65 (OD) _{Standard of} ) Obtaining the lysosome staining stock solution.

2. Lysosome dye solution staining cells

(1) Cell preparation: cells were plated on the day before the experiment, 1.5X10 cells were plated at 100. Mu.L per well of 96 well plate ⁴ The hela cells were cultured overnight (37 ℃ C., 5% CO) in a basal medium (DMEM high-sugar liquid medium) in an incubator ₂ ）；

(2) Preparing a dyeing working solution: diluting the prepared lysosome staining stock solution with a basic culture medium to obtain a staining working solution, and preheating at 37 ℃ for later use; at this time, the concentration of Compound 2 was 0.027. Mu. Mol/L;

(3) Dyeing: removing the cell culture medium, cleaning twice by 1 XPBS, adding 100 mu L of staining working solution into each hole, and incubating for 10 minutes at 37 ℃ in a dark place; after completion, the dye solution was aspirated, washed three times with 1 XPBS, and finally 50. Mu.L of 1 XPBS was added to prevent the cells from drying out, and observed with a fluorescence microscope, see FIG. 8.

Example five

1. Preparing a lysosome staining stock solution: taking a jar with a cover, adding 10 mg compound 3 powder, adding 20 mL DMSO solution, diluting with sterilized water until the OD is 65 (OD) _{Standard of} ) Obtaining the lysosome staining stock solution.

2. Lysosome dye solution staining cells

(2) Preparing a dyeing working solution: diluting the prepared lysosome staining stock solution with a basic culture medium to obtain a staining working solution, and preheating at 37 ℃ for later use; at this time, the concentration of Compound 3 was 0.046. Mu. Mol/L;

(3) Dyeing: removing the cell culture medium, cleaning twice by 1 XPBS, adding 100 mu L of staining working solution into each hole, and incubating for 15 minutes at 37 ℃ in a dark place; after completion, the dye solution was aspirated, washed three times with 1 XPBS, and finally 50. Mu.L of 1 XPBS was added to prevent the cells from drying out, and observed with a fluorescence microscope, see FIG. 9.

Example six

According to the protocol of embodiment III, target cells were stained with Compound 1, washed with PBS buffer, and re-treated with commercially available LysoTracker ™ Green DND-26 (Invitrogen) ^TM Catalog number: L7526) was co-localized stained according to the instructions of its operation, and the result showed that compound 1 and the purchased control were the same function (yellow after co-staining of red and green), see FIG. 10.

Example seven

According to the specific operation scheme of the fifth embodiment, the concentration of the dyeing working solution is 50nM, and the dyeing results of the compound 3 and the existing high-performance dye Lysostracker ™ Deep Red (catalyst number: L12492) are respectively shown in FIG. 11 and FIG. 12, and it can be seen that the compound of the invention has obviously high brightness as a dye under the same concentration of the dyeing solution.

The lysosome probe disclosed by the prior art is mainly blue light and yellow light, can freely penetrate cell membranes, marks living cells, can specifically mark and track acid organelles in the living cells at a low nanomolar concentration, can realize one-step dyeing, specifically marks acid organelle lysosomes in the living cells, and is rapidly introduced at the nanomolar concentration level to detect the cells. Meanwhile, compared with other lysosome dyes, the dye has more obvious dyeing effect, is not easy to quench and has better optical stability, and can be kept in cells for a long time. The excitation emission wavelength of the invention is closer to the near infrared spectrum, and the invention can also meet the requirement of multicolor dyeing.

Claims

1. A lysosomal targeting fluorescent probe, which is one of the following chemical formulas:

；

wherein R is ₁ 、R ₂ Independently selected from hydrogen, alkyl, ether groups; r is R ₃ 、R ₄ Independently selected from hydrogen, alkyl, alkylsulfonic acid ions;

R ₁ 、R ₂ the number of carbon atoms of the alkyl group in the formula (I) is 2-6, R ₃ 、R ₄ The number of carbon atoms of the alkyl group is 2 to 12; the carbon number of the alkyl in the ether group is 2-6; the alkylsulfonic acid is (CH) ₂ )nSO ₃ H, n is 2-8; the alkylsulfonic acid ion is (CH) ₂ )mSO ₃ ^- M is 2 to 8.

2. The lysosomal targeting fluorescent probe of claim 1, wherein the lysosomal targeting fluorescent probe has or does not have a coordinating ion.

3. The method for preparing the lysosome targeted fluorescent probe according to claim 1, wherein an indole compound and a hydroxycyclobutene compound are reacted to obtain the lysosome targeted fluorescent probe; the chemical structural formula of the indole compound is one or two of the following structural formulas:

、/>、/>。

4. the method for preparing a lysosome targeted fluorescent probe according to claim 3, wherein the reaction temperature of the indole compound and the hydroxycyclobutene compound is 120-200 ℃ for 2-6 hours.

5. Use of the lysosomal targeting fluorescent probe according to claim 1 for the preparation of lysosomal probes.

6. Use of the lysosome targeted fluorescent probe of claim 1 in the preparation of a cell developer.

7. The use according to claim 6, wherein the cell development is red development.