CN115073487B

CN115073487B - Rhodamine derivative and preparation method and application thereof

Info

Publication number: CN115073487B
Application number: CN202210860261.5A
Authority: CN
Inventors: 苗荣; 周颖
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2022-07-21
Filing date: 2022-07-21
Publication date: 2023-09-22
Anticipated expiration: 2042-07-21
Also published as: CN115073487A

Abstract

The invention provides a rhodamine derivative, a preparation method and application thereof, wherein the rhodamine derivative has the structural formula:or (b)

Description

Rhodamine derivative and preparation method and application thereof

Technical Field

The invention belongs to the field of fluorescent dyes with specific recognition capability for lysosomes, and particularly relates to a pyrrole or N-phenylpyrrole modified rhodamine derivative, a preparation method thereof and application thereof in lysosome washing-free, high-resolution/super-resolution and fluorescent lifetime imaging.

Background

Lysosomes are used as membrane-coated organelles, the lumen of which contains more than 60 acid hydrolases, and various biological macromolecules are degraded through autophagy and other processes. Because of its role in cell degradation, lysosomes are commonly referred to as a waste disposal system for cells. New studies have found that lysosomes are constantly changing morphology and spatial distribution in order to achieve their function, making them highly dynamic in cells. At the same time, the lysosomes will interact with other organelles to maintain normal cell function. Therefore, in order to further understand lysosomes, it is important to develop fluorescent dyes that can be used for long-term stable visual imaging of lysosomes.

Lysosomes are typically maintained in a low pH (3.5-5.5) environment to maintain optimal biological activity of numerous hydrolytic enzymes and secreted proteins within them. However, some of the currently developed and commercialized lysosomal dyes have some obvious limitations, such as poor photostability, strong background fluorescence of the dye, high pH sensitivity to the acid range of normal lysosomes, and the like, which limit long-term and stable tracking imaging of lysosomes.

Rhodamine molecules are used as a traditional fluorescent dye and are basic tools for biological research due to the following characteristics: 1. the light source has stronger brightness and better light stability; 2. the change in spectral properties can be affected by modification of the molecular structure; 3. the molecule exists in a balance between a colorless, non-fluorescent lactone (L) and a colored, fluorescent zwitterionic (Z). Will K _L/Z The reduction of the value to a lipophilic non-fluorescent lactone form increases the cell permeability of the molecule. However, conventional rhodamine molecules do not have a tic effect and therefore are insensitive to viscosity and polarity and to microenvironment.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a rhodamine derivative, a preparation method and application thereof, wherein the rhodamine derivative has a TICT effect, is sensitive to microenvironment, can realize specific wash-free imaging of lysosomes, comprises high resolution, super resolution and fluorescence lifetime imaging, and has the characteristics of low background fluorescence, good selectivity, excellent light stability and the like.

The invention is realized by the following technical scheme:

a microenvironment sensitive rhodamine derivative has a structure shown in a formula 1 or a formula 2:

the preparation method of the microenvironment sensitive rhodamine derivative comprises the following steps:

the method comprises the following steps:

weighing 4-diethylamino keto acid, mixing with 3-iodophenol and methanesulfonic acid in a reaction bottle, heating, stirring, condensing and refluxing, cooling to room temperature after the reaction is finished, adjusting the pH value of a reaction solution to be 9, adding dichloromethane for extraction to obtain an organic phase, drying, removing a solvent by vacuum filtration, and separating by column chromatography to obtain a pure compound I-RHO;

weighing I-RHO, sequentially adding pyrrole, N-dimethylformamide and triethylamine into a reaction bottle, removing oxygen by a bubbling method, stirring for reaction under 400nm light, removing solvent by vacuum suction filtration after the reaction is finished, and separating by column chromatography to obtain the compound 1 (NH-RHO).

The second method is as follows:

weighing I-RHO, adding 1-phenylpyrrole-2-boric acid pinacol ester, potassium carbonate, palladium acetate, 1, -bis (diphenylphosphine) ferrocene and tetrahydrofuran in sequence into a reaction bottle, stirring under the protection of inert gas, heating for reflux, adding methylene dichloride for extraction after the reaction is finished to obtain an organic phase, drying, removing a solvent by vacuum filtration, and separating by column chromatography to obtain the compound 2 (NPh-RHO).

Preferably, the petroleum ether-ethyl acetate system is selected as the eluent for column chromatography.

Preferably, in method one, the molar ratio of 4-diethylaminoketo acid, 3-iodophenol and methanesulfonic acid is 1: (0.8-3): (150-400), the reaction temperature is 151-156 ℃; the mol ratio of the compound I-RHO, pyrrole, N-dimethylformamide and triethylamine is 1 (50-90): 100-150): 1-4, and the reaction temperature is 20-25 ℃.

Preferably, the molar ratio of I-RHO, 1-phenylpyrrole-2-boric acid pinacol ester, potassium carbonate, palladium acetate, 1, -bis (diphenylphosphine) ferrocene and tetrahydrofuran in the second method is 1 (1-4): 2-8): 0.1-0.2): 0.2-0.5): 300-800, and the reaction temperature is 61-65 ℃.

The microenvironment sensitive rhodamine derivative is used for preparing a lysosome imaging agent, and realizes washing-free imaging of lysosomes in living cells.

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

the invention utilizes pyrrole or derivative modification to construct microenvironment sensitive rhodamine derivative: the pyrrole or N-phenylpyrrole is connected with rhodamine molecule in the form of C-C single bond, and rhodamine derivative modified by pyrrole or derivative thereof has rotatable C-C bondAnd becomes typical TICT type molecules with abundant photophysical properties, the molecules have a D-pi-A structure, and have great sensitivity to microenvironments (polarity, viscosity and the like), and the rotatability of C-C bonds enables the molecules to show more excellent fluorescence performance in the environment with smaller polarity and larger viscosity. The change of substituents on the pyrrole ring affects the photophysical properties of the molecule, making it sensitive to changes in polarity and viscosity of the solvent, while the change of substituents also affects the quantum yield and photostability of the molecule. Specifically, due to the presence of a C-C single bond in the molecule, an increase in solvent viscosity limits the rotation of the single bond, resulting in an increase in fluorescence of the compound; meanwhile, the difference of solvent polarities can also influence the charge distribution situation of molecules, the change of emission wavelength of the compound in solutions with different polarities is shown, the obvious red shift of the emission wavelength occurs in DCM solvents, stokes shift is increased, the quantum yield of the compound in large polar solvents is lower, and the quantum yield of the compound in small polar and medium polar solvents is higher. The compounds themselves are non-fluorescent lactones, to which an appropriate amount of CF is optionally added during the course of the experiment in order to test their fluorescence properties ₃ COOH causes it to open loop and fluoresce visibly red. Because the lysosome contains abundant acid hydrolase, the pH of the lysosome is 3.5-5.5, therefore, when a compound enters cells, a small part of molecules in the lysosome can be opened to form more stable fluorescent zwitterions, the viscosity of an action site of the compound is increased, the polarity is reduced, the fluorescence intensity of the compound is obviously increased, the signal-to-noise ratio and the better light stability are realized, the lysosome can be specifically identified, and meanwhile, long-time washing-free, high resolution/super resolution and fluorescence lifetime imaging of the lysosome can be realized.

The invention has simple synthetic route, and the obtained compound is sensitive to microenvironment (polarity, viscosity and the like), thereby providing a thinking for lysosome dyes with excellent synthetic performance and wide application.

The rhodamine derivative can be used as a lysosome imaging agent, after the rhodamine derivative enters cells in a closed-loop non-fluorescent state, a part of molecules can be opened in an acidic environment of the lysosome to become fluorescent amphoteric ions, and the conversion of the open loop and the closed loop enables the dye to realize the long-term stable specific imaging of the lysosome, so that a good method is provided for real-time monitoring of the lysosome, and more possibilities are provided for the subsequent research of the action of the lysosome in the cells.

Drawings

FIG. 1 and FIG. 2 are respectively a nuclear magnetic hydrogen spectrum and a carbon spectrum of a target compound 1 prepared by the invention;

FIG. 3 and FIG. 4 are respectively a nuclear magnetic hydrogen spectrum and a carbon spectrum of the target compound 2 prepared by the invention;

FIG. 5 and FIG. 6 are the fluorescence spectra of the excitation and emission of the target compounds 1 and 2, respectively;

FIG. 7 is a graph showing fluorescence spectra of the target compounds 1 and 2 prepared by the present invention in solutions of different polarities;

FIG. 9 is a graph showing fluorescence spectra of the target compounds 1 and 2 prepared by the invention in solutions with different viscosities, respectively;

FIGS. 11 and 12 are confocal laser images of a cell in which lysosomes are stained with Compound 1 according to the present invention, after washing and not washed;

FIG. 13 is a super-resolution laser confocal (STED) image taken when compound 1 prepared according to the present invention is used for staining lysosomes in cells;

FIG. 14 is a super-resolution laser confocal fluorescence lifetime (STED-FLIM) image taken after lysosome staining in cells of compound 1 prepared according to the present invention;

FIG. 15 shows the fluorescence lifetime in lysosomes after staining of lysosomes with Compound 1.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

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 invention discloses two microenvironment sensitive rhodamine derivatives modified and constructed by pyrrole groups, which are respectively a compound 1 (NH-RHO) and a compound 2 (NPh-RHO) and rhodamine derivatives I-RHO.

Wherein, the structural formula of the I-RHO is as follows:

the structural formula of NH-RHO is:

the structural formula of NPh-RHO is as follows:

the invention discloses a specific synthesis method of rhodamine derivatives NH-RHO, NPh-RHO and rhodamine derivatives I-RHO constructed by modification of pyrrolyl, which comprises the following steps:

synthesis method of I-RHO

Weighing 4-diethylamino keto acid in a reaction bottle, sequentially adding 3-iodophenol and methanesulfonic acid, stirring, heating, refluxing, cooling the reaction liquid to room temperature after the reaction is completed, adjusting the pH of the reaction liquid to 9 by saturated sodium carbonate solution, extracting by using dichloromethane to obtain an organic phase, and extracting by using Na ₂ SO ₄ The organic phase is dried, the solvent is removed by vacuum suction filtration, and the product I-RHO is obtained by column chromatography separation. In the reaction, 4-diethylaminoketo acid and 3-iodophenolThe feeding ratio of methanesulfonic acid is 1: (0.8-3): (150-400), the reaction temperature is 151-156 ℃, and the petroleum ether-ethyl acetate is used as an eluent for purification by column chromatography separation, wherein the petroleum ether is as follows: ethyl acetate=5:1, structural formula of the product is

Synthesis method of NH-RHO

Weighing I-RHO in a reaction bottle, sequentially adding pyrrole, N-dimethylformamide and triethylamine, removing oxygen by a bubbling method, stirring for reaction under 400nm light, removing solvent by vacuum suction filtration after the reaction is finished, and separating by column chromatography to obtain the product NH-RHO. In the reaction, the feeding ratio of I-RHO, pyrrole, N-dimethylformamide and triethylamine is 1: (50-90): (100-150): (1-4), the reaction temperature is 20-25 ℃, and the petroleum ether-ethyl acetate is used as an eluent for purification by column chromatography separation, wherein the petroleum ether is as follows: ethyl acetate=5:1, structural formula of the product is

Synthesis method of NPh-RHO

Weighing I-RHO, adding 1-phenylpyrrole-2-boric acid pinacol ester, potassium carbonate, palladium acetate, 1, -bis (diphenylphosphine) ferrocene and tetrahydrofuran into a reaction bottle in sequence, stirring under the protection of inert gas, heating for reflux, adding methylene dichloride for extraction after the reaction is finished to obtain an organic phase, drying, removing a solvent by vacuum suction filtration, and separating by column chromatography to obtain the product NPh-RHO. In the reaction, the feeding ratio of I-RHO, 1-phenylpyrrole-2-boric acid pinacol ester, potassium carbonate, palladium acetate, 1, -bis (diphenylphosphine) ferrocene and tetrahydrofuran is 1: (1-4): (2-8): (0.1-0.2): (0.2-0.5): (300-800), the reaction temperature is 61-65 ℃, and the petroleum ether-ethyl acetate is used as an eluent for purification by column chromatography separation, and petroleum ether is used as the eluent: ethyl acetate=10:1, structural formula of the product is

The invention is further illustrated by the following examples:

example 1: synthesis of Compound I-RHO

0.64g of 4-diethyl ketoacid compound is weighed into a reaction bottle, 3-iodophenol and methanesulfonic acid are added, and the mixture is heated and stirred, and then condensed and refluxed at 153 ℃. After the reaction, the mixture was cooled to room temperature, and saturated Na was used ₂ CO ₃ The pH of the reaction solution was adjusted to 9, and then the solution was poured into a separating funnel, and DCM was added to extract the organic phase, na ₂ SO ₄ Drying the organic phase, removing the solvent by vacuum suction filtration, obtaining a product by column chromatography, and selecting petroleum ether: ethyl acetate=5:1 as eluent. Three sets of experiments were performed, each set having a specific material mole ratio as shown in table 1 below.

Table 1 three experimental conditions in example 1

The reaction equation is as follows

Example 2: synthesis of the compound NH-RHO

Weighing 0.3g of I-RHO prepared in example 1, sequentially adding pyrrole, N-dimethylformamide and triethylamine into a reaction bottle, removing oxygen by a bubbling method, stirring for reaction under 400nm light, removing solvent by vacuum filtration after the reaction is finished at 20-25 ℃, separating by column chromatography to obtain a product, and selecting petroleum ether: ethyl acetate=5:1 as eluent. Three sets of experiments were performed, each set having specific material mole ratios as shown in table 2 below. According to a second set of experiments, the target compound is prepared, and the mass spectrum characterization shows that: [ C ₂₈ H ₂₅ N ₂ O ₃ ] ⁺ Is 437.1860 and the actual measurement is 437.1851. Nuclear magnetic hydrogen spectrum and carbon spectrumFig. 1 and 2.

Table 2 three experimental conditions in example 2

The reaction equation is as follows

Example 3: synthesis of the Compound NPh-RHO

Weighing 0.25g of I-RHO prepared in example 1, adding 1-phenylpyrrole-2-boric acid pinacol ester, potassium carbonate, palladium acetate, 1-bis (diphenylphosphine) ferrocene and tetrahydrofuran into a reaction bottle in sequence, stirring under the protection of inert gas, heating and refluxing, adding dichloromethane to extract to obtain an organic phase after the reaction is finished at the temperature of 63 ℃, drying, removing a solvent by vacuum filtration, separating by column chromatography to obtain a product, and selecting petroleum ether: ethyl acetate=10:1 as eluent. Three sets of experiments were performed, each set having a specific material mole ratio as shown in table 3 below. According to a second set of experiments, the target compound is prepared, and the mass spectrum characterization shows that: [ C ₃₄ H ₂₉ N ₂ O ₃ ] ⁺ Is 513.2173 and the actual measurement is 513.2162. The nuclear magnetic hydrogen spectrum and the carbon spectrum are shown in figure 3 and figure 4.

Table 3 three experimental conditions in example 3

The reaction equation is as follows

In order to verify the effect of the present invention, a number of laboratory studies were performed on the pyrrolyl rhodamine derivatives prepared in examples 2 and 3, each of which is as follows:

1. basic fluorescence behavior characterization

The prepared NH-RHO and NPh-RHO are subjected to excitation emission spectrum characterization by adopting an Edinburgh instrument FLS 920 single photon counting fluorescence spectrometer, and the results are shown in fig. 5 and 6. As can be seen from FIG. 5, the maximum excitation wavelength of the compound NH-RHO is 583nm, the maximum emission wavelength is 635nm, and the Stokes shift is 52nm. As can be seen from FIG. 6, the NPh-RHO has a maximum excitation wavelength of 57nm, a maximum emission wavelength of 628nm, and a Stokes shift of 51nm.

2. Polarity sensitivity test

The dichloromethane-methanol system is selected for solvent polarity sensitivity test of the compound, the polarity of the solvent is changed by regulating the volume ratio of dichloromethane to methanol, and the fluorescent spectrograms of the compounds NH-RHO and NPh-RHO in solvents with different polarities are shown in fig. 7 and 8 respectively, and the response effect is represented by the relative magnitudes of the fluorescent intensities. As can be seen, the fluorescence intensity of the compounds all tended to decrease with increasing polarity of the solvent, indicating that the compounds were sensitive to the polarity of the solvent.

3. Viscosity sensitivity test

The methanol-glycerol system is selected for solvent polarity sensitivity test of the compound, the volume ratio of the methanol and the glycerol is regulated to change the viscosity of the solvent, and the fluorescent spectrograms of the compounds NH-RHO and NPh-RHO in solvents with different viscosities are shown in fig. 9 and 10 respectively, and the response effect is represented by the relative magnitude of the fluorescent intensity. As can be seen from the graph, the fluorescence intensity of the compound tends to increase with the increase of the viscosity of the solvent, indicating that the compound has sensitivity to the viscosity of the solvent.

Note that: the CF was added in all the above experiments ₃ COOH ring-opening the compound

4. Lysosome imaging experiments (laser confocal microscope)

Wash-free imaging experiments of lysosomes in cells with the dye NH-RHO prepared in example 2. Preparing 1mg/ml mother liquor, adding 30ul mother liquor into cell culture dish containing 1.5ml culture solution, 37 deg.C, 5% CO ₂ Incubation for 30min, and imaging of lysosomes under a confocal laser microscopeAs shown in fig. 11: the dye NH-RHO can successfully carry out specific dyeing on lysosomes, has low background fluorescence, and has the advantages of high signal-to-noise ratio, good light stability and the like; then washing with PBS, adding the culture medium again, and imaging the lysosome under a confocal laser microscope, wherein as shown in FIG. 12, the PBS can wash away dye which does not enter cells, and compared with FIG. 11, the dye has no difference in dyeing effect, which indicates that the dye can realize wash-free imaging of the lysosome in the cells.

5. Lysosome imaging experiments (ultra-high resolution laser confocal microscope STED)

The dye NH-RHO prepared in example 2 stained lysosomes in cells. Preparing 1mg/ml mother liquor, adding 30ul mother liquor into cell culture dish containing 1.5ml culture solution, 37 deg.C, 5% CO ₂ Incubation was performed for 1h, and then lysosomes were imaged under an ultra-high resolution laser confocal microscope, as shown in fig. 13: after the dye is used for dyeing the lysosome, the dyeing condition of the lysosome can be clearly observed.

6. Fluorescence lifetime imaging experiment (ultra-high resolution laser confocal microscope STED)

The dye NH-RHO prepared in example 2 stained lysosomes in cells. Preparing 1mg/ml mother liquor, adding 30ul mother liquor into cell culture dish containing 1.5ml culture solution, 37 deg.C, 5% CO ₂ Incubation for 1.5h, then observation of lysosomes under an ultra-high resolution confocal laser microscope, as shown in fig. 14, is an image of fluorescence lifetime in cells after staining lysosomes with the dye; as shown in FIG. 15, the lifetime of the dye in the lysosomes of the cells was about 1.42ns.

According to the invention, rhodamine B is taken as a parent fluorophore, and pyrrole and N-phenylpyrrole are modified to obtain novel fluorescent micromolecules, and the novel fluorescent micromolecules are taken as typical TICT type molecules and have great sensitivity to microenvironments (polarity, viscosity and the like). Meanwhile, the molecules can realize specific wash-free imaging of lysosomes, and the dye has the characteristics of low background fluorescence, good selectivity, excellent light stability and the like.

Claims

1. A rhodamine derivative, which is characterized in that the structure is shown as formula 1 or formula 2:

2. the method for preparing rhodamine derivatives according to claim 1, characterized by being one of the following two methods:

the method comprises the following steps: mixing 4-diethylamino keto acid, 3-iodophenol and methanesulfonic acid, stirring, heating for reflux reaction, cooling to room temperature after the reaction is finished, adjusting the pH value of the reaction solution to be=9, adding dichloromethane for extraction to obtain an organic phase, drying the organic phase, removing dichloromethane, and separating by column chromatography to obtain a compound I-RHO;

mixing a compound I-RHO, pyrrole, N-dimethylformamide and triethylamine, removing oxygen, stirring under 400nm light for reaction, removing N, N-dimethylformamide after the reaction is finished, and separating by column chromatography to obtain a compound of formula 1;

the second method is as follows: mixing 4-diethylamino keto acid, 3-iodophenol and methanesulfonic acid, stirring, heating for reflux reaction, cooling to room temperature after the reaction is finished, adjusting the pH value of the reaction solution to be=9, adding dichloromethane for extraction to obtain an organic phase, drying the organic phase, removing dichloromethane, and separating by column chromatography to obtain a compound I-RHO;

mixing the compound I-RHO, 1-phenylpyrrole-2-boric acid pinacol ester, potassium carbonate, palladium acetate, 1' -bis (diphenylphosphine) ferrocene and tetrahydrofuran, stirring under the protection of inert gas, performing secondary heating reflux reaction, adding dichloromethane to extract after the reaction is finished to obtain an organic phase, drying the organic phase, removing dichloromethane, and performing column chromatography separation to obtain the compound of the formula 2.

3. The method for preparing rhodamine derivatives according to claim 2, characterized in that a petroleum ether-ethyl acetate system is used as an eluent during column chromatography.

4. The method for producing rhodamine derivatives according to claim 2, wherein in the method one and the method two, the molar ratio of 4-diethylaminoketo acid, 3-iodophenol and methanesulfonic acid is 1: (0.8-3): (150-400).

5. The process for preparing rhodamine derivatives according to claim 2, wherein in the process for preparing the compound I-RHO in the first and second methods, the temperature of the heating reflux reaction is 151 to 156 ℃.

6. The process for preparing rhodamine derivatives according to claim 2, wherein in the first process, the molar ratio of the compounds I-RHO, pyrrole, N-dimethylformamide and triethylamine is 1 (50-90): 100-150): 1-4.

7. The process for preparing rhodamine derivatives as claimed in claim 2, wherein in the first process, the temperature of the stirring reaction under light is 20 to 25 ℃.

8. The process for preparing rhodamine derivatives according to claim 2, characterized in that in the second process, the molar ratio of the compound I-RHO, 1-phenylpyrrole-2-boronic acid pinacol ester, potassium carbonate, palladium acetate, 1' -bis (diphenylphosphino) ferrocene and tetrahydrofuran is 1 (1-4): 2-8): 0.1-0.2: 0.2-0.5: (300-800).

9. The process for preparing rhodamine derivatives according to claim 2, wherein in the second process, the temperature of the secondary heating reflux reaction is 61-65 ℃.

10. Use of a rhodamine derivative according to claim 1 for the preparation of a lysosomal imaging agent.