CN112500406A - Carbon atom-based lysosome targeted staining reagent of rhodamine derivative skeleton and preparation method and application thereof - Google Patents

Carbon atom-based lysosome targeted staining reagent of rhodamine derivative skeleton and preparation method and application thereof Download PDF

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CN112500406A
CN112500406A CN202011023013.2A CN202011023013A CN112500406A CN 112500406 A CN112500406 A CN 112500406A CN 202011023013 A CN202011023013 A CN 202011023013A CN 112500406 A CN112500406 A CN 112500406A
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李坤
张宏
陈善勇
余孝其
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Abstract

The invention discloses a carbon atom-based lysosome targeted staining reagent of rhodamine derivative frameworks, and a preparation method and application thereof. In addition, the staining reagent has the characteristic of ultra-large Stokes shift, and is basically not interfered by imaging caused by scattered light of exciting light during biological imaging, so that the staining reagent has the specific expression of high signal-to-noise ratio imaging. The preparation method disclosed by the invention is high in yield and mild in reaction conditions, and the prepared dyeing reagent is large in Stokes shift and high in targeting property.

Description

Carbon atom-based lysosome targeted staining reagent of rhodamine derivative skeleton and preparation method and application thereof
Technical Field
The invention relates to the field of biochemistry, in particular to the technical field of lysosome targeted staining, and specifically relates to a carbon atom-based lysosome targeted staining reagent of rhodamine derivative frameworks, and a preparation method and application thereof.
Background
Lysosomes are organelles that break down biological macromolecules such as proteins, nucleic acids, polysaccharides, and the like. Lysosomes have single-layer membranes, are various in shapes, are 0.025-0.8 micron bubble structures, contain a plurality of hydrolases, have the function in cells, decompose substances entering the cells from the outside, and can digest local cytoplasm or organelles of the cells.
Lysosomes are used as 'digestion stations' in cells and can degrade various endogenous and exogenous biological macromolecules, and the acidic microenvironment (pH 4.5-5.5) in lysosomes can ensure the activity of hydrolytic enzymes, so that the digestion and degradation processes in cells can be smoothly carried out. During metabolism, the permeability change of the lysosome membrane stimulates cells to produce substances and reactive oxygen species inducing endogenous cell death, and simultaneously releases hydrolytic enzymes into cytoplasm, finally causing the cells to start apoptosis. Lysosomes, which are also "end-points" of the endocytic process of cells, are involved in three pathways for cell death: apoptosis (Apoptosis), Type II Programmed cell death (Type II Programmed cell death), and Necrosis (Necrosis) are all associated to some extent.
Therefore, the development of a high-selectivity and high-sensitivity lysosome staining technology for accurately imaging lysosomes in living cells in real time has a very profound significance for exploring and solving the basic biomedical problems related to the lysosomes.
The conventional commercially available lysosome staining dye is a fluorescent dye based on a BODIPY structure, the fluorescent dye is poor in light stability, meanwhile, reductive biological thiols such as glutathione in living cells are easy to interfere, the Stokes shift of the dye is too small, the imaging signal-to-noise ratio is not high, the concentration required by imaging is high, the imaging time is long, and a plurality of defects limit the further application of the dye. Therefore, the development of the lysosome targeted fluorescent dye with large Stokes shift, excellent light stability and ultra-fast ultra-low concentration has great significance.
Disclosure of Invention
The invention aims to provide a carbon atom-based rhodamine derivative skeleton lysosome targeted staining reagent to solve the problems of complex operation process, long time consumption and low imaging result accuracy caused by the fact that the existing lysosome targeted dye needs small Stokes shift, high imaging concentration and long imaging time.
The technical scheme for solving the technical problems is as follows:
a carbon atom-based rhodamine derivative framework lysosome targeted staining reagent has a structure shown as (I), (II) or (III):
Figure BDA0002701276180000011
Figure BDA0002701276180000021
wherein R is1Is an alkyl chain or an aromatic group of C1-C10.
In a preferred embodiment of the present invention, R is1Is propyl, phenyl, naphthyl or pyridyl.
In a preferred embodiment of the invention, the above lysosomal targeting staining agent is:
Figure BDA0002701276180000022
a method for preparing a lysosomal targeted staining reagent having a carbon atom-based rhodamine derivative backbone of formula (i), comprising the steps of:
(1.1) dissolving m-bromoaniline and 1, 4-dibromobutane in a first organic solvent, adding a first weak base, heating and stirring to react to obtain an intermediate A1;
(1.2) adding the intermediate A1 into a mixed solution of phosphorus oxychloride and DMF under an inert atmosphere, stirring, adding water to quench and react, and extracting the obtained reaction liquid with dichloromethane or ethyl acetate to obtain an intermediate A2;
(1.3) adding the intermediate A2 and a first reducing agent into a second organic solvent, stirring, adding water, quenching, reacting, and extracting the obtained reaction liquid with dichloromethane or ethyl acetate to obtain an intermediate A3;
(1.4) adding the intermediate A1 and the intermediate A3 into a third organic solvent, adding boron trifluoride diethyl etherate, and stirring to react to obtain an intermediate A4;
(1.5) dissolving the intermediate A4 in a fourth organic solvent, cooling the solution to below 0 ℃, adding a strong base, adding acetone while stirring, adding water to quench the reaction, extracting the reaction solution with dichloromethane or ethyl acetate, adding aluminum trichloride, and reacting at room temperature to obtain an intermediate A5;
(1.6) dissolving the intermediate A5 in a fifth organic solvent, adding potassium permanganate while stirring, and continuing stirring to react to obtain an intermediate A6;
(1.7) dissolving said intermediate A6 in a sixth organic solvent, adding the second weak base with stirring, adding triflic anhydride and adding R with stirring1NH2Preparing a dyeing reagent which is provided with a formula (I) and is based on a phosphorus atom substituted rhodamine derivative framework; wherein R is1NH2Is C1-C10 alkylamine or aromatic amine.
In a preferred embodiment of the present invention, in step (1.1.): stirring for 10-14 h; the first organic solvent is one or two of DMSO and DMF, and the first weak base is one or more of sodium carbonate, potassium phosphate and sodium phosphate; in the step (1.2): stirring at 0 deg.C, heating to 60-80 deg.C, and stirring overnight; in the step (1.3): stirring for 6-10h at room temperature; the first reducing agent is one or more of sodium borohydride, potassium borohydride and lithium aluminum hydride, and the second organic solvent is one or more of methanol, ethanol, dioxane and tetrahydrofuran; in step (1.4): stirring for 5-7h at room temperature; the third organic solvent is one or two of dichloromethane and trichloromethane; in the step (1.5): further cooling the solution to-70-80 ℃, stirring for 45-80min, and reacting for 10-14h at room temperature; the fourth organic solvent is one or the combination of two of tetrahydrofuran and diethyl ether, and the strong base is n-butyllithium, sec-butyllithium or tert-butyllithium; in the step (1.6): before adding potassium permanganate, cooling the system to 0 ℃, and then adding potassium permanganate and stirring for 5-7 h; the fifth organic solvent is one or the combination of two of acetone and acetonitrile; in step (1.7): cooling the system to 0 deg.C before adding trifluoromethanesulfonic anhydride, stirring for 45-80min, adding R1NH2Then stirring for 5-7h at room temperature; the sixth organic solvent is one or more of dichloromethane, trichloromethane, acetonitrile and DMF, and the second weak base is one or more of pyridine, triethylamine and 4-dimethylaminopyridine.
A method for preparing a lysosomal targeted staining reagent having a carbon atom-based rhodamine derivative backbone of formula (ii), comprising the steps of:
(2.1) dissolving 6-bromoindole in a seventh organic solvent, adding methyl iodide and a first weak base under the stirring condition, and reacting to obtain an intermediate B1;
(2.2) dissolving the intermediate B1 in an eighth organic solvent and adding a second reducing agent under stirring to obtain intermediate B2;
(2.3) adding the intermediate B2 into a mixed solution of phosphorus oxychloride and DMF, and stirring to react to obtain an intermediate B3;
(2.4) adding the intermediate B3 and a first reducing agent into a second organic solvent, and stirring to react to obtain an intermediate B4;
(2.5) adding the intermediate B2 and the intermediate B4 into a third organic solvent, adding boron trifluoride diethyl etherate, and stirring to react to obtain an intermediate B5;
(2.6) dissolving the intermediate B5 in a fourth organic solvent, cooling the solution to below 0 ℃, adding strong base, adding acetone while stirring, adding water to quench the reaction, extracting the obtained reaction solution with dichloromethane after the reaction is completed, adding aluminum trichloride, and reacting at room temperature to obtain an intermediate B6;
(2.7) dissolving the intermediate B6 in a fifth organic solvent, adding potassium permanganate while stirring, and continuing stirring to react to obtain an intermediate B7;
(2.8) dissolving said intermediate B7 in a sixth organic solvent, adding the second weak base with stirring, adding triflic anhydride and adding R with stirring1NH2Preparing a dyeing reagent which is provided with a formula (II) and is based on a phosphorus atom substituted rhodamine derivative framework; wherein R is1NH2Is C1-C10 alkylamine or aromatic amine.
In a preferred embodiment of the present invention, in step (2.1): the seventh organic solvent is one or more of tetrahydrofuran, DMF, diethyl ether and acetonitrile, and the first weak base is one or more of sodium carbonate, potassium phosphate and sodium phosphate; in the step (2.2): the eighth organic solvent is one or more of acetic acid, propionic acid and water, and the second reducing agent is sodium cyanoborohydride; in the step (2.4): the first reducing agent is one or more of sodium borohydride, potassium borohydride and lithium aluminum hydride, and the second organic solvent is one or more of methanol, ethanol, dioxane and tetrahydrofuran; in the step (2.5): the third organic solvent is one or two of dichloromethane and trichloromethane; in the step (2.6): the fourth organic solvent is one or the combination of two of tetrahydrofuran and diethyl ether, and the strong base is n-butyllithium, sec-butyllithium or tert-butyllithium; in step (2.7): the fifth organic solvent is one or the combination of two of acetone and acetonitrile; in the step (2.8): the sixth organic solvent is one or more of dichloromethane, trichloromethane, acetonitrile and DMF, and the second weak base is one or more of pyridine, triethylamine and 4-dimethylaminopyridine.
A method of making a lysosomal targeting staining reagent having a carbon atom-based rhodamine derivative backbone of formula (iii), comprising the steps of:
(3.1) dissolving m-bromoaniline, acetone and iodine in a ninth organic solvent, adding a first weak base, heating and stirring to react to obtain an intermediate C1;
(3.2) dissolving the intermediate C1 in a seventh organic solvent, adding the first weak base and methyl iodide while stirring, and reacting to obtain an intermediate C2;
(3.3) adding the intermediate C2 into a mixed solution of phosphorus oxychloride and DMF, and stirring to react to obtain an intermediate C3;
(3.4) adding the intermediate C3 and a first reducing agent into a second organic solvent, and stirring to react to obtain an intermediate C4;
(3.5) adding the intermediate C2 and the intermediate C4 into a third organic solvent, adding boron trifluoride diethyl etherate, and stirring to react to obtain an intermediate C5;
(3.6) dissolving the intermediate C5 in a fourth organic solvent, cooling the solution to below 0 ℃, adding a strong base, adding acetone under stirring, adding water for quenching reaction, extracting the obtained reaction solution with dichloromethane, adding aluminum trichloride, and reacting at room temperature to obtain an intermediate C6;
(3.7) dissolving the intermediate C6 in a fifth organic solvent, adding potassium permanganate while stirring, and continuing stirring to react to obtain an intermediate C7;
(3.8) dissolving said intermediate C7 in a sixth organic solvent, adding the second weak base with stirring, adding triflic anhydride and adding R with stirring1NH2Preparing a dyeing reagent based on a phosphorus atom substituted rhodamine derivative skeleton with a formula (III); wherein R is1NH2Is C1-C10 alkylamine or aromatic amine.
In a preferred embodiment of the present invention, in step (3.1): the ninth organic solvent is one or more of tetrahydrofuran, acetonitrile and DMF, and the first weak base is one or more of sodium carbonate, potassium phosphate and sodium phosphate; in the step (3.2): the seventh organic solvent is one or a combination of tetrahydrofuran, DMF, diethyl ether and acetonitrile; in the step (3.4): the first reducing agent is one or more of sodium borohydride, potassium borohydride and lithium aluminum hydride, and the second organic solvent is one or more of methanol, ethanol, dioxane and tetrahydrofuran; in the step (3.5): the third organic solvent is one or two of dichloromethane and trichloromethane; in the step (3.6): the fourth organic solvent is one or the combination of two of tetrahydrofuran and diethyl ether, and the strong base is n-butyllithium, sec-butyllithium or tert-butyllithium; in step (3.7): the fifth organic solvent is one or the combination of two of acetone and acetonitrile; in the step (3.8): the sixth organic solvent is one or more of dichloromethane, trichloromethane, acetonitrile and DMF, and the second weak base is one or more of pyridine, triethylamine and 4-dimethylaminopyridine.
The application of a staining reagent based on phosphorus atom substituted rhodamine derivative skeleton in lysosome fluorescence imaging.
The invention has the following beneficial effects:
the carbon atom-substituted rhodamine derivative is used as a basic skeleton, and different forms of aromatic amine modification are carried out, so that the lysosome staining reagent with large Stokes shift near infrared, ultra-fast speed, ultra-low concentration, high targeting and high stability is designed and synthesized, and the lysosome staining reagent can be used for lysosome staining of in vitro cultured cells and histiocytes. The prepared dyeing reagent has larger Stokes shift (more than 150nm) and can effectively avoid the interference of background light. The staining reagent disclosed by the invention has the characteristics of ultra-fast staining and ultra-low concentration imaging on lysosome staining, can effectively reduce the interference of background fluorescence, improves the accuracy of cell imaging results, and makes long-term monitoring of biological processes possible.
Drawings
FIG. 1(a) is a synthesis scheme of example 1 of the present invention. FIG. 1(b) is a synthetic scheme of example 2 of the present invention. FIG. 1(c) is a synthetic scheme of example 3 of the present invention.
FIG. 2(a) is a hydrogen spectrum of the staining reagent of example 1. FIG. 2(b) is a carbon spectrum of the staining reagent of example 1. FIG. 2(c) is a high resolution mass spectrum of the staining reagent of example 1.
FIG. 3(a) is a hydrogen spectrum of the staining reagent of example 2. FIG. 3(b) is a carbon spectrum of the staining reagent of example 2. FIG. 3(c) is a high resolution mass spectrum of the staining reagent of example 2.
FIG. 4(a) is a hydrogen spectrum of the staining reagent of example 3. FIG. 4(b) is a carbon spectrum of the staining reagent of example 3. FIG. 4(c) is a high resolution mass spectrum of the staining reagent of example 3.
FIG. 5 is a graph showing the UV absorption spectrum of the staining reagent of example 1 in a PBS solution.
FIG. 6 is a UV absorption spectrum of the staining reagent of example 2 in a PBS solution.
FIG. 7 is a UV absorption spectrum of the staining reagent of example 3 in a PBS solution.
FIG. 8 shows fluorescence emission spectra of the staining reagent of example 1 in PBS solution.
FIG. 9 shows fluorescence emission spectra of the staining reagent of example 2 in PBS solution.
FIG. 10 shows fluorescence emission spectra of the staining reagent of example 3 in PBS solution.
FIG. 11 shows MTS cytotoxicity assays as staining reagents in examples 1, 2 and 3.
FIG. 12 is a laser confocal experiment of lysosomal staining in HepG2 cells with the staining reagent of example 1.
FIG. 13 shows laser confocal experiments of lysosomal staining in HepG2 cells with the staining reagent of example 2.
FIG. 14 is a laser confocal experiment of lysosomal staining in HepG2 cells with the staining reagent of example 3.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the embodiment of the invention, m-bromoaniline, butyl lithium, phosphorus oxychloride, various solvents, catalysts and alkali are purchased from Allantin technologies, Inc., cell strains are purchased from ATCC (American Type Culture Collection), 10% Fetal Bovine Serum (FBS) is purchased from Hyclone, and 1640 medium is purchased from Gibco, USA.
It should be noted that the raw materials of the organic solvent, the reducing agent, the weak base, the strong base, and the like of the present invention include, but are not limited to, specific substances described in the following examples, and those skilled in the art can select the following alternative technical schemes:
the first organic solvent is one or two of Dimethyl sulfoxide (DMSO) and Dimethylformamide (DMF). The first organic solvent is high boiling point DMSO and DMF, and the solvent is prevented from participating in the reaction.
The second organic solvent is one or more of methanol, ethanol, dioxane and tetrahydrofuran. The reaction of the second organic solvent involves a strong reducing agent, so that methanol, ethanol, dioxane and tetrahydrofuran which do not participate in the reaction are selected, and common solvents which participate in the reaction, such as halogenated hydrocarbon, DMSO and DMF, are avoided.
The third organic solvent is one or two of dichloromethane and trichloromethane. The reaction participated by the third organic solvent is electrophilic substitution reaction catalyzed by boron trifluoride diethyl etherate, and dichloromethane or trichloromethane is used as the solvent to stabilize the formation of the intermediate, promote the reaction, avoid the failure of the reaction caused by other organic solvents and prevent the target compound from being obtained.
The fourth organic solvent is one or two of tetrahydrofuran and diethyl ether. The fourth organic solvent involves organic strong base, such as n-butyl lithium, so that only two organic solvents, namely tetrahydrofuran and diethyl ether, which cannot react with the organic strong base can be selected, and the problem that the reaction with the organic strong base can be caused by the addition of other organic solvents, which causes great danger, is avoided.
The fifth organic solvent is one or the combination of two of acetone and acetonitrile. The reaction participated by the fifth organic solvent relates to a strong oxidant potassium permanganate, so that acetone and acetonitrile are used as solvents, and the problem that the target compound cannot be obtained due to reaction failure caused by the reaction of the organic solvent and the strong oxidant is avoided.
The sixth organic solvent is one or more of dichloromethane, trichloromethane, acetonitrile and DMF. The sixth organic solvent participates in the reaction and relates to trifluoromethanesulfonic anhydride, so that aprotic solvents such as dichloromethane, trichloromethane, acetonitrile or DMF are adopted, and the problems that protic solvents participate in the reaction and cause poor solubility of intermediates are avoided.
The seventh organic solvent is one or more of tetrahydrofuran, DMF, diethyl ether and acetonitrile. The reaction participated by the seventh organic solvent is electrophilic substitution reaction, and tetrahydrofuran, DMF, diethyl ether or acetonitrile is taken as a solvent to avoid participating in the reaction.
The eighth organic solvent is one or a combination of two of acetic acid and propionic acid. The reaction with the eighth organic solvent requires a weakly acidic environment and must have good solubility for the substrate, so that only two solvents can be selected.
The ninth organic solvent is one or more of tetrahydrofuran, acetonitrile and DMF. The ninth organic solvent involves aromatization of the substrate, so that halogenated hydrocarbons which participate in the reaction and protic solvents cannot be selected, and only tetrahydrofuran, acetonitrile or DMF can be selected.
The first weak base is one or more of sodium carbonate, potassium phosphate and sodium phosphate. The first weak base is used as an acid-binding agent to remove hydrogen halide generated in the reaction, and the first weak base cannot be too strong to perform the elimination reaction, so that only the combination of the first weak base and the second weak base can be selected.
The second weak base is one or more of pyridine, triethylamine and 4-dimethylamino pyridine. The second weak base is involved in the formation of an active intermediate with triflic anhydride and acts as an acid-binding agent, therefore, only a few combinations can be selected.
The strong base is n-butyllithium, sec-butyllithium or tert-butyllithium. These lithium metal reagents are necessary to remove the bromine atom from the aromatic hydrocarbon, and only three of these options are available.
The first reducing agent is one or more of sodium borohydride, potassium borohydride and lithium aluminum hydride. This reaction involves the reduction of the aldehyde group and does not remove the halogen atom, so that only these reducing agents can be selected.
The second reducing agent is sodium cyanoborohydride. The reaction is reductive amination under acidic conditions, so that only sodium cyanoborohydride can be selected as a reducing agent.
The carbon atom-based rhodamine derivative skeleton lysosome targeted staining reagent disclosed by the embodiment of the invention is obtained by reacting a carbon atom-substituted xanthone intermediate with propylamine. The synthetic route is shown in fig. 1(a), fig. 1(b) and fig. 1(c), and the synthetic processes of the three compounds are approximately the same, except that:
FIG. 1(a) is a synthetic route of a lysosome targeted staining reagent with a carbon atom-based rhodamine derivative skeleton shown in formula (I), wherein m-bromoaniline is used as a starting material, and an electrophilic substitution reaction is carried out on the m-bromoaniline and 1, 4-dibromobutane to obtain an intermediate A1. FIG. 1(B) is a synthetic route of a dyeing reagent based on phosphorus atom substituted rhodamine derivative skeleton with a formula (II), wherein a starting material of the compound with the formula (II) is 6-bromoindole, and the compound is subjected to electrophilic substitution reaction with iodomethane to obtain an intermediate B1, and then is subjected to reduction reaction with sodium cyanoborohydride to obtain an intermediate B2. Fig. 1(C) shows a synthetic route of a dyeing reagent based on a phosphorus atom substituted rhodamine derivative skeleton, which has a formula (iii), wherein a starting material of a compound of the formula (iii) is m-bromoaniline, and the intermediate C1 is prepared by performing aromatization reaction with acetone, and then a second intermediate C2 is prepared by performing electrophilic substitution reaction with methyl iodide. Subsequently, the same reaction was carried out using each of the prepared forms of the intermediate, respectively, to prepare the final three different forms of the staining reagent.
The present invention will be further described with reference to the following examples.
Example 1:
the preparation method of the embodiment comprises the following steps:
(1) synthesis of intermediate A1
The synthetic route is as follows:
Figure BDA0002701276180000071
m-bromoaniline (1.0mmol), 1, 4-dibromobutane (1.5mol) and carbonic acidPotassium (3.0mmol) was mixed in acetonitrile (5mL) with stirring for 12 h, followed by addition of 100mL of water. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (30 mL. times.3). The organic extracts were washed with brine and Na was used2SO4And (5) drying. The solvent is removed, decompressed and distilled, and then purified by a 200-300 mesh silica gel column chromatography. Elution with petroleum ether/ethyl acetate (20: 1) gave intermediate a1 as a pale yellow liquid in 68% yield.
(2) Synthesis of intermediate A2
The synthetic route is as follows:
Figure BDA0002701276180000072
a1(1mmol) was added to a mixed solution of phosphorus oxychloride (2g, 21mmol, 60% dispersed in mineral oil) and DMF under nitrogen, the resulting solution was stirred at 0 ℃ for 1 hour, then the mixture was slowly heated to 70 ℃ and stirred overnight. The reaction was then quenched by the addition of water. The organic layer was separated and the aqueous layer was extracted with dichloromethane (30 mL. times.3). The organic extracts were washed with brine and Na was used2SO4And (5) drying. The solvent is removed, decompressed and distilled, and then purified by a 200-300 mesh silica gel column chromatography. Elution with petroleum ether/ethyl acetate (6:1) gave intermediate A2 as a white solid in 82% yield.
(3) Synthesis of intermediate A3
The synthetic route is as follows:
Figure BDA0002701276180000081
compound A2(1mmol), sodium borohydride (2mmol) were added to 10.0mL of methanol and reacted at room temperature for 8 hours, and after completion of the reaction was monitored by thin layer chromatography, the reaction mixture was poured into 100mL of water and extracted with dichloromethane. The organic layer was washed with brine, water and Na2SO4And (5) drying. Removal of the solvent by distillation under reduced pressure gave intermediate a3 as a white solid in 98% yield.
(4) Synthesis of intermediate A4
The synthetic route is as follows:
Figure BDA0002701276180000082
after adding compound A1(1mmol) and A3(1mmol) to dichloromethane (10mL), a boron trifluoride ether solution (2mmol) was added dropwise to the stirred solution. The mixture was then stirred at room temperature for about 6 hours, after monitoring the completion of the reaction by thin layer chromatography, the solvent was removed by rotary evaporation and the crude product was purified by 200-mesh 300-mesh silica gel column chromatography eluting with petroleum ether/ethyl acetate (5:1) to give intermediate A4 as a pale yellow solid in 67% yield.
(5) Synthesis of intermediate A5
The synthetic route is as follows:
Figure BDA0002701276180000083
under the protection of nitrogen, adding A4(10mmol) into a reaction bottle, adding anhydrous tetrahydrofuran (40mL) to dissolve a compound A1(1mmol), dropping an n-butyl lithium solution (11mmol) into a stirring solution when a system is cooled to minus seventy-eight ℃, stirring the mixture at minus seventy-eight ℃ for about 1 hour, adding acetone (5.5mmol), heating to room temperature for 12 hours, monitoring the reaction completion by thin layer chromatography, adding 100mL water into the system, extracting with dichloromethane, and adding aluminum trichloride (50mmol) under stirring to obtain a light yellow liquid intermediate A5.
(6) Synthesis of intermediate A6
The synthetic route is as follows:
Figure BDA0002701276180000084
compound A5(1mmol) was added to acetone (50mL) and potassium permanganate (3mmol) was added in portions as the system cooled to zero degrees. The mixture was then stirred at room temperature for about 6 hours, after monitoring the completion of the reaction by thin layer chromatography, manganese dioxide was removed by suction filtration, the solvent was removed by rotary evaporation, and the crude product was purified by 200-mesh and 300-mesh silica gel column chromatography, eluting with petroleum ether/ethyl acetate (2:1) to give the pale yellow solid intermediate A6 in 32% yield.
(7) Synthesizing a lysosomal targeting staining agent having a carbon atom-based rhodamine derivative backbone of formula (i): compound A7
The synthetic route is as follows:
Figure BDA0002701276180000091
compound A6(1mmo) and pyridine (8mmol) were added to dichloromethane (50mL) and when the system cooled to zero, triflic anhydride (3mmol) was added and stirring was continued for 1 h. Then propylamine (10mmol) was added to the system, stirred at room temperature for about 6 hours, after monitoring the completion of the reaction by thin layer chromatography, the solvent was removed by rotary evaporation, and the crude product was purified by 200-mesh and 300-mesh silica gel column chromatography, and eluted with dichloromethane/methanol (60:1) to give a wine red solid compound A7 with a yield of 57%.
The hydrogen spectrum, the carbon spectrum and the high-resolution mass spectrum of the ultra-fast and ultra-low concentration lysosome targeted staining reagent based on the carbon atom substituted rhodamine derivative skeleton, which is prepared in the embodiment, are respectively shown in fig. 2(a) -2 (c).
Example 2:
this example is essentially the same as example 1 except that the starting material was replaced with a B intermediate prepared from 6-bromoindole, the synthetic route of which is shown in figure 1(B), which comprises the following steps:
(1) synthesis of intermediate B1
The synthetic route is as follows:
Figure BDA0002701276180000092
6-bromoindole (1.0mmol), methyl iodide (1.5mol) and potassium carbonate (2.0mmol) were mixed in acetonitrile (5mL) and stirred for 12 h, followed by the addition of 100mL of water. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (30 mL. times.3). The organic extracts were washed with brine and Na was used2SO4And (5) drying. Decompression by solvent removalAfter distillation, the product was purified by 200-mesh 300-mesh silica gel column chromatography. Elution with petroleum ether/ethyl acetate (10: 1) gave intermediate B1 as a pale yellow liquid in 75% yield.
(2) Synthesis of intermediate B2
The synthetic route is as follows:
Figure BDA0002701276180000093
b1(1.0mmol) and sodium cyanoborohydride (5.0mmol) were mixed in acetic acid (5mL) and stirred for 12 h, followed by the addition of 100mL of water. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (30 mL. times.3). The organic extracts were washed with brine and Na was used2SO4And (5) drying. The solvent is removed, decompressed and distilled, and then purified by a 200-300 mesh silica gel column chromatography. Elution with petroleum ether/ethyl acetate (10: 1) provided intermediate B2 in 58% yield.
(3) Synthesis of intermediate B3
The synthetic route is as follows:
Figure BDA0002701276180000101
b2(1mmol) was added to a mixed solution of phosphorus oxychloride (2g, 21mmol, 60% dispersed in mineral oil) and DMF under nitrogen, the resulting solution was stirred at 0 ℃ for 1 hour, then the mixture was slowly heated to 70 ℃ and stirred overnight. The reaction was then quenched by the addition of water. The organic layer was separated and the aqueous layer was extracted with dichloromethane (30 mL. times.3). The organic extracts were washed with brine and Na was used2SO4And (5) drying. The solvent is removed, decompressed and distilled, and then purified by a 200-300 mesh silica gel column chromatography. Elution with petroleum ether/ethyl acetate (6:1) provided intermediate B3.
(4) Synthesis of intermediate B4
The synthetic route is as follows:
Figure BDA0002701276180000102
compound B3(1mmol), sodium borohydride (2mmol) were added to 10.0mL of methanol and reacted at room temperature for 8 hours, and after completion of the reaction was monitored by thin layer chromatography, the reaction mixture was poured into 100mL of water and extracted with dichloromethane. The organic layer was washed with brine, water and Na2SO4And (5) drying. The solvent was removed and distilled under reduced pressure to give intermediate B4.
(5) Synthesis of intermediate B5
The synthetic route is as follows:
Figure BDA0002701276180000103
after adding compound B2(1mmol) and B4(1mmol) to dichloromethane (10mL), a boron trifluoride ether solution (2mmol) was added dropwise to the stirred solution. The mixture was then stirred at room temperature for about 6 hours, after monitoring the completion of the reaction by thin layer chromatography, the solvent was removed by rotary evaporation, and the crude product was purified by 200-mesh 300-mesh silica gel column chromatography eluting with petroleum ether/ethyl acetate (5:1) to give intermediate B5.
(6) Synthesis of intermediate B6
The synthetic route is as follows:
Figure BDA0002701276180000104
under the protection of nitrogen, adding B5(10mmol) into a reaction bottle, adding anhydrous tetrahydrofuran (40mL) to dissolve a compound B5(1mmol), dropping an n-butyl lithium solution (11mmol) into a stirring solution when a system is cooled to minus seventy-eight ℃, stirring the mixture at minus seventy-eight ℃ for about 1 hour, adding acetone (5.5mmol), heating to room temperature for 12 hours, monitoring the reaction completion by thin layer chromatography, adding 100mL of water into the system, extracting by dichloromethane, and adding aluminum trichloride (50mmol) under stirring to obtain an intermediate B6.
(7) Synthesis of intermediate B7
The synthetic route is as follows:
Figure BDA0002701276180000111
compound B6(1mmol) was added to acetone (50mL) and potassium permanganate (3mmol) was added in portions as the system cooled to zero degrees. The mixture was then stirred at room temperature for about 6 hours, after monitoring the completion of the reaction by thin layer chromatography, manganese dioxide was removed by suction filtration, the solvent was removed by rotary evaporation, the crude product was purified by 200-mesh 300-mesh silica gel column chromatography, eluting with petroleum ether/ethyl acetate (2:1) to give intermediate B7.
(8) Synthesizing a lysosome targeted staining reagent having a carbon atom-based rhodamine derivative backbone of formula (ii): compound B8
The synthetic route is as follows:
Figure BDA0002701276180000112
compound B7(1 mmol) and pyridine (8mmol) were added to dichloromethane (50mL) and when the system cooled to zero, triflic anhydride (3mmol) was added and stirring was continued for 1 h. Then propylamine (10mmol) was added to the system, stirred at room temperature for about 6 hours, after monitoring the completion of the reaction by thin layer chromatography, the solvent was removed by rotary evaporation, and the crude product was purified by 200-mesh 300-mesh silica gel column chromatography, eluting with methylene chloride/methanol (60:1) to give compound B8.
The hydrogen spectrum, the carbon spectrum and the high-resolution mass spectrum of the phosphorirhodamine derivative staining reagent prepared in the embodiment, which can monitor the lysosome-mediated lipid droplet autophagy process in real time, are respectively shown in fig. 3(a) -3 (c).
Example 3:
this example is essentially the same as example 1, except for the C intermediate prepared by substituting the starting material with m-bromoaniline, and the synthetic route is as follows:
(1) synthesis of intermediate C1
The synthetic route is as follows:
Figure BDA0002701276180000113
m-bromoaniline (1.0mmol), acetone (10mol) and elemental iodine (0.01mmol) were mixed in acetonitrile (5mL) and stirred for 12 hours, followed by the addition of 100mL of water. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (30 mL. times.3). The organic extracts were washed with brine and Na was used2SO4And (5) drying. The solvent is removed, decompressed and distilled, and then purified by a 200-300 mesh silica gel column chromatography. Elution with petroleum ether/ethyl acetate (10: 1) gave intermediate C1 as a pale yellow liquid in 47% yield.
(2) Synthesis of intermediate C2
The synthetic route is as follows:
Figure BDA0002701276180000121
c1(1mol), methyl iodide (1mol) and potassium carbonate (3.0mmol) were mixed in acetonitrile (5mL) and stirred for 12 h, followed by the addition of 100mL of water. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (30 mL. times.3). The organic extracts were washed with brine and Na was used2SO4And (5) drying. The solvent is removed, decompressed and distilled, and then purified by a 200-300 mesh silica gel column chromatography. Elution with petroleum ether/ethyl acetate (20: 1) provided intermediate C2.
(3) Synthesis of intermediate C3
The synthetic route is as follows:
Figure BDA0002701276180000122
c2(1mmol) was added to a mixed solution of phosphorus oxychloride (2g, 21mmol, 60% dispersed in mineral oil) and DMF under nitrogen, the resulting solution was stirred at 0 ℃ for 1 hour, then the mixture was slowly heated to 70 ℃ and stirred overnight. The reaction was then quenched by the addition of water. The organic layer was separated and the aqueous layer was extracted with dichloromethane (30 mL. times.3). The organic extracts were washed with brine and Na was used2SO4And (5) drying. The solvent is removed, decompressed and distilled, and then purified by a 200-300 mesh silica gel column chromatography. Elution with petroleum ether/ethyl acetate (6:1) gave intermediate C3.
(4) Synthesis of intermediate C4
The synthetic route is as follows:
Figure BDA0002701276180000123
compound C3(1mmol), sodium borohydride (2mmol) were added to 10.0mL of methanol and reacted at room temperature for 8 hours, and after completion of the reaction was monitored by thin layer chromatography, the reaction mixture was poured into 100mL of water and extracted with dichloromethane. The organic layer was washed with brine, water and Na2SO4And (5) drying. The solvent was removed and distilled under reduced pressure to give intermediate C4.
(5) Synthesis of intermediate C5
The synthetic route is as follows:
Figure BDA0002701276180000124
after adding compound C2(1mmol) and C4(1mmol) to dichloromethane (10mL), a boron trifluoride ether solution (2mmol) was added dropwise to the stirred solution. The mixture was then stirred at room temperature for about 6 hours, after monitoring the completion of the reaction by thin layer chromatography, the solvent was removed by rotary evaporation, and the crude product was purified by 200-mesh 300-mesh silica gel column chromatography eluting with petroleum ether/ethyl acetate (5:1) to give intermediate C5.
(6) Synthesis of intermediate C6
The synthetic route is as follows:
Figure BDA0002701276180000131
under the protection of nitrogen, C5(10mmol) is added into a reaction bottle, then anhydrous tetrahydrofuran (40mL) is added to dissolve a compound C5(1mmol), when the system is cooled to minus seventy-eight ℃, n-butyl lithium solution (11mmol) is dripped into a stirring solution, then the mixture is stirred for about 1 hour at minus seventy-eight ℃, acetone (5.5mmol) is added, the temperature is raised to room temperature for reaction for 12 hours, after the reaction is monitored by thin layer chromatography, 100mL of water is added into the system, dichloromethane is used for extraction, and aluminum trichloride (50mmol) is added under stirring to obtain an intermediate C6.
(7) Synthesis of intermediate C7
The synthetic route is as follows:
Figure BDA0002701276180000132
compound C6(1mmol) was added to acetone (50mL) and potassium permanganate (3mmol) was added in portions as the system cooled to zero degrees. The mixture was then stirred at room temperature for about 6 hours, after monitoring the completion of the reaction by thin layer chromatography, manganese dioxide was removed by suction filtration, the solvent was removed by rotary evaporation, the crude product was purified by 200-mesh 300-mesh silica gel column chromatography, eluting with petroleum ether/ethyl acetate (2:1) to give intermediate C7.
(8) Synthesizing a lysosomal targeting staining reagent having a carbon atom-based rhodamine derivative backbone of formula (iii): compound C8
The synthetic route is as follows:
Figure BDA0002701276180000133
compound C7(1 mmol) and pyridine (8mmol) were added to dichloromethane (50mL) and when the system cooled to zero, triflic anhydride (3mmol) was added and stirring was continued for 1 h. Propylamine (10mmol) was then added to the system and stirred at room temperature for about 6 hours, after monitoring the completion of the reaction by thin layer chromatography, the solvent was removed by rotary evaporation, and the crude product was purified by 200-mesh 300-mesh silica gel column chromatography eluting with methylene chloride/methanol (60:1) to give compound C8.
The hydrogen spectrum, the carbon spectrum and the high-resolution mass spectrum of the ultra-fast and ultra-low concentration lysosome targeted staining reagent based on the carbon atom substituted rhodamine derivative skeleton, which is prepared in the embodiment, are respectively shown in fig. 4(a) -4 (c).
Test example 1 ultraviolet absorption Spectroscopy
The ultra-fast and ultra-low concentration lysosome targeted staining reagent based on the carbon atom substituted rhodamine derivative skeleton prepared in the above examples 1-3 is respectively prepared into 10mM DMSO mother liquor. The solutions were mixed with PBS (1, 2, 4, 6, 8, 10 uL), and the UV absorption values were scanned and plotted. The ultraviolet absorption spectrum of the staining reagent of example 1 is shown in FIG. 5, the ultraviolet absorption spectrum of the staining reagent of example 2 is shown in FIG. 6, and the ultraviolet absorption spectrum of the staining reagent of example 3 is shown in FIG. 7. As shown, examples 1 and 2 have two absorption peaks. One of these occurs at around 375nm and the other at around 480nm, and example 3 has three absorption peaks at 325nm, 375nm and 510nm, respectively.
Experimental example 2 fluorescence Spectroscopy
The staining reagents prepared in examples 1, 2 and 3 were prepared as 10mM DMSO stock solutions. And respectively adding PBS solution to measure the fluorescence spectrum of the mixture to obtain a fluorescence emission curve. In the solution of PBS, the maximum emission wavelength of the samples in examples 1, 2 and 3 is significantly red-shifted, especially in examples 2 and 3, the maximum emission wavelength is within the near infrared emission region. The fluorescence intensity of the staining reagent of example 1 is shown in FIG. 8, the fluorescence intensity of the staining reagent of example 2 is shown in FIG. 9, and the fluorescence intensity of the staining reagent of example 3 is shown in FIG. 10.
In conclusion, the carbon atom-substituted aminorhodamine derivative skeleton is used as the basis of the lysosomal dye, and the lysosomal targeted staining reagents with different emission wavelengths based on the carbon atom-substituted aminorhodamine derivative skeleton are obtained through reasonable regulation and control design of aromatic amines with different forms. In addition, the staining reagent has the characteristic of ultra-large Stokes shift, and is basically not interfered by imaging caused by scattered light of exciting light during biological imaging, so that the staining reagent has the specific expression of high signal-to-noise ratio imaging. The preparation method disclosed by the invention is high in yield and mild in reaction conditions, and the prepared dyeing reagent is large in Stokes shift and high in targeting property.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. A carbon atom-based rhodamine derivative skeleton lysosome targeted staining reagent is characterized by having a structure shown as (I), (II) or (III):
Figure FDA0002701276170000011
wherein R is1Is an alkyl chain or an aromatic group of C1-C10.
2. The lysosomal targeted staining reagent of claim 1, wherein R is1Is propyl, phenyl, naphthyl or pyridyl.
3. The lysosomal targeted staining reagent of claim 1, wherein the lysosomal targeted staining reagent is:
Figure FDA0002701276170000012
4. a method for preparing a lysosomal targeting staining reagent having a carbon atom-based rhodamine derivative scaffold according to claim 1 comprising the steps of:
(1.1) dissolving m-bromoaniline and 1, 4-dibromobutane in a first organic solvent, adding a first weak base, heating and stirring to react to obtain an intermediate A1;
(1.2) adding the intermediate A1 into a mixed solution of phosphorus oxychloride and DMF under an inert atmosphere, stirring, adding water to quench and react, and extracting the obtained reaction liquid with dichloromethane or ethyl acetate to obtain an intermediate A2;
(1.3) adding the intermediate A2 and a first reducing agent into a second organic solvent, stirring, adding water, quenching, reacting, and extracting the obtained reaction liquid with dichloromethane or ethyl acetate to obtain an intermediate A3;
(1.4) adding the intermediate A1 and the intermediate A3 into a third organic solvent, adding boron trifluoride diethyl etherate, and stirring to react to obtain an intermediate A4;
(1.5) dissolving the intermediate A4 in a fourth organic solvent, cooling the solution to below 0 ℃, adding a strong base, adding acetone while stirring, adding water to quench the reaction, extracting the reaction solution with dichloromethane or ethyl acetate, adding aluminum trichloride, and reacting at room temperature to obtain an intermediate A5;
(1.6) dissolving the intermediate A5 in a fifth organic solvent, adding potassium permanganate while stirring, and continuing stirring to react to obtain an intermediate A6;
(1.7) dissolving said intermediate A6 in a sixth organic solvent, adding the second weak base with stirring, adding triflic anhydride and adding R with stirring1NH2Preparing a dyeing reagent which is provided with a formula (I) and is based on a phosphorus atom substituted rhodamine derivative framework; wherein R is1NH2Is C1-C10 alkylamine or aromatic amine.
5. The method of claim 4,
in step (1.1.): stirring for 10-14 h; the first organic solvent is one or two of DMSO and DMF, and the first weak base is one or more of sodium carbonate, potassium phosphate and sodium phosphate;
in the step (1.2): stirring at 0 deg.C, heating to 60-80 deg.C, and stirring overnight;
in the step (1.3): stirring for 6-10h at room temperature; the first reducing agent is one or more of sodium borohydride, potassium borohydride and lithium aluminum hydride, and the second organic solvent is one or more of methanol, ethanol, dioxane and tetrahydrofuran;
in the step (1.4): stirring for 5-7h at room temperature; the third organic solvent is one or two of dichloromethane and trichloromethane;
in the step (1.5): further cooling the solution to-70-80 ℃, stirring for 45-80min, and reacting for 10-14h at room temperature; the fourth organic solvent is one or the combination of two of tetrahydrofuran and diethyl ether, and the strong base is n-butyllithium, sec-butyllithium or tert-butyllithium;
in the step (1.6): before adding potassium permanganate, cooling the system to 0 ℃, and then adding potassium permanganate and stirring for 5-7 h; the fifth organic solvent is one or the combination of two of acetone and acetonitrile;
in step (1.7): cooling the system to 0 deg.C before adding trifluoromethanesulfonic anhydride, stirring for 45-80min, adding R1NH2Then stirring for 5-7h at room temperature; the sixth organic solvent is one or more of dichloromethane, trichloromethane, acetonitrile and DMF, and the second weak base is one or more of pyridine, triethylamine and 4-dimethylaminopyridine.
6. A method for preparing a lysosomal targeted staining reagent having a carbon atom-based rhodamine derivative scaffold according to claim 1 comprising the steps of:
(2.1) dissolving 6-bromoindole in a seventh organic solvent, adding methyl iodide and a first weak base under the stirring condition, and reacting to obtain an intermediate B1;
(2.2) dissolving the intermediate B1 in an eighth organic solvent and adding a second reducing agent under stirring to obtain intermediate B2;
(2.3) adding the intermediate B2 into a mixed solution of phosphorus oxychloride and DMF, and stirring to react to obtain an intermediate B3;
(2.4) adding the intermediate B3 and a first reducing agent into a second organic solvent, and stirring to react to obtain an intermediate B4;
(2.5) adding the intermediate B2 and the intermediate B4 into a third organic solvent, adding boron trifluoride diethyl etherate, and stirring to react to obtain an intermediate B5;
(2.6) dissolving the intermediate B5 in a fourth organic solvent, cooling the solution to below 0 ℃, adding strong base, adding acetone while stirring, adding water to quench the reaction, extracting the obtained reaction solution with dichloromethane after the reaction is completed, adding aluminum trichloride, and reacting at room temperature to obtain an intermediate B6;
(2.7) dissolving the intermediate B6 in a fifth organic solvent, adding potassium permanganate while stirring, and continuing stirring to react to obtain an intermediate B7;
(2.8) dissolving said intermediate B7 in a sixth organic solvent, adding the second weak base with stirring, adding triflic anhydride and adding R with stirring1NH2Preparing a dyeing reagent which is provided with a formula (II) and is based on a phosphorus atom substituted rhodamine derivative framework; wherein R is1NH2Is C1-C10 alkylamine or aromatic amine.
7. The method of claim 6,
in step (2.1.): the seventh organic solvent is one or more of tetrahydrofuran, DMF, diethyl ether and acetonitrile, and the first weak base is one or more of sodium carbonate, potassium phosphate and sodium phosphate;
in the step (2.2): the eighth organic solvent is one or more of acetic acid, propionic acid and water, and the second reducing agent is sodium cyanoborohydride;
in the step (2.4): the first reducing agent is one or more of sodium borohydride, potassium borohydride and lithium aluminum hydride, and the second organic solvent is one or more of methanol, ethanol, dioxane and tetrahydrofuran;
in the step (2.5): the third organic solvent is one or two of dichloromethane and trichloromethane;
in the step (2.6): the fourth organic solvent is one or the combination of two of tetrahydrofuran and diethyl ether, and the strong base is n-butyllithium, sec-butyllithium or tert-butyllithium;
in step (2.7): the fifth organic solvent is one or the combination of two of acetone and acetonitrile;
in the step (2.8): the sixth organic solvent is one or more of dichloromethane, trichloromethane, acetonitrile and DMF, and the second weak base is one or more of pyridine, triethylamine and 4-dimethylaminopyridine.
8. A method for preparing a lysosomal targeted staining reagent having a carbon atom-based rhodamine derivative scaffold according to claim 1, characterized in that it comprises the steps of:
(3.1) dissolving m-bromoaniline, acetone and iodine in a ninth organic solvent, adding a first weak base, heating and stirring to react to obtain an intermediate C1;
(3.2) dissolving the intermediate C1 in a seventh organic solvent, adding the first weak base and methyl iodide while stirring, and reacting to obtain an intermediate C2;
(3.3) adding the intermediate C2 into a mixed solution of phosphorus oxychloride and DMF, and stirring to react to obtain an intermediate C3;
(3.4) adding the intermediate C3 and a first reducing agent into a second organic solvent, and stirring to react to obtain an intermediate C4;
(3.5) adding the intermediate C2 and the intermediate C4 into a third organic solvent, adding boron trifluoride diethyl etherate, and stirring to react to obtain an intermediate C5;
(3.6) dissolving the intermediate C5 in a fourth organic solvent, cooling the solution to below 0 ℃, adding a strong base, adding acetone under stirring, adding water for quenching reaction, extracting the obtained reaction solution with dichloromethane, adding aluminum trichloride, and reacting at room temperature to obtain an intermediate C6;
(3.7) dissolving the intermediate C6 in a fifth organic solvent, adding potassium permanganate while stirring, and continuing stirring to react to obtain an intermediate C7;
(3.8) dissolving said intermediate C7 in a sixth organic solvent, adding the second weak base with stirring, adding triflic anhydride and adding R with stirring1NH2Preparing a dyeing reagent based on a phosphorus atom substituted rhodamine derivative skeleton with a formula (III); wherein R is1NH2Is C1-C10 alkylamine or aromatic amine.
9. The method of claim 8,
in step (3.1.): the ninth organic solvent is one or more of tetrahydrofuran, acetonitrile and DMF, and the first weak base is one or more of sodium carbonate, potassium phosphate and sodium phosphate;
in the step (3.2): the seventh organic solvent is one or a combination of tetrahydrofuran, DMF, diethyl ether and acetonitrile;
in the step (3.4): the first reducing agent is one or more of sodium borohydride, potassium borohydride and lithium aluminum hydride, and the second organic solvent is one or more of methanol, ethanol, dioxane and tetrahydrofuran;
in the step (3.5): the third organic solvent is one or two of dichloromethane and trichloromethane;
in the step (3.6): the fourth organic solvent is one or the combination of two of tetrahydrofuran and diethyl ether, and the strong base is n-butyllithium, sec-butyllithium or tert-butyllithium;
in step (3.7): the fifth organic solvent is one or the combination of two of acetone and acetonitrile;
in the step (3.8): the sixth organic solvent is one or more of dichloromethane, trichloromethane, acetonitrile and DMF, and the second weak base is one or more of pyridine, triethylamine and 4-dimethylaminopyridine.
10. Use of a staining reagent based on a phosphorus atom substituted rhodamine derivative scaffold as claimed in any one of claims 1 to 3 in lysosomal fluorescence imaging.
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