CN108516950B - Tetraphenylethylene-based subcellular organelle viscosity probe and preparation method and application thereof - Google Patents
Tetraphenylethylene-based subcellular organelle viscosity probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a tetraphenylethylene-based subcellular organelle viscosity probe, a preparation method and application thereof, wherein the subcellular organelle viscosity probe comprises a mitochondrial viscosity probe and a lysosome viscosity probe, and the preparation method mainly comprises the steps of (1) synthesizing a compound TPE-Br, (2) synthesizing a compound TPE-CHO, (3) synthesizing a mitochondrial viscosity probe, and (4) synthesizing the lysosome viscosity probe; the synthesized viscosity probe can be used for detecting the viscosity of subcellular organelles. The preparation method of the tetraphenylethylene-based subcellular organelle viscosity probe is simple, the synthesized viscosity probe has good water solubility, has no influence on the state of a researched cell within a working concentration range, has weak self-fluorescence background, improves the imaging contrast ratio of subcellular organelle viscosity monitoring, is sensitive to the reaction of viscosity, has extremely strong anti-interference performance, and has good targeting specificity to subcellular organelles.
Description
Technical Field
The invention belongs to the technical field of organic biology, and particularly relates to a tetraphenylethylene-based subcellular organelle viscosity probe, and a preparation method and application thereof.
Background
Intracellular viscosity essentially controls all diffusion-mediated processes including mass transport, signal transduction, biomolecular interactions, diffusion of metabolites and electron transport. Mitochondria and lysosomes are two important organelles in eukaryotic cells, are characterized by specific viscosity and further play an important role in functions such as ATP generation, biomolecule degradation and the like. Abnormal viscosity may reflect the state of dysfunction and is closely related to a variety of diseases. It is recognized that abnormal mitochondrial viscosity is associated with neurodegenerative diseases, diabetes and cellular malignancies, while abnormal lysosomal viscosity is a hallmark of lysosomal presence diseases, inflammation and even cancer. Thus, intracellular viscosity is a potential biomarker for different diseases. Due to the heterogeneity of intracellular viscosity, organ-specific probes must be developed for monitoring viscosity or its kinetics during different biological processes such as apoptosis and autophagy.
The molecular rotor is free of emission waves in a low viscosity medium and has strong fluorescence in the viscous medium, thereby providing a useful method for detecting intracellular viscosity. Most probes have non-specific localization in cells and are not suitable for detecting the micro-viscosity of specific organs. Recent studies have successfully demonstrated a method for molecular rotors to track micro-viscosity in mitochondria or lysosomes. However, these probes rely on Twisted Intramolecular Charge Transfer (TICT) excited states or pH-responsive organic molecular targeting, which is susceptible to interference from local polarity changes or microenvironment pH changes. Polymerization induced emission (AIE) provides an alternative investigation method for viscosity measurement. The AIE mechanism is the enhancement of fluorescence due to restriction of movement within the molecule in an aggregated or viscous microenvironment. In addition, AIE light sources typically have excellent light stability and large stokes shift. These properties make AIE luminescent materials important tools for widespread applications in the fields of biosensing and biomedical imaging. Existing probes have insufficient water solubility and suffer from reduced imaging contrast for viscosity monitoring due to their high fluorescent background, and furthermore, supplementation of the cell culture medium with organic solvents may slightly alter the state of the cells under investigation. Therefore, in a specific organelle, it is important to design a novel fluorescent probe with better water solubility for better water solubility.
Disclosure of Invention
In view of the above, the present invention is directed to a tetraphenylethylene-based subcellular organelle viscosity probe, and a preparation method and an application thereof, so as to solve the problems of insufficient water solubility of the probe, low imaging contrast of viscosity monitoring, and the like.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a tetraphenylethylene-based subcellular organelle viscosity probe comprising a mitochondrial viscosity probe and a lysosomal viscosity probe, wherein the mitochondrial viscosity probe has the following structural formula:
wherein the structure of the lysosomal viscosity probe is as follows:
further, the preparation method comprises the following steps:
(1) synthesis of Compound TPE-Br
Dissolving 4-7 g of (4-bromophenyl) (4-methoxyphenyl) ketone in 10mL of tetrahydrofuran, adding 1.5-4.5 g of zinc powder, placing a reaction bottle at-40 ℃, dropwise adding 1-4 mL of titanium tetrachloride into the reaction bottle, reacting for 1-2 h at room temperature, refluxing for 12h, extracting with ethyl acetate, washing an extracted organic phase with concentrated brine twice, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating a crude product by column chromatography to obtain TPE-Br;
(2) synthesis of Compound TPE-CHO
Dissolving 350-700 mg of TPE-Br in 8mL of anhydrous tetrahydrofuran, stirring at-40 ℃, carrying out nitrogen protection, adding 0.5-2 mL of 2.5M n-butyllithium, continuing to react for 0.5-2 h, then adding 0.1-0.4 mL of DMF, continuing to react for 2-5 h, sequentially adding a 10% HCl solution and a saturated sodium bicarbonate solution, washing with saturated saline water, drying with anhydrous sodium sulfate, filtering, and carrying out column chromatography separation on the filtrate to obtain TPE-CHO;
(3) synthesis of mitochondrial viscosity probes
a. Synthesis of mitochondrial Probe Mito-AIE1
And (3) dissolving 320-380 mg of TPE-Py in 10mL of dichloromethane, and stirring in an ice bath. Dropwise adding 0.1-0.3 mL of boron bromide, continuously reacting for 1-3 h, after the reaction is finished, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing the extracted organic phase with saturated salt water, collecting the organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating the crude product by column chromatography to obtain Mito-AIE 1;
b. synthesis of mitochondrial Probe Mito-AIE2
Dissolving 170-220 mg of compound TPE-M-Id in 10mL of dichloromethane, dropwise adding 0.1-0.4 mL of boron bromide under an ice bath condition, continuing to react for 1-3 h, after the reaction is finished, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing an organic phase with saturated saline, collecting an organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating a crude product by column chromatography to obtain a mitochondrion probe Mito-AIE 2;
(4) synthesis of lysosomal viscosity probes
a. Synthesis of lysosomal viscosity probe Lyso-AIE1
Dissolving 0.8-2 g of TPE-M-Py in 10mL of dichloromethane, stirring in an ice bath, then dropwise adding 0.2-0.8 mL of boron bromide, continuing to react for 1-3 h, after the reaction is finished, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing an organic phase with saturated salt water, collecting an organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating a crude product by column chromatography to obtain a lysosome viscosity probe Lyso-AIE 1;
b. synthesis of lysosomal viscosity probe Lyso-AIE2
Dissolving 550-650 mg of compound TPE-Id in 10mL of dichloromethane, stirring in an ice bath, then dropwise adding 0.2-0.8 mL of boron bromide, continuing to react for 2 hours, adding a saturated sodium bicarbonate solution after the reaction is finished, extracting with ethyl acetate, washing an organic phase with saturated saline, collecting an organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating a crude product by column chromatography to obtain a lysosome viscosity probe Lyso-AIE 2.
Further, the preparation method of (4-bromophenyl) (4-methoxyphenyl) methanone in the step (1) is as follows: dissolving 2.0-3.5 g of 4-bromobenzoyl chloride and 4-8 mL of anisole in 20mL of dichloromethane, and stirring for 20-40 min under ice bath. And then adding 1.2-2.0 g of aluminum chloride into the reaction solution, stirring at room temperature for 9-15 h, pouring ice water into the mixture, extracting with water for three times, filtering, distilling under reduced pressure, and separating the crude product by column chromatography to obtain a compound (4-bromophenyl) (4-methoxyphenyl) ketone.
Further, the synthesis method of the TPE-Py in the step (3) is as follows:
adding 300-600 mg of TPE-Br, 20-50 mg of palladium acetate, 130-220 mg of (o-td)3P, 1.2-3.1 g of potassium carbonate and 12-20 mL of N-methyl-2-pyrrolidone into a reaction bottle, stirring for 20-45 min under the protection of nitrogen, then adding 0.2-0.6 mL of 4-vinylpyridine, heating and refluxing in an oil bath at 130 ℃ for 24-48 h, cooling to room temperature after the reaction is finished, adding dichloromethane into the reaction solution, washing with water for 3 times, filtering, distilling under reduced pressure, and separating the crude product by column chromatography to obtain an orange solid compound TPE-Py.
Further, the synthesis method of the TPE-M-Id in the step (3) is as follows:
dissolving 75-100 mg of TPE-Id and 40-85 mg of 1,2,3, 3-tetramethyl-3H-indoxyl iodide in absolute ethyl alcohol, heating to 60-100 ℃, carrying out reflux reaction for 8-14H under the protection of argon, washing with saturated saline solution after the reaction is finished, drying with anhydrous sodium sulfate, filtering, carrying out reduced pressure distillation, and carrying out column chromatography separation on a crude product to obtain the compound TPE-M-Id.
Further, the preparation method of the 1,2,3, 3-tetramethyl-3H-indoxyl iodide comprises the following steps: adding methyl iodide into an acetonitrile solution dissolved in 2,3, 3-trimethyl-3H-indole, refluxing in an oil bath for 8-14H, carrying out vacuum filtration, washing with ethyl acetate, and drying to obtain a product.
Further, the synthesis method of the TPE-Id comprises the following steps:
reacting 1.2-2.1 g of 2,3, 3-trimethyl-3H-indole with 0.5-2 mL of concentrated hydrochloric acid at room temperature for 1.5-2.5H, then adding an absolute ethanol solution in which 75-100 mg of TPE-CHO is dissolved, reacting at 80 ℃, refluxing for 9-15H under the protection of argon, after the reaction is finished, sequentially adding a saturated sodium bicarbonate solution and a saturated saline solution, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating and purifying a crude product by column chromatography to obtain a compound TPE-Id;
further, the synthesis method of the TPE-M-Py in the step (4) is as follows:
dissolving 0.5-1.8 g of TPE-Py and 0.3-1.0 mL of iodomethane in 10mL of dichloromethane, reacting for 15-36 h, distilling under reduced pressure after the reaction is finished, and separating the crude product by column chromatography to obtain the compound TPE-M-Py.
The application of the tetraphenylethylene-based subcellular organelle viscosity probe or the tetraphenylethylene-based subcellular organelle viscosity probe prepared by the preparation method in detecting the subcellular organelle viscosity.
Compared with the prior art, the tetraphenylethylene-based subcellular organelle viscosity probe and the preparation method and application thereof have the following advantages:
(1) the preparation method of the tetraphenylethylene-based subcellular organelle viscosity probe is simple, the synthesized viscosity probe has good water solubility, does not influence the state of a researched cell within a working concentration range, has weak self-fluorescence background of the viscosity probe, and improves the imaging contrast ratio of subcellular organelle viscosity monitoring;
(2) the tetraphenylethylene-based subcellular organelle viscosity probe and the preparation method and application thereof have the advantages that the viscosity probe is very sensitive to the reaction of viscosity, has extremely strong anti-interference performance and good targeting specificity to the subcellular organelle, and the probe provides a research method with wide application prospect for the in-situ and real-time research of the physiological and pathological processes related to the subcellular organelles such as mitochondria and lysosomes.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is an experimental schematic diagram of the present invention;
wherein panel a is four tetraphenylethylene-derived fluorophores;
FIG. B is a schematic diagram of mitochondrial and lysosomal viscosity detection imaging;
FIG. 2 is a scheme for the synthesis of related compounds of the present invention;
wherein the reaction condition a is 4-bromobenzoyl chloride, anisole and dichloromethane at 0 ℃;
the reaction condition b is-40 ℃;
reaction conditions c were n-butyllithium, 10% HCl, DMF, nitrogen, -40 ℃;
reaction condition d is palladium acetate, (o-td)3P, potassium carbonate, N-methyl-2-pyrrolidone, 4-vinylpyridine, nitrogen, 130 ℃;
the reaction condition e is 2,3, 3-trimethyl-3H-indole, concentrated hydrochloric acid, ethanol and argon;
the reaction condition f is 1,2,3, 3-tetramethyl-3H-indoxyl iodide, ethanol, argon and argon;
the reaction conditions g are boron bromide and dichloromethane;
FIG. 3 is a graph of the fluorescence response curve and cell imaging experiment of MitoAIE1 in different solutions according to the present invention;
wherein panel a is a plot of the fluorescence of MitoAIE1 in PBS buffer;
FIG. b is a graph of the fluorescence of MitoAIE1 in a 99% glycerol + 1% DMSO solution system;
FIG. c1-c4 shows confocal imaging experiments performed after HeLa cells are selected and incubated with probes MitoAIE1 and Mito-Tracker green respectively;
FIG. 4 shows the fluorescence response curves of LysoAIE2 in different solutions according to the present invention
Wherein panel a is a plot of the fluorescence of LysoAIE2 in PBS buffer;
FIG. b is a graph of the fluorescence of LysoAIE2 in a 99% glycerol + 1% DMSO solution system;
FIGS. c1-c4 are graphs of confocal fiber imaging experiments performed after incubation of the probes LysoAIE2 and Lyso-Tracker green with HeLa cells, respectively;
FIG. d1-d4 graphs depicting real-time tracking of lysosome imaging by introducing the probe LysoAIE2 into cells;
FIG. 5 is a graph showing the viscosity change in the autophagy process of mitochondria and lysosomes according to an embodiment of the invention;
wherein, the graph a1-a4 is a graph of the change of confocal fluorescence imaging observed when Mito-AIE1 is added into Hela cells and the cells are subjected to 0min,30min,60min and120 min;
FIG. b1-b4 is a graph showing the change of confocal fluorescence imaging of Hela cells when Mito-AIE1 is added to the cells and observed at 0min,30min,60min and120 min;
Detailed Description
The present invention will be described in detail with reference to the following examples and accompanying drawings.
Example 1
A tetraphenylethylene-based subcellular organelle viscosity probe comprising a mitochondrial viscosity probe and a lysosomal viscosity probe, wherein the mitochondrial viscosity probe has the following structural formula:
wherein the structure of the lysosomal viscosity probe is as follows:
the synthetic route for the viscosity probe is shown in FIG. 2.
1. Synthesis of (4-bromophenyl) (4-methoxyphenyl) methanone
2.52g of 4-bromobenzoyl chloride and 6mL of anisole were dissolved in 20mL of dichloromethane and stirred for 25min under ice bath. Then, 1.57g of aluminum chloride was added to the reaction solution, and stirred at room temperature for 13 hours, then, ice water was poured into the mixture, extracted with water three times, filtered, distilled under reduced pressure, and the crude product was separated by column chromatography to obtain the compound (4-bromophenyl) (4-methoxyphenyl) methanone with a yield of 53.2%.1H-NMR(400MHz,CDCl3)(ppm):7.796(d,J＝8.8Hz,2H),7.624(s,4H)；6.968(d,J＝8.4,2H)；3.891(s,3H)13C-NMR(100MHz,CDCl3):194.45,163.44,137.01,132.49,131.52,131.31,129.73,126.86,113.71,55.56。
2. Synthesis of Compound TPE-Br
Dissolving 5.6g of (4-bromophenyl) (4-methoxyphenyl) ketone in 10mL of tetrahydrofuran, adding 2.8g of zinc powder, placing the reaction bottle at-40 ℃, dropwise adding 2.3mL of titanium tetrachloride into the reaction bottle, reacting for 1h at room temperature, refluxing for 12h, extracting with ethyl acetate, washing the extracted organic phase twice with concentrated brine, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating the crude product by column chromatography [ V (petroleum ether): V (ethyl acetate): 20: 1]. Compound (6.52g) was obtained as a white solid in 59.5% yield. 1H-NMR (400MHz, CDCl3) (ppm):7.220(d, J ═ 8.4Hz,4H),6.882(d, J ═ 8.4Hz, 8H); 6.652(d, J ═ 8.4, 4H); 3.749(s,6H)13C-NMR (100MHz, CDCl3) 158.34,142.99,139.03,135.41,132.98,132.51,130.92,120.39,113.37,55.51.
3. Synthesis of Compound TPE-CHO
Dissolving 559mg of TPE-Br in 8mL of anhydrous tetrahydrofuran, stirring at-40 ℃, adding 1.2mL of 2.5M n-butyllithium under the protection of nitrogen, continuing to react for 1h, then adding 0.2mL of DMF, continuing to react for 3h, sequentially adding a 10% HCl solution, a saturated sodium bicarbonate solution, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, and separating the filtrate by column chromatography [ V (petroleum ether): V (ethyl acetate): 4:1]. The compound TPE-CHO (71mg) was obtained as a yellow solid in 15.8% yield.1H-NMR(400MHz,CDCl3)(ppm):9.913(d,J＝6.8,2H),7.221-7.169(m,4H)；6.936-6.874(m,4H)；6.698-6.644(m,4H)；3.757(d,J＝7.2Hz 6H)13C-NMR(100MHz,CDCl3):191.89,158.76,150.45,140.56,134.72,134.45,132.58,132.49,131.03,131.95,129.27,113.56,113.49,55.16。
4. Synthesis of Compound TPE-Py
475.1mg of TPE-Br, 41mg of palladium acetate, 182.6mg of (o-td)3P, 2.2g of potassium carbonate and 15mL of N-methyl-2-pyrrolidone are added into a reaction bottle, stirred for 30min under the protection of nitrogen, then 0.43mL of 4-vinylpyridine is added, oil bath heating reflux is carried out at 130 ℃ for 36h, after the reaction is finished, the mixture is cooled to room temperature, dichloromethane is added into the reaction solution, washing is carried out for 3 times by water, filtering and reduced pressure distillation are carried out, and the crude product is separated by column chromatography [ V (petroleum ether): v (ethyl acetate) ═ 2:1]The compound TPE-Py (398mg) was obtained as an orange solid in a yield of 45.2%.1H-NMR(400MHz,DMSO-d6)(ppm):8.454(s,4H),7.245-7.198(m,8H)；7.127(d,J＝16.4,2H)；6.978(d,J＝7.6Hz,4H)；6.890-6.869(m,6H)；6.584(d,J＝8.0,4H),3.656(s,6H)13C-NMR(100MHz,DMSO-d6):158.32,149.88,144.98,144.95,135.93,134.01,133.09,132.66,131.93,126.48,125.46,120.84,113.29,55.12。
5. The synthetic method of TPE-M-Py is as follows:
1.2g TPE-Py and 0.6mL methyl iodide were dissolved in 10mL dichloromethane and reacted for 24h, after the reaction was completed, distillation was performed under reduced pressure, and the crude product was isolated by column chromatography [ V (dichloromethane): v (methanol) 10:1]The compound TPE-M-Py (957mg) was obtained as a red solid in 80% yield.1H-NMR(400MHz,DMSO-d6)(ppm):8.850(d,J＝6.0 4H)；8.850(d,J＝6.0 4H)；8.195(d,J＝6.4,4H)；7.941(d,J＝16Hz,2H)；7.563(d,J＝8.0,4H)；7.468(d,J＝16.4,2H)；7.071(d,J＝7.6,4H)；6.918(d,J＝8.4,4H)；6.749(d,J＝8.4,4H)；4.259(s,6H)；3.689(s,6H)13C-NMR(100MHz,DMSO-d6):158.53,152.90,146.40,145.51,140.61,140.25,135.53,133.78,132.65,131.99,128.21,123.90,113.99,55.51,47.43。
6. Synthesis of Compound TPE-Id:
reacting 1.59g of 2,3, 3-trimethyl-3H-indole with 1mL of concentrated hydrochloric acid at room temperature for 2H, then adding an absolute ethyl alcohol solution in which 89.6mg of TPE-CHO is dissolved, reacting at 80 ℃, refluxing for 12H under the protection of argon, after the reaction is finished, sequentially adding a saturated sodium bicarbonate solution, a saturated saline solution, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating and purifying a crude product by column chromatography [ V (petroleum ether): v (ethyl acetate) ═ 1:1]The compound TPE-Id was obtained as a yellow solid (5.55g) in 76% yield.1H-NMR(400MHz,DMSO-d6)(ppm):7.691-7.447(m,10H),7.342-7.907(m,2H)；7.242-7.201(m,4H)；7.031(d,J＝6.0Hz 4H)；6.939(d,J＝6.8Hz 4H)；6.760(d,J＝7.6Hz 4H)；3.706(s,6H)；1.394(d,J＝8.0Hz 12H)13C NMR(100MHz,DMSO-d6):183.58,158.42,154.19,147.23,145.39,139.94,139.90,137.50,135.95,135.83,134.53,134.42,132.65,132.59,131.91,131.84,128.08,127.75,125.88,121.99,120.49,120.32,120.18,113.93,55.41,52.75,23.42。
7. The TPE-M-Id is synthesized by the following method:
adding methyl iodide into acetonitrile solution of 2,3, 3-trimethyl-3H-indole, refluxing in oil bath for 12 hr, vacuum filtering, washing with ethyl acetate, and oven drying to obtain 1,2,3, 3-tetramethyl-3H-indololium iodideA compound (I) is provided.1H-NMR(400MHz,DMSO-d6)(ppm):7.923(d,J＝7.6Hz 1H)；7.841(d,J＝6.8Hz 1H)；7.637-7.616(m,2H)；3.987(s,3H)；2.788(s,3H)；1.539(s,6H)13C-NMR(100MHz,DMSO-d6):196.48,142.59,142.09,129.79,129.30,123.79,115.63,54.42,35.28,22.20,14.74.
Dissolving 89.6mg of TPE-Id and 59.2mg of 1,2,3, 3-tetramethyl-3H-indoxyl iodide in absolute ethyl alcohol, heating to 75 ℃, carrying out reflux reaction for 11H under the protection of argon, washing with saturated saline after the reaction is finished, drying with anhydrous sodium sulfate, filtering, carrying out reduced pressure distillation, and carrying out column chromatography separation on a crude product [ V (dichloromethane): v (methanol) 10:1]The red solid compound TPE-M-Id (73mg) was obtained in 36% yield.1H-NMR(400MHz,DMSO-d6)(ppm):8.342(d,J＝16.4Hz 2H),8.044(d,J＝7.2Hz 4H)；7.899(s,4H)；7.630(m,6H)；7.198(t,J＝8.4Hz 4H)；6.961(t,J＝8.0Hz 4H)；6.799(d,J＝8.8Hz 4H)；4.142(d,J＝8.4Hz 6H)；3.723(d,J＝4.0Hz 6H)；1.782(d,J＝6.4Hz 12H)13C-NMR(100MHz,DMSO-d6):156.56,156.44,150.46,145.21,144.84,144.45,141.66,137.72,134.58,134.58,134.51,134.21,133.15,132.55,131.74,131.35,128.30,127.00,126.58,126.05,121.24,115.16,115.05。
8. Synthesis of mitochondrial viscosity probes
a. Synthesis of mitochondrial Probe Mito-AIE1
353mg of TPE-Py was dissolved in 10mL of dichloromethane and stirred in an ice bath. Then dropwise adding 0.2mL of boron bromide, continuing to react for 2h, after the reaction is finished, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing the extracted organic phase with saturated sodium chloride solution, collecting the organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating the crude product by column chromatography [ V (methanol): v (dichloromethane) ═ 1:4]The compound Mito-AIE1 was obtained as a red solid (256mg) in 85% yield.1H-NMR(400MHz,DMSO-d6) (ppm). Mito-AIE1 was dissolved in PBS buffer, 99% glycerol + 1% DMSO solution, respectively, Mito-AIE1 produced almost no fluorescence signal in PBS buffer, its maximum absorption was 625nm in 99% glycerol + 1% DMSO solution system, and to test the sensitivity of MitoAIE1 to viscosity, it was changedVolume fraction of glycerol to investigate the response performance of the water/glycerol binary system to viscosity, at 25 ℃, as the viscosity increases from 0.894cP (water) to 942.5cP (99% glycerol), the fluorescence intensity of the probe AIEMITO1 at 625nm gradually increases, and the response time ratio is about 15 times, which shows that the probe is a fluorescent probe sensitive to viscosity, and the specific results are shown in FIG. 3a and FIG. 3 b. In order to test the targeting property of the probe, HeLa cells are selected for cell imaging experiments, probes MitoAIE1 and Mito-Tracker green are respectively incubated with the HeLa cells for confocal imaging experiments, images show that the PCC coefficients of MitoAIE1 and Mito-Tracker green are 0.93, and the probes MitoAIE1 can well target mitochondria, which is shown in fig. 3c1-c 4.
b. Synthesis of mitochondrial Probe Mito-AIE2
Dissolving 202mg of compound TPE-M-Id in 10mL of dichloromethane, dropwise adding 0.2mL of boron bromide under an ice bath condition, continuing to react for 2h, after the reaction is finished, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing an organic phase with saturated saline, collecting the organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating a crude product by column chromatography [ V (methanol): v (dichloromethane) ═ 1: 5. to give the compound Mito-AIE2(140mg) as an orange solid in 71% yield. 1H-NMR (400MHz, DMSO-d6) (ppm).
9. Synthesis of lysosomal viscosity probes
a. Synthesis of lysosomal viscosity probe Lyso-AIE1
Dissolving 1.2g of TPE-M-Py in 10mL of dichloromethane, stirring in an ice bath, then dropwise adding 0.6mL of boron bromide, continuing to react for 2 hours, adding a saturated sodium bicarbonate solution after the reaction is finished, extracting with ethyl acetate, washing an organic phase with saturated saline, collecting the organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating a crude product by column chromatography [ V (petroleum ether): v (ethyl acetate) ═ 1:1]The compound LysoAIE1(705mg) was obtained as an orange solid in 80% yield.1H-NMR(400MHz,DMSO-d6)(ppm):9.396(s,2H)；8.518(s,4H)；7.518-7.398(m,10H)；7.191-7.125(m,2H)；6.993(d,J＝8.0,4H)；6.782(d,J＝8.0,4H)；6.551(t,J＝6.4,4H)13C-NMR(100MHz,DMSO-d6):156.55,156.44,150.38,145.08,145.00,144.83,144.78,139.76,139.69,133.10,132.63,132.55,131.89,131.81,127.01,126.95,126.18,126.14,115.32,115.24。
b. Synthesis of lysosomal viscosity probe Lyso-AIE2
587mg of the compound TPE-Id is dissolved in 10mL of dichloromethane, the mixture is stirred in an ice bath, then 0.5mL of boron bromide is added dropwise, the reaction is continued for 2 hours, after the reaction is finished, a saturated sodium bicarbonate solution is added, ethyl acetate is used for extraction, an organic phase is washed by saturated saline solution, the organic phase is collected, dried by anhydrous sodium sulfate, filtered and distilled under reduced pressure, and a crude product is separated by column chromatography [ V (petroleum ether): v (ethyl acetate) ═ 3: 1. LysoAIE2(421mg) was obtained as an orange solid in 60% yield. 1H-NMR (400MHz, DMSO-d6) (ppm):9.413(s,2H),7.677-7.430(m, 10H); 7.310-7.274(m, 2H); 7.213-7.170(m, 4H); 7.025(d, J ═ 6.8Hz 4H); 6.785(d, J ═ 7.2Hz 4H); 6.560(d, J ═ 8.0Hz 4H); 1.381(d, J ═ 9.6Hz 12H)13C-NMR (100MHz, DMSO-d6):183.58,158.62,156.48,154.22,154.20,147.23,145.81,145.70,139.79,137.50,134.45,134.37,134.32,134.26,132.68,132.59,131.95,131.87,128.08,127.75,127.65,125.87,125.85,121.98,120.48,120.12,120.03,115.38,115.26,52.75, 23.41. Mito-AIE1 was dissolved in PBS buffer, 99% glycerol + 1% DMSO solution, respectively, and Mito-AIE1 produced almost no fluorescence signal in PBS buffer, and its emission peak at 570nm was significantly increased in 99% glycerol + 1% DMSO solution system, as shown in FIG. 4 a. To test the sensitivity of LysoAIE2 to viscosity, the volume fraction of glycerol was varied to explore the response of the water/glycerol binary system to viscosity, with increasing viscosity from 0.894cP (water) to 942.5cP (99% glycerol) at 25 ℃, the fluorescence intensity of probe LysoAIE2 at 570nm increased gradually, with a response fold ratio of approximately 12, indicating that it is a highly viscous fluorescent dye. HeLa cells are selected for cell imaging experiments, probes LysoAIE2 and Lyso-Tracker green are respectively incubated with the HeLa cells, then confocal fiber imaging experiments are carried out, and images c1-c4 show that the PCC coefficient of the overlap of the LysoAIE2 and the Lyso-Tracker green is 0.92. The results indicate that probe MitoAIE1 is able to target lysosomes well.
The LysoAIE2 probe was introduced into cells to perform real-time tracking lysosomal imaging experiments, dexamethasone was added to the cells as a stabilizer for lysosomal membranes and an inhibitor of lysosomal enzyme release, and confocal images showed a gradual increase in fluorescence within 20min, indicating an increase in viscosity. This suggests that the increase in viscosity may be due to a decrease in lysosomal degradation efficiency. LysoAIE2 was also used to track lysosomes moving due to dexamethasone induction of lysosomes, see fig. 4d1-d 4.
Mitochondrial and lysosomal viscosity monitoring
We added MitoAIE1 and LysoAIE2 to monitor changes in viscosity of autophagy processes in mitochondria and lysosomes, respectively. HeLa cells were cultured in serum-free medium, with the addition of MitoAIE1 or LysoAIE2, respectively, and fluorescence images were obtained at different time points of 2h, with confocal images showing the transition of mitochondria from tubular to globular structures and a gradual increase in fluorescence signal over time. In contrast, the fluorescence signal of cells cultured in normal medium is almost constant, and lysosomes are the same, as shown in FIG. 5. These results indicate that the viscosity of mitochondria and lysosomes is indeed increased during autophagy, probably due to the greater viscosity of autophagosomes, and the increase in lysosomal viscosity probably due to the increased burden of protein degradation.
Example 2
The preparation method of the subcellular organelle viscosity probe comprises the following steps:
1. synthesis of (4-bromophenyl) (4-methoxyphenyl) methanone
2.80g of 4-bromobenzoyl chloride and 8mL of anisole were dissolved in 20mL of dichloromethane and stirred for 40min under ice bath. Then, 2.0g of aluminum chloride was added to the reaction solution, and stirred at room temperature for 9 hours, then, ice water was poured into the mixture, extracted with water three times, filtered, distilled under reduced pressure, and the crude product was separated by column chromatography to obtain the compound (4-bromophenyl) (4-methoxyphenyl) methanone with a yield of 50.2%.1H-NMR(400MHz,CDCl3)(ppm):7.796(d,J＝8.8Hz,2H),7.624(s,4H)；6.968(d,J＝8.4,2H)；3.891(s,3H)13C-NMR(100MHz,CDCl3):194.45,163.44,137.01,132.49,131.52,131.31,129.73,126.86,113.71,55.56。
2. Synthesis of Compound TPE-Br
Dissolving 4.22g of (4-bromophenyl) (4-methoxyphenyl) ketone in 10mL of tetrahydrofuran, adding 1.5g of zinc powder, placing the reaction bottle at-40 ℃, dropwise adding 1mL of titanium tetrachloride into the reaction bottle, reacting at room temperature for 2h, refluxing for 12h, extracting with ethyl acetate, washing the extracted organic phase twice with concentrated brine, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating the crude product by column chromatography [ V (petroleum ether): V (ethyl acetate): 20: 1]. Compound (6.52g) was obtained as a white solid in 52.7% yield. 1H-NMR (400MHz, CDCl3) (ppm):7.220(d, J ═ 8.4Hz,4H),6.882(d, J ═ 8.4Hz, 8H); 6.652(d, J ═ 8.4, 4H); 3.749(s,6H)13C-NMR (100MHz, CDCl3) 158.34,142.99,139.03,135.41,132.98,132.51,130.92,120.39,113.37,55.51.
3. Synthesis of Compound TPE-CHO
Dissolving 350mg of TPE-Br in 8mL of anhydrous tetrahydrofuran, stirring at the temperature of minus 40 ℃, carrying out nitrogen protection, adding 0.5mL of 2.5M n-butyllithium, continuing to react for 0.5h, then adding 0.1mL of DMF, continuing to react for 5h, sequentially adding a 10% HCl solution and a saturated sodium bicarbonate solution, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, and carrying out column chromatography separation on the filtrate [ V (petroleum ether): V (ethyl acetate): 4:1]. Yellow solid compound TPE-CHO (49mg) was obtained in 13.2% yield.1H-NMR(400MHz,CDCl3)(ppm):9.913(d,J＝6.8,2H),7.221-7.169(m,4H)；6.936-6.874(m,4H)；6.698-6.644(m,4H)；3.757(d,J＝7.2Hz 6H)13C-NMR(100MHz,CDCl3):191.89,158.76,150.45,140.56,134.72,134.45,132.58,132.49,131.03,131.95,129.27,113.56,113.49,55.16。
4. Synthesis of Compound TPE-Py
Adding 450mg of TPE-Br, 20mg of palladium acetate, 130mg (o-td)3P, 1.2g of potassium carbonate and 20mL of N-methyl-2-pyrrolidone into a reaction bottle, stirring for 45min under the protection of nitrogen, then adding 0.2mL of 4-vinylpyridine, heating and refluxing in an oil bath at 130 ℃ for 24h, cooling to room temperature after the reaction is finished, adding dichloromethane into the reaction solution, washing with water for 3 times, filtering, distilling under reduced pressure, and separating a crude product by column chromatography [ V (petroleum ether): v (ethyl acetate) ═ 2:1]The compound TPE-Py (372mg) was obtained as an orange solid in 44.5% yield.1H-NMR(400MHz,DMSO-d6)(ppm):8.454(s,4H),7.245-7.198(m,8H)；7.127(d,J＝16.4,2H)；6.978(d,J＝7.6Hz,4H)；6.890-6.869(m,6H)；6.584(d,J＝8.0,4H),3.656(s,6H)13C-NMR(100MHz,DMSO-d6):158.32,149.88,144.98,144.95,135.93,134.01,133.09,132.66,131.93,126.48,125.46,120.84,113.29,55.12。
5. The synthetic method of TPE-M-Py is as follows:
0.8g of TPE-Py and 0.5mL of methyl iodide are dissolved in 10mL of dichloromethane and reacted for 20h, after the reaction is finished, reduced pressure distillation is carried out, and the crude product is separated by column chromatography [ V (dichloromethane): v (methanol) 10:1]The red solid compound TPE-M-Py (759mg) was obtained in 77% yield.1H-NMR(400MHz,DMSO-d6)(ppm):8.850(d,J＝6.0 4H)；8.850(d,J＝6.0 4H)；8.195(d,J＝6.4,4H)；7.941(d,J＝16Hz,2H)；7.563(d,J＝8.0,4H)；7.468(d,J＝16.4,2H)；7.071(d,J＝7.6,4H)；6.918(d,J＝8.4,4H)；6.749(d,J＝8.4,4H)；4.259(s,6H)；3.689(s,6H)13C-NMR(100MHz,DMSO-d6):158.53,152.90,146.40,145.51,140.61,140.25,135.53,133.78,132.65,131.99,128.21,123.90,113.99,55.51,47.43。
6. Synthesis of Compound TPE-Id:
reacting 1.2g of 2,3, 3-trimethyl-3H-indole with 0.5mL of concentrated hydrochloric acid at room temperature for 2.5H, then adding an absolute ethyl alcohol solution in which 75mg of TPE-CHO is dissolved, reacting at 80 ℃, refluxing for 9H under the condition of argon protection, after the reaction is finished, sequentially adding a saturated sodium bicarbonate solution, a saturated saline solution and anhydrous sodium sulfate, drying, filtering and distilling under reduced pressure, and separating and purifying a crude product by column chromatography [ V (petroleum ether): v (ethyl acetate) ═ 1:1]The compound TPE-Id was obtained as a yellow solid (5.14g) in 73.2% yield.1H-NMR(400MHz,DMSO-d6)(ppm):7.691-7.447(m,10H),7.342-7.907(m,2H)；7.242-7.201(m,4H)；7.031(d,J＝6.0Hz 4H)；6.939(d,J＝6.8Hz 4H)；6.760(d,J＝7.6Hz 4H)；3.706(s,6H)；1.394(d,J＝8.0Hz 12H)13C NMR(100MHz,DMSO-d6):183.58,158.42,154.19,147.23,145.39,139.94,139.90,137.50,135.95,135.83,134.53,134.42,132.65,132.59,131.91,131.84,128.08,127.75,125.88,121.99,120.49,120.32,120.18,113.93,55.41,52.75,23.42。
7. The TPE-M-Id is synthesized by the following method:
adding methyl iodide into acetonitrile solution dissolved in 2,3, 3-trimethyl-3H-indole, refluxing in oil bath for 8H, vacuum filtering, washing with ethyl acetate, and oven drying to obtain 1,2,3, 3-tetramethyl-3H-indoxyl iodide.1H-NMR(400MHz,DMSO-d6)(ppm):7.923(d,J＝7.6Hz 1H)；7.841(d,J＝6.8Hz 1H)；7.637-7.616(m,2H)；3.987(s,3H)；2.788(s,3H)；1.539(s,6H)13C-NMR(100MHz,DMSO-d6):196.48,142.59,142.09,129.79,129.30,123.79,115.63,54.42,35.28,22.20,14.74。
Dissolving 75mg of TPE-Id and 40mg of 1,2,3, 3-tetramethyl-3H-indoxyl iodide in absolute ethyl alcohol, heating to 60 ℃, carrying out reflux reaction for 14H under the protection of argon, washing with saturated saline solution after the reaction is finished, drying with anhydrous sodium sulfate, filtering, carrying out reduced pressure distillation, and carrying out column chromatography separation on a crude product to obtain [ V (dichloromethane): v (methanol) 10:1]The red solid compound TPE-M-Id (66mg) was obtained in 34% yield.1H-NMR(400MHz,DMSO-d6)(ppm):8.342(d,J＝16.4Hz 2H),8.044(d,J＝7.2Hz 4H)；7.899(s,4H)；7.630(m,6H)；7.198(t,J＝8.4Hz 4H)；6.961(t,J＝8.0Hz 4H)；6.799(d,J＝8.8Hz 4H)；4.142(d,J＝8.4Hz 6H)；3.723(d,J＝4.0Hz 6H)；1.782(d,J＝6.4Hz 12H)13C-NMR(100MHz,DMSO-d6):156.56,156.44,150.46,145.21,144.84,144.45,141.66,137.72,134.58,134.58,134.51,134.21,133.15,132.55,131.74,131.35,128.30,127.00,126.58,126.05,121.24,115.16,115.05。
8. Synthesis of mitochondrial viscosity probes
a. Synthesis of mitochondrial Probe Mito-AIE1
320mg of TPE-Py was dissolved in 10mL of dichloromethane and stirred in an ice bath. Then dropwise adding 0.1mL of boron bromide, continuing to react for 3h, after the reaction is finished, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing the extracted organic phase with saturated sodium chloride solution, collecting the organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating the crude product by column chromatography [ V (methanol): v (dichloromethane) ═ 1:4]The compound Mito-AIE1 was obtained as a red solid (186mg) in 82% yield.1H-NMR(400MHz,DMSO-d6)(ppm)。
b. Synthesis of mitochondrial Probe Mito-AIE2
Dissolving 170mg of compound TPE-M-Id in 10mL of dichloromethane, dropwise adding 0.1mL of boron bromide under an ice bath condition, continuing to react for 3h, after the reaction is finished, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing an organic phase with saturated saline, collecting the organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating a crude product by column chromatography [ V (methanol): v (dichloromethane) ═ 1: 5. give the compound Mito-AIE2(95mg) as an orange solid in 67% yield. 1H-NMR (400MHz, DMSO-d6) (ppm).
9. Synthesis of lysosomal viscosity probes
a. Synthesis of lysosomal viscosity probe Lyso-AIE1
Dissolving 2g of TPE-M-Py in 10mL of dichloromethane, stirring in an ice bath, then dropwise adding 0.8mL of boron bromide, continuing to react for 1h, after the reaction is finished, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing an organic phase with saturated saline, collecting an organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating a crude product by column chromatography [ V (petroleum ether): v (ethyl acetate) ═ 1:1]The compound LysoAIE1(763mg) was obtained as an orange solid in 75% yield.1H-NMR(400MHz,DMSO-d6)(ppm):9.396(s,2H)；8.518(s,4H)；7.518-7.398(m,10H)；7.191-7.125(m,2H)；6.993(d,J＝8.0,4H)；6.782(d,J＝8.0,4H)；6.551(t,J＝6.4,4H)13C-NMR(100MHz,DMSO-d6):156.55,156.44,150.38,145.08,145.00,144.83,144.78,139.76,139.69,133.10,132.63,132.55,131.89,131.81,127.01,126.95,126.18,126.14,115.32,115.24。
b. Synthesis of lysosomal viscosity probe Lyso-AIE2
Dissolving 550mg of compound TPE-Id in 10mL of dichloromethane, stirring in an ice bath, then dropwise adding 0.2mL of boron bromide, continuing to react for 2 hours, after the reaction is finished, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing an organic phase with saturated saline, collecting the organic phase, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure, and separating a crude product by column chromatography [ V (petroleum ether): v (ethyl acetate) ═ 3: 1. the compound LysoAIE2(392mg) was obtained as an orange solid in 58% yield. 1H-NMR (400MHz, DMSO-d6) (ppm):9.413(s,2H),7.677-7.430(m, 10H); 7.310-7.274(m, 2H); 7.213-7.170(m, 4H); 7.025(d, J ═ 6.8Hz 4H); 6.785(d, J ═ 7.2Hz 4H); 6.560(d, J ═ 8.0Hz 4H); 1.381(d, J ═ 9.6Hz 12H)13C-NMR (100MHz, DMSO-d6):183.58,158.62,156.48,154.22,154.20,147.23,145.81,145.70,139.79,137.50,134.45,134.37,134.32,134.26,132.68,132.59,131.95,131.87,128.08,127.75,127.65,125.87,125.85,121.98,120.48,120.12,120.03,115.38,115.26,52.75, 23.41.
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 (2)
1. A tetraphenylethylene-based subcellular organelle viscosity probe characterized by: the subcellular organelle viscosity probe includes a mitochondrial viscosity probe and a lysosomal viscosity probe, wherein the mitochondrial viscosity probe has a structural formula as follows:
wherein the structure of the lysosomal viscosity probe is as follows:
2. use of the tetraphenylethylene-based subcellular organelle viscosity probe of claim 1 for detecting subcellular organelle viscosity.
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