CN114105879A - Preparation of novel isophorone derivative and application of novel isophorone derivative in fields of organelle marking and viscosity detection - Google Patents

Preparation of novel isophorone derivative and application of novel isophorone derivative in fields of organelle marking and viscosity detection Download PDF

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CN114105879A
CN114105879A CN202111388806.9A CN202111388806A CN114105879A CN 114105879 A CN114105879 A CN 114105879A CN 202111388806 A CN202111388806 A CN 202111388806A CN 114105879 A CN114105879 A CN 114105879A
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isophorone
derivative
isophorone derivative
organelle
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葛健锋
喻情
倪靖阳
孙如
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Suzhou University
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Abstract

The invention discloses preparation of a novel isophorone derivative and application of the novel isophorone derivative in the fields of organelle marking and viscosity detection, wherein a compound (3,5, 5-trimethylcyclohex-2-eneylidene) malononitrile reacts with different building blocks containing aldehyde groups to obtain four isophorone derivatives, and conjugation degree is increased by introducing the building blocks to enable the compound to reach a near infrared region. The compound obtained by the invention can mark mitochondria, lysosomes, endoplasmic reticulum or lipid droplets. The invention discloses an isophorone derivative fluorescent dye with organelle targeting ability for the first time, and solves the problem that the isophorone derivative fluorescent dye is undefined in organelle targeting ability, and the compound is sensitive to viscosity and remarkably enhances fluorescence in a viscous environment. The invention discloses that isophorone derivatives can regulate organelle targeting ability, making it possible to design fluorescent markers with different organelle targeting ability by using a single basic backbone structure.

Description

Preparation of novel isophorone derivative and application of novel isophorone derivative in fields of organelle marking and viscosity detection
Technical Field
The invention belongs to the technical field of fluorescence labeling, and particularly relates to preparation of a novel isophorone derivative and application of the novel isophorone derivative in the fields of organelle labeling and viscosity detection.
Background
Changes in the intracellular microenvironment (including pH, temperature, polarity, viscosity, etc.) disrupt the homeostasis and function of organelles (see: chem. sci.,2020,11, 596-. In particular, studies have demonstrated that mitochondrial viscosity abnormalities are associated with neurodegenerative diseases, arteriosclerosis, and diabetes (see: chem. Commun.,2019,55, 7410-. Abnormal lysosomal physiological states can lead to lysosomal storage diseases, tuberculosis, metabolic disorders, inflammation, neurodegenerative diseases (see: j. mater. chem. b,2018,6, 6592-. Disorders of the endoplasmic reticulum can easily cause memory deterioration, heart disease and kidney disease (see: J. Mater. chem. B,2021,9, 5664-. Many metabolic diseases such as obesity, fatty liver, cardiovascular diseases and diabetes, and neutral lipid storage diseases are accompanied by lipid droplet dysfunction (see: ACS Sens.2021,6, (1), 22-26). The proper viscosity environment is an important factor for maintaining the smooth operation of each organelle, and the viscosity change of the organelle is also considered as an important index reflecting the working state thereof.
To date, many small molecule viscosity probes have been developed based on the Twisted Intramolecular Charge Transfer (TICT) mechanism, in which intramolecular rotation is inhibited, nonradiative decay is reduced, and fluorescence is significantly enhanced in a viscous environment (see: anal. chem.2020,92, (5), 3517-. However, they still have much room for improvement. First, many viscosity probes are susceptible to interference by nucleophiles and are not ideally selective. Second, most viscosity probes emit at shorter wavelengths and do not reach the near infrared region. Almost all near-infrared viscosity probes are salt compounds, and although the emission wavelength reaches the near-infrared region, the high toxicity of the salt compounds cannot be well solved. Third, there is no clear organelle targeting function. Viscosity probes with well-defined organelle targeting are currently reported to be mostly cationic viscosity probes, which mostly enter mitochondria due to their negatively charged membrane potential, making them easy to enter mitochondria, however this inevitably destroys the microenvironment of the organism (see: anal. chem.2019,91, (13), 8415-8421). Although viscosity probes have developed rapidly in recent years, reports on other organelle viscosity probes have remained quite limited.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of a novel isophorone derivative and an application of the isophorone derivative in the fields of organelle marking and viscosity detection.
An isophorone derivative, the derivative having the structural formula:
Figure BDA0003367964380000021
wherein R is selected from the following structural formulae (1) to (4):
Figure BDA0003367964380000022
the process for producing an isophorone derivative of claim 1, comprising the steps of:
dissolving (3,5, 5-trimethylcyclohex-2-eneylidene) malononitrile and a compound M in an organic solvent, and reacting at 80-120 ℃ for 12 hours under the action of a catalyst to obtain the isophorone derivative;
wherein the compound M is 1H-indazole-5-formaldehyde, 4- (4-hydroxypiperidin-1-yl) benzaldehyde, 4-hydroxy-1-naphthaldehyde or 1-pyrene formaldehyde.
In one embodiment of the invention, the molar ratio of the (3,5, 5-trimethylcyclohex-2-enylidene) malononitrile to the compound M is 1:0.7 to 1: 2.0.
In one embodiment of the invention, the organic solvent is ethanol or DMF.
In one embodiment of the invention, the catalyst comprises a basic catalyst and an acidic catalyst for aldol condensation;
in one embodiment of the invention, the acidic catalyst is piperidine.
In one embodiment of the invention, the basic catalyst is trimethylchlorosilane.
The isophorone derivative is applied to preparation of a cell fluorescent reagent.
The isophorone derivative is applied to organelle fluorescence imaging for non-diagnosis and treatment purposes.
In one embodiment of the invention, the organelle is a lysosome, a mitochondrion, an endoplasmic reticulum, or a lipid droplet.
The invention also provides a method for non-diagnostic non-therapeutic purpose cellular fluorescence imaging, comprising the following steps: and co-culturing the cells and the isophorone derivative in a culture medium, and then performing cell imaging to finish cell fluorescence imaging. In the invention, the cells and the culture medium are conventional products, and the technical effect of the invention is not influenced.
In one embodiment of the invention, the concentration of the isophorone derivative in a culture medium is 10-20 mu M.
The invention also provides a method for non-diagnostic non-therapeutic purpose cellular fluorescence imaging, comprising the following steps: co-culturing cells, the isophorone derivatives and the ionophore in a culture medium, and then performing cell imaging to finish cell fluorescence imaging, wherein the isophorone derivatives can perform fluorescence labeling of different organelles.
In one embodiment of the invention, the mitochondrial, lysosome, endoplasmic reticulum and lipid droplet fluorescent markers can be added into the culture medium respectively to obtain culture media containing the mitochondrial, lysosome, endoplasmic reticulum and lipid droplet fluorescent markers respectively; mitochondria, lysosomes, endoplasmic reticulum, lipid drop fluorescent markers and ionophores (monensin, dexamethasone and nystatin) can be added into a culture medium respectively to obtain a culture medium containing the fluorescent markers and the ionophores; preferably, the concentration of the fluorescent marker in the culture medium is 10 μ M, 14 μ M, 10 μ M and 20 μ M respectively; in the culture medium containing the fluorescent marker and the ionophore, the concentration of the fluorescent marker in the culture medium is 2 mu M, and the concentration of the ionophore is 10 mu M.
In one embodiment of the present invention, the cell imaging can be performed by using a confocal laser microscope, for example, the green light channel is excited by 488nm wavelength to collect the fluorescence signal in the wavelength range of 468-550nm, the red light channel is excited by 561nm wavelength to collect the fluorescence signal in the wavelength range of 570-800 nm; cell imaging results show that the four isophorone derivative fluorescent dyes contained in the invention can well mark mitochondria, lysosomes, endoplasmic reticulum or lipid droplets respectively, and can be used as a green mitochondrial marker, a red lysosomal marker, a red endoplasmic reticulum marker and a red lipid droplet marker. After the cell viscosity is increased by adding the ionophore, the fluorescence of the fluorescent dye in its corresponding organelle is significantly enhanced.
In one embodiment of the invention, the CO-cultivation is carried out at 5% CO at saturated humidity, 37 ℃2Co-culturing for 5 minutes in an incubator; preferably, the fluorescent markers of mitochondria, lysosomes, endoplasmic reticulum and lipid droplets are added respectively, and then the mixture is cultured for 5 minutes, washed by PBS buffer solution and subjected to cell imaging by using a laser confocal microscope.
In one embodiment of the invention, the ionophore is monensin, dexamethasone, or nystatin.
In one embodiment of the invention, the concentration of the isophorone derivative in a culture medium is 1-2 mu M; the concentration of the ionophore is 5-20 mu M.
According to the change of the structure of the isophorone derivative, the isophorone derivative has the capability of fluorescently marking organelles at high concentration; and the viscosity of the organelle is detected by the fluorescence intensity at low concentration.
The invention also provides application of the isophorone derivative in viscosity detection.
In one embodiment of the invention, the organelle viscosity is measured by the fluorescence intensity of the isophorone derivative.
The invention discloses a series of isophorone derivative fluorescent dyes, which not only have different organelle targeting capabilities, but also can detect viscosity. Most of these four neutral fluorescent dyes still reach the Near Infrared (NIR) region due to the strong Intramolecular Charge Transfer (ICT) effect. Thanks to the neutral fluorescent dyes, the cytotoxicity is significantly reduced compared to the salt fluorescent dyes. The method solves the problem of undefined targeting capability of an isophorone derivative fluorescent dye organelle for the first time, and can monitor viscosity at the same time. The invention enables the design of viscosity probes with different organelle targeting capabilities by using a single basic framework structure through the synthesis of cheap building blocks, obviously reduces the cost and the difficulty, and has important scientific significance and commercial value.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the invention discloses four kinds of isophorone derivative fluorescent dyes, which can be respectively marked with different organelles after being co-cultured with cells; meanwhile, the four fluorescent dyes are sensitive to viscosity, and after the ionophore, the fluorescent dyes and the cells are cultured together, the fluorescence of the fluorescent dyes in the corresponding organelles is obviously enhanced, so that the viscosity of the corresponding organelles can be detected. The invention uses a basic structure for improved design, improves the optical property and the biological activity of the fluorescent marker by participating in synthesis through different cheap building blocks, and can adjust the targeting capability of organelles, thereby having important scientific significance and commercial value.
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In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a synthetic route of the dye designed by the present invention.
FIG. 2 shows UV-visible absorption spectra and fluorescence spectra of the dyes 1a to 1d according to the invention in glycerol and dimethyl sulfoxide, respectively. Wherein (a) is the uv-vis absorption spectrum and the fluorescence spectrum of 1a in glycerol, (b) is the uv-vis absorption spectrum and the fluorescence spectrum of 1a in dimethyl sulfoxide, (c) is the uv-vis absorption spectrum and the fluorescence spectrum of 1b in glycerol, (d) is the uv-vis absorption spectrum and the fluorescence spectrum of 1b in dimethyl sulfoxide, (e) is the uv-vis absorption spectrum and the fluorescence spectrum of 1c in glycerol, (f) is the uv-vis absorption spectrum and the fluorescence spectrum of 1c in dimethyl sulfoxide, (g) is the uv-vis absorption spectrum and the fluorescence spectrum of 1d in glycerol, (f) is the uv-vis absorption spectrum and the fluorescence spectrum of 1d in dimethyl sulfoxide;
FIG. 3 is a photograph showing the images of the dyes 1a to 1d of the present invention in HeLa cells, respectively. Wherein (a1-a6) is an imaging picture of 1a in HeLa cells, (b1-b6) is an imaging picture of 1b in HeLa cells, (c1-c6) is an imaging picture of 1c in HeLa cells, and (d1-d6) is an imaging picture of 1d in HeLa cells.
FIG. 4 is an image of cells before and after the addition of ionophore to HeLa cells with dyes 1a-1d according to the present invention. Wherein (A) is an image of the cell before and after the addition of the ionophore to the HeLa cell in 1a, (B) is an image of the cell before and after the addition of the ionophore to the HeLa cell in 1B, (C) is an image of the cell before and after the addition of the ionophore to the HeLa cell in 1C, and (D) is an image of the cell before and after the addition of the ionophore to the HeLa cell in 1D.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1
Compound 1((3,5, 5-trimethylcyclohex-2-enylidene) malononitrile, 1.7 mmol, 254 mg), compound a (1H-indazole-5-carbaldehyde, 1.7 mmol, 200 mg) was dissolved in 10 ml of anhydrous ethanol, and 100 μ l of piperidine was added thereto. The reaction was refluxed at 80 ℃ for 12 hours under nitrogen. After the reaction was completed and cooled to room temperature, the organic solvent was removed by a rotary evaporator. The pure product is obtained after column chromatography separation, and the eluent: dichloromethane, orange solid dye 1a, 317.83 mg, 57% yield.
Nuclear magnetic resonance hydrogen spectrum (300MHz, DMSO-d) of dye 1a6)1H NMR(400MHz,DMSO-d6)δ(ppm)13.24(s,1H,NH),8.13(d,J＝20.77Hz 2H,CH),7.82(d,J＝8.48Hz,1H,Ar-H),7.58(d,J＝7.77Hz,1H,Ar-H),7.43(s,1H,Ar-H),5.76(s,1H,CH),2.62(d,J＝11.74Hz,CH3),1.04(s,1H,CH2). Nuclear magnetic resonance carbon spectrum of dye 1a (600MHz, DMSO-d)6),13C NMR(600MHz,DMSO-d6)δ(ppm)143.24,129.33,113.13,111.70,107.41,101.66,100.60,98.05,96.23,94.87,86.94,86.12,83.67,48.28,27.81,15.22,11.12,4.61.HRMS(ESI+) Calculated m/z: c20H20N4 +[M+H]+315.1604, found: 315.1707.
example 2
Compound 1((3,5, 5-trimethylcyclohex-2-enylidene) malononitrile, 1.0 mmol, 186 mg), compound b (4- (4-hydroxypiperidin-1-yl) benzaldehyde, 1.0 mmol, 205 mg) was dissolved in 10 ml of anhydrous ethanol, 100. mu.l of piperidine was added thereto, and the reaction was refluxed for 12 hours under nitrogen. After the reaction was completed and cooled to room temperature, the organic solvent was removed by a rotary evaporator. The pure product is obtained after column chromatography separation, and the eluent: dichloromethane, the red brown solid dye 1b, 194.40 mg, 52% yield.
Nuclear magnetic resonance hydrogen spectrum (300MHz, DMSO-d) of dye 1b6)1H NMR(400MHz,DMSO-d6)δ(ppm)7.56(d,J＝8.36Hz,2H,Ar-H),7.21(m,J＝16.02Hz,2H,CH),6.96(d,J＝8.50Hz,2H,Ar-H),6.77(s,1H,CH),4.72(d,J＝3.75Hz,1H,OH),3.71(d,J＝9.77Hz,2H,CH2),3.03(t,J＝10.44Hz,2H,CH2),2.58(d,4H,CH2),1.81(d,J＝9.48Hz,2H,CH2),1.43(m,J＝9.50Hz,2H,CH2),1.01(s,3H,CH3). Nuclear magnetic resonance carbon spectrum of dye 1b (600MHz, DMSO-d)6),13C NMR(600MHz,DMSO-d6)δ(ppm)142.57,129.70,124.08,111.33,102.26,97.59,97.52,93.21,86.93,86.11,46.11,38.40,17.56,14.87,10.76,6.08,4.18.HRMS(ESI+) Calculated m/z: c24H28N3O+[M+H]+374.2227, found: 374.2498.
example 3
Compound 1((3,5, 5-trimethylcyclohex-2-enylidene) malononitrile, 1.0 mmol, 186 mg) and compound c (4-hydroxy-1-naphthaldehyde, 1.0 mmol, 172 mg) were dissolved in 10 ml of anhydrous ethanol, 100 μ l of piperidine was added thereto, and the reaction was refluxed for 12 hours under nitrogen. After the reaction was completed and cooled to room temperature, the organic solvent was removed by a rotary evaporator. The pure product is obtained after column chromatography separation, and the eluent: dichloromethane, brown red solid dye 1c, 270.4 mg, 61% yield.
Nuclear magnetic resonance Hydrogen Spectroscopy (300MHz, CDCl) of dye 1c3)1H NMR(400MHz,CDCl3)δ(ppm)8.29(d,J＝7.82Hz,1H,Ar-H),8.14(d,J＝8.14Hz,1H,Ar-H),7.82(d,J＝15.95Hz,2H,CH),7.72(d,J＝7.88Hz,1H,Ar-H),7.62(m,J＝8.00Hz,2H,Ar-H),7.02(d,J＝15.72Hz,1H,Ar-H),6.90(m,J＝7.99Hz,2H,Ar-H),5.93(s,1H,OH),2.62(d,J＝11.42Hz,4H,CH2),1.12(s,6H,CH3). Nuclear magnetic resonance carbon Spectroscopy (600MHz, DMSO-d) of dye 1c6),13C NMR(600MHz,DMSO-d6)δ(ppm)164.73,142.78,129.43,128.21,106.49,104.90,101.96,100.88,99.72,98.78,97.47,96.94,95.71,95.16,94.26,86.69,85.86,81.17,80.13,47.34,14.92,10.66,4.23.HRMS(ESI+) Calculated m/z: c23H20N2NaO+[M+Na]+363.1468, found: 363.1662.
example 4
Compound 1((3,5, 5-trimethylcyclohex-2-enylidene) malononitrile, 0.86 mmol, 161 mg) and compound d (1-pyrenecarboxaldehyde, 0.86 mmol, 200 mg) were dissolved in 10 ml of anhydrous ethanol, 100. mu.l of piperidine was added thereto, and the reaction was refluxed for 12 hours under nitrogen. After the reaction is finished and the temperature is reduced to room temperature, a large amount of precipitate is separated out, the precipitate is filtered out by suction and washed by absolute ethyl alcohol, and the final pure product, namely the red solid dye 1d, 178.0 mg and the yield of 52 percent, is obtained.
Nuclear magnetic resonance hydrogen spectrum of dye 1d (300MHz, DMSO-d)6)1H NMR(400MHz,DMSO-d6)δ(ppm)8.80(d,J＝9.17Hz,1H,Ar-H),8.66(d,J＝8.27Hz,1H,Ar-H),8.39(m,J＝8.14Hz,4H,Ar-H),8.24(J＝5.04Hz,2H,Ar-H),8.14(t,J＝7.95Hz,1H,Ar-H),7.78(d,J＝16.41Hz,2H,CH),7.02(s,1H,CH),2.81(2H,CH2),2.62(2H,CH2),1.09(6H,CH3. The nuclear magnetic resonance carbon spectrum could not be obtained due to the poor solubility of 1 d. HRMS (ESI)+) Calculated m/z: c29H22N2Na+[M+Na]+421.1675, found: 421.2039.
test example 1
The dyes prepared in examples 1 to 4 (each at a concentration of 10. mu.M) were tested for their ultraviolet absorption and fluorescence emission spectra in glycerol and dimethylsulfoxide, with the abscissa representing the wavelength and the ordinate representing the absorbance or fluorescence intensity, respectively, and the results are shown in FIG. 2.
In the UV-visible absorption spectrum, dye 1a has an absorption maximum at 424 nm; in the fluorescence emission spectrum, dye 1a had the highest fluorescence intensity at 565nm and the highest intensity in glycerol, at which the excitation wavelength was 424nm and the slit width was 5nm/5nm, as shown in FIGS. 2- (a) (b), respectively.
Dye 1b has an absorption maximum at 493nm in the UV-visible absorption spectrum; in the fluorescence emission spectrum, dye 1b has the highest fluorescence intensity at 685nm and the highest intensity in glycerol, when the excitation wavelength is 545nm and the slit width is 10nm/1.5nm, as shown in FIGS. 2- (c) (d), respectively.
In the UV-visible absorption spectrum, dye 1c has an absorption maximum at 473 nm; in the fluorescence emission spectrum, dye 1c had the highest fluorescence intensity at 659nm and the highest intensity in glycerol, at which the excitation wavelength was 506nm and the slit width was 10nm/3nm, as shown in FIG. 2- (e) (f), respectively.
Dye 1d has an absorption maximum at 476nm in the UV-visible absorption spectrum; in the fluorescence emission spectrum, dye 1d had the highest fluorescence intensity at 660nm and the highest intensity in glycerol, at which the excitation wavelength was 507nm and the slit width was 5nm/5nm, as shown in FIG. 2(g) (h), respectively.
Test example 2
Dye 1a was prepared as a stock solution using DMSO (dimethyl sulfoxide), and then added to a conventional cell culture medium so that the concentration of dye 1a in the cell culture medium was 10. mu.M, followed by mixing with HeLa cells at saturated humidity, 37 ℃ and 5% CO2Incubators were co-incubated (same experiment below) for 10 minutes, followed by addition of the commercial mitochondrial Red marker Mito Tracker Red CMXRos (2. mu.M) for an additional 10 minutes; then washed three times by PBS buffer solution, and then cell imaging is carried out by using a laser confocal microscope. The green light channel is excited by 488nm wavelength, and the fluorescence signal in the range of 468-550nm is collected. The red light channel is excited by using a wavelength of 561nm, and the fluorescence signals in the range of 600-750nm are collected. Cell imaging shows that the dye 1a can well mark mitochondria in HeLa cells and can be used as a green marker of the mitochondria. The results are shown in FIG. 3, in which (a1) is an image of bright field, (a2) is an image of dye 1a, (a3) is an image of a commercial mitochondrial red marker, (a4) is an overlay of green and red channels, (a5) is a co-localization experiment, co-localization coefficient is 0.92, and (a6) is fluorescence intensity of ROI line in the overlay.
Test example 3
Dye 1b was at a concentration of 14 μ M in cell culture medium, followed by addition of the commercial lysosomal Green marker Lyso Tracker Green (2 μ M) for another 10 min incubation; after three washes with PBS buffer, cells were imaged using confocal laser microscopy. The red light channel is excited by 561nm wavelength to collect the fluorescence signal in the range of 600-750nm, the green light channel is excited by 488nm wavelength to collect the fluorescence signal in the range of 468-550 nm. Cell imaging shows that the dye 1b can well mark lysosomes in HeLa cells and can be used as a lysosome red marker. The results are shown in fig. 3, where (b1) is an image of bright field, (b2) is an image of a commercial lysosomal green marker, (b3) is an image of dye 1b, (b4) is a overlay of green and red channels, (b5) is a co-localization experiment with co-localization coefficient of 0.94, and (b6) is the fluorescence intensity of the ROI line in the overlay.
Test example 4
Dye 1c was present at a concentration of 10. mu.M in cell culture medium, followed by addition of the commercial endoplasmic reticulum Green marker ER Tracker Green (6. mu.M) for another 10 min; after three washes with PBS buffer, cells were imaged using confocal laser microscopy. The red light channel is excited by 561nm wavelength to collect the fluorescence signal in the range of 600-750nm, the green light channel is excited by 488nm wavelength to collect the fluorescence signal in the range of 468-550 nm. Cell imaging shows that the dye 1c can well mark endoplasmic reticulum in HeLa cells and can be used as an endoplasmic reticulum red marker. The results are shown in FIG. 3, where (c1) is an image of bright field, (c2) is an image of a cell of a commercial endoplasmic reticulum green marker, (c3) is an image of a cell of dye 1c, (c4) is an overlay of green and red channels, (c5) is a co-localization experiment, co-localization coefficient is 0.96, and (c6) is the fluorescence intensity of the ROI line in the overlay.
Test example 5
Dye 1d was at a concentration of 20 μ M in cell culture medium, followed by addition of commercial lipid droplet Green marker LDs Tracker Green (4 μ M) for another 10 min incubation; after three washes with PBS buffer, cells were imaged using confocal laser microscopy. The red light channel is excited by 561nm wavelength to collect the fluorescence signal in the range of 600-750nm, the green light channel is excited by 488nm wavelength to collect the fluorescence signal in the range of 468-550 nm. Cell imaging shows that the dye 1d can well mark lipid droplets in HeLa cells and can be used as a lipid droplet red marker. The results are shown in FIG. 3, where (d1) is an image of bright field, (d2) is an image of a cell of a commercial lipid droplet green marker, (d3) is an image of a cell of dye 1d, (d4) is a superimposed image of green and red channels, (d5) is a co-localization experiment, co-localization coefficient is 0.90, and (d6) is the fluorescence intensity of the ROI line in the superimposed image.
Test example 6
The concentration of the dye 1a in the cell culture medium is 2 μ M, the concentration of the ionophore monensin is 10 μ M, the cells are co-cultured for 30 minutes, washed three times with PBS buffer, and then imaged by a confocal laser microscope. The green light channel is excited by 488nm wavelength, and the fluorescence signal in the range of 500-550nm is collected. Cellular imaging showed that the fluorescence signal of dye 1a was significantly enhanced after mitochondrial viscosity was increased by addition of monensin. The results are shown in FIG. 4(A), wherein (a) is a bright field image, (b) is a cell image of dye 1a, (c) is a superimposed image of (a) and (b), (d) is a bright field image, (e) is a cell image of dye 1a and monensin, (f) is a superimposed image of (d) and (e), and (g) is the mean fluorescence intensity.
Test example 7
The concentration of the dye 1b in the cell culture medium is 2 mu M, the concentration of the ion carrier dexamethasone is 10 mu M, the cells are cultured for 30 minutes together, and after washing with PBS buffer solution for three times, cell imaging is carried out by using a laser confocal microscope. The red light channel is excited by using a wavelength of 561nm, and the fluorescence signal in the range of 600-800nm is collected. Cell imaging showed that the fluorescence signal of dye 1b was significantly enhanced after increasing the lysosomal viscosity by the addition of dexamethasone. The results are shown in FIG. 4(B), wherein (a) is a bright field image, (B) is a cell image of dye 1B, (c) is a superimposed image of (a) and (B), (d) is a bright field image, (e) is a cell image of dye 1B and dexamethasone, (f) is a superimposed image of (d) and (e), and (g) is the mean fluorescence intensity.
Test example 8
The concentration of the dye 1c in the cell culture medium is 2 μ M, the concentration of the ionophore monensin is 10 μ M, the cells are co-cultured for 30 minutes, washed three times with PBS buffer, and then imaged by a confocal laser microscope. The red light channel is excited by using a wavelength of 561nm, and the fluorescence signal in the range of 600-800nm is collected. Cellular imaging showed that the fluorescence signal of dye 1c was significantly enhanced after increasing the viscosity of the endoplasmic reticulum by the addition of monensin. The results are shown in FIG. 4(C), wherein (a) is a bright field image, (b) is a cell image of dye 1C, (C) is a superimposed image of (a) and (b), (d) is a bright field image, (e) is a cell image of dye 1C and monensin, (f) is a superimposed image of (d) and (e), and (g) is the mean fluorescence intensity.
Test example 9
The concentration of the dye 1d in the cell culture medium is 2 muM, the concentration of the ionophore nystatin is 10 muM, the cells are cultured for 30 minutes together, and after washing with PBS buffer solution for three times, the cells are imaged by a laser confocal microscope. The red light channel is excited by using a wavelength of 561nm, and the fluorescence signal in the range of 600-800nm is collected. Cellular imaging showed that the fluorescence signal of dye 1d was significantly enhanced after lipid droplet viscosity was increased by the addition of nystatin. The results are shown in FIG. 4(D), wherein (a) is a bright field image, (b) is a cell image of dye 1D, (c) is a superimposed image of (a) and (b), (D) is a bright field image, (e) is a cell image of dye 1D and nystatin, (f) is a superimposed image of (D) and (e), and (g) is the mean fluorescence intensity.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims (10)

1. An isophorone derivative, wherein the derivative has the structural formula:
Figure FDA0003367964370000011
wherein R is selected from the following structural formulae (1) to (4):
Figure FDA0003367964370000012
2. the process for producing an isophorone derivative of claim 1, which comprises the steps of:
dissolving (3,5, 5-trimethylcyclohex-2-eneylidene) malononitrile and a compound M in an organic solvent, and heating to react under the action of a catalyst to obtain the isophorone derivative;
wherein the compound M is 1H-indazole-5-formaldehyde, 4- (4-hydroxypiperidin-1-yl) benzaldehyde, 4-hydroxy-1-naphthaldehyde or 1-pyrene formaldehyde.
3. The preparation method according to claim 2, wherein the molar ratio of the (3,5, 5-trimethylcyclohex-2-enylidene) malononitrile to the compound M is 1:0.7 to 1: 2.0.
4. Use of the isophorone derivative of claim 1 in the preparation of a cytofluorescent agent.
5. Use of an isophorone derivative of claim 1 in the fluorescence imaging of organelles for non-diagnostic therapeutic purposes.
6. The use according to claim 5, wherein the organelle is a lysosome, a mitochondrion, an endoplasmic reticulum, or a lipid droplet.
7. A method of fluorescence imaging of cells of non-diagnostic, non-therapeutic interest, comprising the steps of: co-culturing cells with the isophorone derivative of claim 1 in a culture medium, and imaging the cells to complete fluorescence imaging of the cells, wherein the isophorone derivative can be used for fluorescence labeling of different organelles.
8. A method of fluorescence imaging of cells of non-diagnostic, non-therapeutic interest, comprising the steps of: co-culturing cells with the isophorone derivative of claim 1 and an ionophore in a culture medium, and then performing cell imaging to complete cell fluorescence imaging.
9. The method according to claim 8, wherein the ionophore is monensin, dexamethasone, or nystatin.
10. Use of the isophorone derivative of claim 1 to detect viscosity.
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