CN115197112B - Active oxygen-tolerant dicyanoisophorone fluorescent probe, preparation method and application thereof - Google Patents
Active oxygen-tolerant dicyanoisophorone fluorescent probe, preparation method and application thereof Download PDFInfo
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- CN115197112B CN115197112B CN202210928814.6A CN202210928814A CN115197112B CN 115197112 B CN115197112 B CN 115197112B CN 202210928814 A CN202210928814 A CN 202210928814A CN 115197112 B CN115197112 B CN 115197112B
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- 238000010992 reflux Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000006561 solvent free reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- UXLVQUDJEAWBPQ-UHFFFAOYSA-N trimethylsilyl carbamate Chemical compound C[Si](C)(C)OC(N)=O UXLVQUDJEAWBPQ-UHFFFAOYSA-N 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 229910001868 water Inorganic materials 0.000 description 1
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- C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
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Abstract
The invention relates to the technical field of fluorescent probes, in particular to an active oxygen tolerant dicyanoisophorone fluorescent probe, a preparation method and application thereof, wherein dicyanoisophorone is taken as a fluorophore, and two small molecules of thioketone and formic acid are selected to respectively react with dicyanoisophorone through condensation reaction to prepare probes DCN-SS-R and DCN-C. The fluorescence emission of the fluorescent probes DCN-SS-R and DCN-C is not influenced by the polarity of the solvent, is only in positive correlation with the environmental viscosity, and has the characteristics of high sensitivity, wide linear range, strong pH stability, good biocompatibility and the like, and shows remarkable tolerance to active oxygen; in a mitochondrial/lysosome co-localization experiment, the probes DCN-SS-R and DCN-C have good targeting characteristics on mitochondria, can monitor viscosity fluctuation in mitochondria in real time, and have important application prospects in the aspects of biological detection and diagnosis of mitochondrial viscosity-related diseases.
Description
Technical Field
The invention relates to the technical field of fluorescent probes, in particular to an active oxygen-tolerant dicyanoisophorone fluorescent probe, a preparation method and application thereof.
Background
Intracellular viscosity is an important parameter for studying normal physiological activities of cells and is closely related to the occurrence and development of various diseases (e.g., diabetes, alzheimer's disease, hypertension, and various tumors, etc.). At the cellular level, fluctuations in viscosity can significantly affect the migration and diffusion rates of intracellular substances, and thus the intracellular signaling and interactions between biological macromolecules. Various detection tools have been used to detect intracellular bulk viscosity, such as capillary viscometers, falling ball viscometers, rotational viscometers for viscosity measurement, and viscosity responsive fluorescent probes. However, since cells are a highly heterogeneous microenvironment, the local viscosity differences are significant in different regions (especially in different organelles). Thus, monitoring from time to time for a particular organelle viscosity within a cell can more accurately elucidate the correlation of viscosity and normal physiological activity of the cell. Mitochondria serve as energy metabolism sites of cells, and are involved in regulating various functions of cells. When the viscosity in the mitochondrial matrix changes, the diffusion of the metabolites is changed by the osmotic pressure inside and outside the mitochondria, thereby affecting the normal metabolism of the mitochondria. Therefore, developing a tool that can effectively monitor the intra-granular viscosity change of the wire has important biomedical value.
The fluorescence imaging technology has been developed rapidly in recent years, and has been widely used for detecting the viscosity in mitochondria due to the advantages of high resolution, high selectivity, high sensitivity, real-time monitoring and the like. However, the currently reported fluorescent probes for monitoring mitochondrial viscosity from time to time still have certain drawbacks in terms of stability. This is mainly due to the fact that mitochondria are the sites of energy metabolism, during which large amounts of Reactive Oxygen Species (ROS) are produced, which often lead to a number of fluorescent probe structures that change and thus a significant decrease in fluorescence intensity, a phenomenon known as fluorescence quenching. Therefore, the development of the fluorescent probe with good ROS tolerance, high quantum yield and excellent mitochondrial targeting has important application prospect in the aspects of diagnosis and mechanism research of mitochondrial viscosity related diseases.
Disclosure of Invention
The invention aims to solve the problem of dynamically measuring the viscosity in a linear particle body through a fluorescent probe based on dicyanoisophorone, and provides an active oxygen tolerant dicyanoisophorone fluorescent probe, a preparation method and application thereof.
In order to achieve the above object, the present invention discloses an active oxygen-tolerant dicyanoisophorone fluorescent probe (DCN-SS-R and DCN-C) which is (E) -2- ((2- ((2-hydroxyethyl) thio) propan-2-yl) thio) ethyl- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) carbamate (DCN-SS-R) or (E) -N- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) carboxamide (DCN-C), the DCN-SS-R having the following structural formula:
the structural formula of the DCN-C is as follows:
the invention also discloses a preparation method of the DCN-SS-R in the active oxygen tolerant dicyanoisophorone fluorescent probe, and the synthetic route is as follows:
under the action of condensing agent and acid binding agent, compound dicyanoisophorone and 2, 2-bis- (2-hydroxyethyl sulfonamide) -propane are prepared into a mixed system, and the mixed system and 2, 2-bis- (2-hydroxyethyl sulfonamide) -propane react in an organic solvent to obtain the target probe DCN-SS-R.
The acid binding agent may be independently selected from N, N-Diisopropylethylamine (DIPEA), triethylamine, ethylenediamine, anhydrous potassium carbonate, anhydrous sodium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, or 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU), with preference given to: n, N-Diisopropylethylamine (DIPEA), triethylamine.
The condensing agent in the synthesis step may be independently selected from the group consisting of 2, 2-trichloroethyl chloroformate, bis (trichloromethyl) carbonate, trimethylsilyl carbamate, trimethylsilyl (trimethylsilyl) carbamate, isopropyl trimethylsilyl carbonate, or trimethylsilyl N- (trimethylsilyloxy) carbamate, with preference given to: bis (trichloromethyl) carbonate, 2-trichloroethyl chloroformate.
The organic solvent may be independently selected from: toluene, methanol, acetonitrile, chloroform, dichloromethane, tetrahydrofuran, DMF or DMSO, with toluene, dichloromethane, acetonitrile being preferred.
The reaction temperature in the step of preparing the mixed system of the Dicyanoisophorone (DCN) and the condensing agent is 0-200 ℃, preferably 100-120 ℃.
The molar ratio of the Dicyanoisophorone (DCN) to the acid-binding agent is 1:1-1:20, preferably 1:1-1:5.
The reaction temperature of the mixed system prepared by Dicyanoisophorone (DCN) and condensing agent and 2, 2-bis- (2-hydroxyethyl sulfonamide) -propane in the synthesis step is 0-100 ℃, preferably 0-30 ℃.
The invention also discloses a preparation method of DCN-C in the active oxygen tolerant dicyanoisophorone fluorescent probe, and the synthetic route is as follows:
the compound Dicyanoisophorone (DCN) and formic acid directly react under the heating condition to obtain the target probe DCN-C.
In the preparation method, the synthesis step is a solvent-free reaction, and the selected formic acid is a reaction reagent and is also a solvent for the reaction.
In the above preparation method, the reaction temperature of Dicyanoisophorone (DCN) and formic acid in the synthesis step is 0 to 200 ℃, and among them, 100 to 150 ℃ is preferable.
In the preparation method, the purification method of the fluorescent probe DCN-C in the synthesis step adopts silica gel column chromatography, and the lotion is petroleum ether: ethyl acetate.
The invention also discloses application of the active oxygen tolerant dicyanoisophorone fluorescent probe (DCN-SS-R and DCN-C) in monitoring the viscosity of living cells in real time under the interference of Reactive Oxygen Species (ROS) in the tolerant living cells.
The invention also discloses application of the active oxygen tolerant dicyanoisophorone fluorescent probe (DCN-SS-R and DCN-C) in visual real-time monitoring of mitochondrial viscosity and/or physiological activity in living cells.
According to the invention, dicyano-phorone fluorophore is used as a parent body, and the viscosity fluorescent probes DCN-C and DCN-SS-R are successfully synthesized through thioketone and formic acid small molecules, so that the push-pull system of the fluorophore is changed through the reaction of thioketone and formic acid with amino, the electron donating capability of the amino after amide bond formation is reduced, and the fluorescence emission wavelength of the probes is blue shifted, so that the two fluorescent probes emit yellow fluorescence.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
1. the invention adopts dicyanoisophorone fluorophore as a matrix, designs the dicyanoisophorone-based fluorescent probes DCN-C and DCN-SS-R obtained by the probe structure, and can be applied to the measurement of intracellular viscosity;
2. the fluorescence emission of the small molecular fluorescent probes DCN-C and DCN-SS-R in the invention is not influenced by the polarity of the solvent, is only positively correlated with the environmental viscosity, has the characteristics of high sensitivity, wide linear range, strong pH stability, good biocompatibility and the like, and has remarkable ROS tolerance compared with the currently reported mitochondrial viscosity fluorescent probes, thus being more suitable for mitochondrial dynamic imaging;
3. the small molecular fluorescent probe DCN-C in the invention has excellent selectivity to the line granule, can track the normal physiological process (autophagy process) of mitochondria in cells, and has very important biomedical significance.
4. The probe provided by the invention is simple and easy to obtain, and can be prepared by one-step condensation reaction.
Drawings
FIG. 1 is an ultraviolet-visible absorption diagram of probes DCN-SS-R (A) and DCN-C (B) in solvents of different polarities;
FIG. 2 shows fluorescence emission of probe DCN-SS-R in solvents of different polarities, (A) fluorescence emission pattern of probe in solvents of different polarities (B) fluorescence emission of probe DCN-SS-R in solvents of different polarities at 565nm (n=3);
FIG. 3 fluorescence emission of probe DCN-C in solvents of different polarities (A) fluorescence emission profile of probe in solvents of different polarities (B) fluorescence emission of probe in solvents of different polarities at 558nm (n=3);
fig. 4 is a graph of fluorescence intensity versus viscosity for DCN-C in 40% -99% water-glycerol solution (n=3);
fig. 5 (a) is a graph of fluorescence emission of probe DCN-C in water-glycerol solutions of different proportions, and (B) is a graph of fluorescence intensity in 0% -70% water-glycerol solution versus viscosity (n=3);
FIG. 6 (A) shows the pH stability of probe DCN-C in a 50% water-glycerol system, and (B) shows the light stability of probe DCN-C under conditions of PBS and glycerol under continuous irradiation at 400nm for 60 min;
FIG. 7 (A) is a fluorescence lifetime spectrum (E) of a fluorescent probe DCN-C (5. Mu.M) in a different water-glycerol viscosity system ex ＝400nm，E em =558 nm), fig. 7 (B) is a linear relationship between log (η) and log (τ) (R 2 ＝0.98944)；
FIG. 8 is a graph showing the effect of metal ions, amino acids, active oxygen, and reducing agents on the fluorescence intensity of probe DCN-C (5. Mu.M), metal ions: k (K) + 、Na + 、Ca 2+ 、Mg 2+ 、Zn 2+ 、Cu 2+ Amino acid: glycine (Gly), methionine (Met), serine (Ser), L-cysteine (Cys) active oxygen species: 1 O 2 、H 2 O 2 、ClO - 、·OH、ONOO - 、O 2 ；HSO 3 - the method comprises the steps of carrying out a first treatment on the surface of the The method comprises the steps of carrying out a first treatment on the surface of the Reduced Glutathione (GSH): the above substances are 500 μm;
FIG. 9 shows the viability of (A) A549 cells (B) HeLa cells (n=3) after 24h treatment with different concentrations of probe DCN-C;
FIG. 10 (A) is a CLSM image of A549 cells co-labeled for 30min with DCN-C (5. Mu.M) and commercial organelle localization dyes (Mito-Tracker and Lyso-Tracker) (200 nM), (a, d) yellow channel, probe DCN-C (B, e) green channel: commercial organelle localization dyes (C, f) green and clear channel superimposed images, FIG. 10 (B) is a plot of mitochondrial and lysosome co-localization coefficients (n=3) for probe DCN-C incubated with Mito-Tracker and Lyso-Tracker, respectively, for 30min, scale bar equal to 20 μm;
FIG. 11 is a laser confocal image of A549 cells co-stained with probe DCN-C (5. Mu.M) and Mito-Tracker Green (200 nM), (A) superimposed yellow and Green channel, and (B) intensity profile of (ROI) of A549 cells;
FIG. 12 (A) is a pseudo-color laser confocal plot of A549 cells incubated with DCN-C (5. Mu.M) for 30min and (a-C) nystatin (10. Mu.M), monensin (10. Mu.M), rapamycin (30. Mu.M) for 30min, and (B) is the average fluorescence intensity in the corresponding image, scale bar 20. Mu.m;
FIG. 13 is a graph of co-labeling pseudo-color laser confocal images of A549 cells incubated with DCN-C (10. Mu.M) and Lyso-Tracker (200 nM) followed by incubation for different times with serum-free and CCCP (20. Mu.M), (A) a549 cell lysosome control group, serum-free incubation group, CCCP group pseudo-color laser confocal images, (a 1-e 1) yellow channel, (a 2-e 2) bright field, (a 3-e 3) green channel, (A5-e 5) co-localization coefficient histogram of probe DCN-C and Lyso-Tracker lysosome, and (B) co-localization coefficient histogram of probe DCN-C and Lyso-Tracker lysosome, scale bar equal to 20 μm;
FIG. 14 is a graph of pseudo-color laser confocal images of A549 cells co-labeled for different times after incubation with DCN-C (10. Mu.M) and Mito-Tracker (200 nM) using serum-free and CCCP (20. Mu.M), (A) a549 cell lysosome control group, serum-free incubation group, CCCP group pseudo-color laser confocal images; (a 1-e 1) yellow channel, (a 2-e 2) bright field; (a 3-e 3) green channel, (a 5-e 5) probe DCN-C and Mito-Tracker lysosome co-localization map, (B) probe DCN-C and Mito-Tracker lysosome co-localization coefficient histogram, scale bar equal to 20 μm.
Detailed Description
The above and further technical features and advantages of the present invention are described in more detail below with reference to the accompanying drawings.
Example 1: preparation of fluorescent probe DCN-SS-R
To a 250mL three-necked round bottom flask was added Dicyanoisophorone (DCN) (69.5 mg,0.24 mmol), N-diisopropylethylamine DIPEA (0.288 mmol,49.5 μL), and 100mL anhydrous toluene. After the addition, the mixture was cooled to 0 ℃. Under the protection of nitrogen, an anhydrous toluene solution (ca.4M, 60mL,0.24 mmol) of bis (trichloromethyl) carbonate is slowly added dropwise, and after the dropwise addition, stirring is continued for about 30min at 0 ℃, the temperature is slowly increased to 120 ℃ and the reaction is continued for about 3h. After the reaction, the reaction solution was cooled to 0℃in an ice bath and a methylene chloride solution (ca.4M, 12mL,0.48 mmol) of 2, 2-bis- (2-hydroxyethylsulfonamido) -propane was slowly added dropwise thereto, and the mixture was warmed to room temperature and stirred overnight. After the reaction, the solvent is removed by a rotary evaporator, the sample is loaded by a wet method, and is separated and purified by a column, wherein the eluent is ethyl acetate: dichloromethane=1:50, giving red viscous solid compound 6 about 20mg, i.e. fluorescent probe DCN-SS-R, in 17.9% yield.
HRMS(ESI):calculated for C 27 H 33 NaN 3 O 3 S 2 + ,(M+Na) + ,534.1856found 534.1853. 1 HNMR(600MHz,DMSO–d 6 )δ 1 H NMR(600MHz,DMSO-d 6 )δ0.98(s,7H),1.54(s,7H),2.49(s,2H),2.56(s,2H),2.66(t,J＝7.0Hz,3H),2.86(t,J＝6.7Hz,3H),3.51(q,J＝6.4Hz,2H),4.22(t,J＝6.9Hz,3H),4.80(t,J＝5.4Hz,1H),6.79(s,1H),7.21(q,J＝16.1Hz,2H),7.49(d,J＝8.6Hz,2H),7.60(d,J＝8.4Hz,2H),9.90(s,1H). 13 C NMR(151MHz,DMSO-d 6 )δ170.63,156.62,153.52,140.95,137.94,129.20,128.17,122.49,118.69,114.41,113.60,76.03,64.14,61.05,56.12,42.78,38.66,33.24,32.08,31.30,29.46,27.87。
Example 2: preparation of fluorescent probe DCN-C:
DCN (60 mg) was added to a 25mL round bottom flask, 2.5mL formic acid was removed by pipetting with a pipette, reflux was condensed under argon for 21h, after the reaction, the solvent was removed by rotary evaporation, loaded onto wet silica gel, and purified by wet loaded silica gel column to give the product, the eluent was petroleum ether: ethyl acetate = 1:1, the product was an orange solid.
HRMS(ESI):calculated for C 20 H 20 N 3 O 3 + ,(M+H) + ,318.1601found 318.1596, 1 H NMR(600MHz,dmso)δ0.98(s,6H),2.50(s,2H),2.57(s,2H),6.81(s,1H),7.20(d,J＝16.5Hz,2H),7.27(d,J＝16.1Hz,1H),7.62(q,J＝8.3Hz,4H),8.28(s,1H),8.88(d,J＝10.9Hz,0.23H),10.27(d,J＝10.8Hz,0.36H),10.34(s,1H). 13 C NMR(151MHz,DMSO-d 6 )δ170.64,160.19,156.47,139.90,137.66,129.20,128.70,122.76,119.72,114.36,113.56,76.31,42.77,38.66,32.09,27.88。
Example 3: probe mother liquor configuration
Configuration of 0.5mM probe DCN-SS-R stock: 12.78mg of sample is accurately weighed on an analytical balance and transferred into a 4mL centrifuge tube, a pipette is used for transferring a dimethyl sulfoxide (DMSO) dissolved sample into a 25mL brown volumetric flask, the volume is fixed, the flask is sealed by a sealing film and then is stored at 4 ℃ in a refrigerator, and the final concentration of the used probe is 5 mu unless otherwise noted in subsequent experiments.
Configuration of 0.5mM DCN-C Probe stock solution: and (3) accurately weighing 7.94mg of a sample on an analytical balance, transferring the sample to a 4mL centrifuge tube, transferring the solution to a 25mL brown volumetric flask after dissolving the sample in dimethyl sulfoxide DMSO, fixing the volume, sealing the solution by using a sealing film, and storing the solution in a refrigerator at 4 ℃, wherein the final concentration of the probe is 5 mu unless otherwise noted in subsequent experiments.
Example 4: determination of probe ultraviolet absorption spectrum and fluorescence emission spectrum under different solvent polarities
In the experiment, dichloromethane (DCM), ethyl Acetate (EA), dimethyl sulfoxide (DMSO), methanol (MeOH), ethanol (Ethanol), acetonitrile (MeCN), N-Dimethylformamide (DMF) and glycerol (Gly) are selected as test systems, 20 mu L of probe DCN-SS-R and DCN-C solutions are respectively removed and added into a 2mL centrifuge tube, 1980 mu L of organic solvents with different polarities are removed, and the ultraviolet absorption spectrum and the fluorescence emission spectrum of the probe are measured.
As shown in FIG. 1, the probes DCN-SS-R and DCN-C showed a red shift in ultraviolet absorption only in glycerol, possibly in a high viscosity environment, inhibiting the free rotation of the probe, resulting in an increase in the degree of conjugation. As also shown in FIGS. 2 and 3, probes DCN-SS-R and DCN-C showed a significant increase in fluorescence emission in glycerol systems as compared to other polar solvents, while probes DCN-SS-R and DCN-C showed weak fluorescence emission in other polar solvents, indicating that solvent polarity did not significantly interfere with probes DCN-SS-R and DCN-C, indicating that probes DCN-SS-R and DCN-C both responded to viscosity, and can be used in viscosity measurements.
Example 5: determination of sensitivity of fluorescent probes to viscosity
The method comprises the steps of selecting a phosphate buffer solution (PBS, which is prepared from disodium hydrogen phosphate (Na 2HPO 4), monopotassium hydrogen phosphate (KH 2PO 4), sodium chloride (NaCl) and potassium chloride (KCl) with certain mass) and glycerin, changing the viscosity of the test system through changing the mass fraction ratio of the glycerin to the PBS, wherein the volume fractions of the glycerin are increased by 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 99% respectively in sequence; adding 1980 mu 9 glycerol into a centrifuge tube, sucking 20 mu L of probe mother liquor into the centrifuge tube by using a pipette, adding the probe mother liquor into the centrifuge tube, performing ultrasonic treatment for 2min, and standing at room temperature for 30min; scanning ultraviolet absorption spectrum in 220-800nm wavelength range, and setting 5nm/5nm slit of fluorescent instrument.
As shown in FIGS. 4A, B, probe DCN-SS-R (E ex ＝463nm，E em =565 nm), and the linear interval of the fluorescence intensity and viscosity response thereof is 40% -99%, and the correlation coefficient r=0.994; as shown in FIGS. 5A, B, probe DCN-C (E x ＝400nm，E m =558 nm) increases with the viscosity of the solvent, and the linear interval of the fluorescence intensity and viscosity response is 0% -70%, and the correlation coefficient r=0.997.
The fluorescence intensity changes of the probe DCN-SS-R and the probe DCN-C have good linear positive correlation on the viscosity of the solvent, and compared with the probe DCN-C has more excellent sensitivity at low viscosity.
Example 6: determination of pH stability and light stability of fluorescent Probe DCN-C
pH stability experiment: the experiment selects a phosphate buffer system (0.2M PB, pH=4-11), and the specific preparation method is as follows: 2.1852mg of disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O) and 0.5382mg of NaH2PO4.H2O are accurately weighed into a beaker, dissolved by adding a proper amount of ultrapure water, the pH value of the solution is regulated by using 0.1MHCl and 0.1M NaOH solution, and then the solution is transferred into a brown volumetric flask for constant volume. Preparing a solution with the mass fraction of glycerin of 50% and 70% by adopting buffer solutions with different pH values, carrying out ultrasonic treatment on the prepared solution on an ultrasonic instrument for 2min, and standing at room temperature for 30min. The final probe concentration was 5. Mu.M, and the fluorescence intensities were measured on a fluorometer, respectively, with slit widths set at 5nm.
The probe DCN-C is selected as an experimental object, and a 50% glycerol system is selected for research, and the result is shown in a figure 6, wherein the probe has good stability at pH=5-10, which indicates that the probe has good stability in physiological and pathological pH environments;
example 7: fluorescence lifetime determination of fluorescent probe DCN-C
The solution viscosity was increased from 1.0cP (100% pbs) to 999cP (100% glycerol) by adjusting the glycerol ratio. Setting the maximum emission wavelength of 558nm, selecting 365nm exciter, setting the slit width of 5nm, photon intensity of 3000, and calculating the fluorescence life time as follows:
Log(τ)＝c+x log(η)
where η is the solvent viscosity, c is a constant, τ is the fluorescence lifetime, and x is the sensitivity of the fluorescent probe to viscosity.
As shown in FIG. 7, the fluorescence decay curve of the probe can be seen to be in the range of 22.5cp-1410cp, and the service life of the probe is gradually prolonged as the viscosity of the test system is increased. According to the Forster-Hoffmann equation, the luminescence lifetime (log (τ)) of the probe is in good linear relation (R) to the viscosity (log (η)) on a logarithmic scale 2 =0.99, x=0.05). The results show that the DCN-C life viscosity curve of the probe can also be used as a calibration curve for in vitro quantitative determination of viscosity. The experimental results further verify the principle of the probe DCN-C and viscosity responseBased on the molecular rotation principle, the molecules can rotate freely in the environment with low viscosity, the fluorescence intensity is lower, and the molecular rotation is inhibited in the environment with high viscosity, the fluorescence intensity is increased, and the fluorescence lifetime is prolonged.
Example 8: determination of the selectivity of the fluorescent probe DCN-C and ROS tolerance
Selective experiments of probe DCN-C are carried out to obtain metal ions, amino acid, active oxygen, reduced glutathione and HSO 3 - Four classes of interferents. The metal ions include K + 、Na + 、Ca 2+ 、Mg 2+ 、Zn 2+ 、Cu 2+ And (3) carrying out the process of (1) carrying out the process of (2. Amino acids include glycine (Gly), methionine (Met), serine (Ser), L-cysteine (Cys). Active oxygen comprising ONOO - 、H2O2、ClO - 、·OH、 1 O 2 、O 2 ·- 。
Preparing a metal ion solution: accurately weigh proper amount of NaCl, KCl, znCl 2 、CaCl 2 、MgCl 2 The solid was dissolved in ultrapure water to prepare a solution having a concentration of 0.1M, and the solution was placed in a refrigerator for refrigeration and diluted as required in the use.
Preparation of amino acid solution: and (3) weighing a proper amount of glycine, methionine and serine solids, dissolving the glycine, methionine and serine solids with ultrapure water to prepare a solution with the concentration of 0.1M, and placing the solution in a refrigerator for refrigeration for later use, wherein the solution is diluted according to the experiment requirement when in use.
Preparing glutathione and sodium bisulphite solution: accurately weighing a proper amount of sodium bisulphite powder, dissolving with ultrapure water to prepare a 0.1M solution, diluting according to the experimental requirement when in use, accurately weighing Cys solid powder, dissolving with ultrapure water to prepare a 0.1M solution, and diluting according to the experimental requirement. Precisely weighing glutathione GSH powder, dissolving with ultrapure water to prepare a solution with the concentration of 0.1M, and placing in a refrigerator for refrigeration for later use, and diluting for use when the experiment needs.
Hypochlorite ion (ClO) - ) The calibration method of the solution comprises measuring absorbance of sodium hypochlorite solution at 292nm, and determining extinction coefficient of 350M -1 cm -1 To calibrate ClO - Is a concentration of (3). Hydrogen peroxide (H) 2 O 2 ) Determination of the solution concentration according to its absorbance at 240nm, the extinction coefficient was ε=43.6M -1 cm -1 The concentration of hydrogen peroxide was calculated according to lambert-beer's law. Peroxynitrite ion (ONOO) - ) The preparation steps and the calibration method of the (a): in a 25mL round bottom flask, first, hydrogen chloride (0.6M) acidified hydrogen peroxide solution (0.7M) was mixed with sodium nitrite solution (0.6M), sodium hydroxide solution (3M) was added immediately after 1-2s reaction, the reacted solution was stored in a-20 ℃ refrigerator, and the concentration was measured by dissolving the solution in 0.1M sodium hydroxide solution, measuring 302nm absorbance according to lambert-beer's law and extinction coefficient epsilon=1670M, and heating rapidly before use -1 cm -1 Thus obtaining ONOO - Concentration of the solution. Preparation and calibration methods of superoxide anions; carefully weigh the superoxide (KO 2 ) Dissolving in DMSO, performing ultrasonic treatment with an ultrasonic instrument for 15min to obtain superoxide anion solution, and determining ultraviolet absorption value at 550nm to obtain standard concentration (ε=21.6 mM) -1 cm -1 )， 1 O 2 Is prepared by mixing sodium hypochlorite and hydrogen peroxide in the same proportion; the preparation of the hydroxyl radical (. OH) is carried out according to the Fenton reaction.
As shown in FIG. 8, in the case of low-viscosity 50% glycerol and high-viscosity 70% glycerol, there was no significant interference of the metal ions (500. Mu.M in each case) with the probe, and in the interference test of amino acid, bisulfite ion, reduced glutathione, and active oxygen, there was no significant interference with the fluorescence of the probe DCN-C, particularly in the case of OH. Cndot., 1 O 2 、ONOO - 、H 2 O 2 ，ClO - 、O 2 - Under the condition that the concentration of the six active oxygen is 500 mu M, the fluorescence intensity of the probe DCN-C is not obviously reduced, and the probe DCN-C has better active oxygen resistance.
Example 9: determination of cytotoxicity of fluorescent Probe DCN-C
Resuscitating HeLa and A549 cell strains, culturing with high sugar culture medium, placing into a cell culture box, observing cell growth condition every other day with an inverted microscope, and waiting for cell growthDigesting with trypsin when the growth is 80%, replacing fresh culture medium, counting with full-automatic cell counter, inoculating digested cells into 96-well plate (8000-1000 cells/well per well), arranging 9 auxiliary wells, arranging blank group, experimental group, and culturing in 5% CO 2 Culturing in a cell culture incubator at 37deg.C for 24 hr, observing whether cells adhere by using an inverted microscope, sucking and discarding culture medium in culture holes of 96-well plate after cells adhere, adding culture medium and probe DCN-C200 μl of different concentrations, culturing in cell culture incubator for 24 hr, and adding 20 μl of 5mg mL under dark condition -1 After the addition of the 96-well plate, placing the mixture into a cell culture box for continuous culture for 4 hours, sucking out the culture medium in the culture holes by a liquid transfer gun, adding 150 mu L of DMSO, shaking and mixing the mixture uniformly for 10 minutes by a shaking table at 37 ℃ under the condition of avoiding light, dissolving substances generated by living cell reaction, measuring the absorbance of each culture hole at 490nm on an enzyme-labeled instrument, and calculating the cell viability according to the following formula:
Cell survival(％)＝(OD Experiment -OD Blank )/(OD Control -OD Blank )×100％，
wherein OD Experiment Refers to the absorbance value and OD after adding the probe DCN-C Blank Refers to the absorbance value, OD, of DCN-C without probe Blank Refers to the absorbance of cells not added to the medium, and the final probe concentration used in this experiment was a cell viability greater than 85%.
As shown in FIG. 9, when the concentration of the probe DCN-C reaches 30 mu M, the survival rate of the A549 cells is still higher, and the probe DCN-C has low cytotoxicity and high compatibility; the cytotoxicity to Hela cells was also examined by the same method, and as shown in the results of FIG. B, when the concentration of the probe was 20. Mu.M, the survival rate of the cells was still higher than 85%, indicating that the probe was less toxic to cells and highly compatible, indicating that the probe DCN-C was highly biocompatible and can be used in tumor cell fluorescence imaging.
Example 10: mitochondrial targeting effect determination of fluorescent probe DCN-C
And (3) selecting A549 cells to perform a co-localization experiment on mitochondria and lysosomes of the probe DCN-C, and adopting a commercial lysosome localization dye Lyso-Tracker Green and a mitochondrial localization dye Mito-Tracker Green as control probes. The excitation wavelength of the settings of the Lyso-Tracker Green and Mito-Tracker Green were 488nm, and the Green channel (450-535 nm) was observed. The probe DCN-C was set to an excitation wavelength of 405nm and observed for yellow channels (550-650 nm).
Culturing A549 cells, observing the growth condition of the cells by an inverted microscope every other day, when the cells grow to the bottom of a confluent cell culture bottle, firstly discarding old culture medium in the cell culture bottle, washing 3 times by using PBS solution, then digesting for 2min by using trypsin digestion solution, sucking and discarding trypsin digestion solution, re-suspending uniform cells by using fresh culture medium, diluting the re-dispersed cell suspension by using culture medium, after diluting by a proper multiple, sucking 1mL, carefully slowly adding along a glass bottom culture dish (diameter of 15 mm), putting into a carbon dioxide cell culture box, culturing to the wall, sucking and discarding the culture medium by a control group, carefully washing 2-3 times by using PBS, adding 1mL of culture medium, performing mitochondrial co-localization experiment group, carefully washing a localization dye Mito-Tracker Green (final concentration of 200 nm) and a probe DCN-C (final concentration of 5 mu M) by using PBS, incubating for 30min, incubating for 2-3 times, finally adding 1mL of PBS, dissolving, and performing laser confocal imaging on a laser confocal instrument. Lysosomal co-localization experimental groups were operated as above.
The results are shown in FIGS. 10 (a) - (f), wherein (a) and (d) are graphs of laser confocal imaging of probe DCN-C incubated with A549 for 30min, and (b) are graphs of laser confocal imaging of Lyso-tracker incubated with A549 for 30min, and (e) are graphs of superposition of the three channels. The results of the A549 cell mitochondrial fluorescence confocal imaging are shown in fig. 10 (d) - (f), wherein (d) is a laser confocal imaging image of the probe DCN-C incubated with the A549 for 30min, (e) is a laser confocal imaging image of the Mito-tracker incubated with the A549 for 30min, and (f) is a superposition of the three channels. By contrast, the probe DCN-C and Mito-tracker have high superposition degree of fluorescence imaging, the localization coefficient of mitochondria is 0.84, and the co-localization coefficient of lysosomes is lower than that of mitochondria. As shown in FIG. 11, the fluorescence intensity curve of the probe DCN-C has high coincidence with the fluorescence intensity of Mito-Tracker as shown in the results of the selected areas (A) and (B) on the A549 cell confocal imaging chart, and in conclusion, the probe DCN-C has good positioning property on mitochondria of the A549 cell.
Example 11: viscosity imaging of fluorescent probe DCN-C in cells
Setting a control group and an experimental group, culturing A549 cells in a carbon dioxide cell incubator until the cells are fully filled with the culture medium, digesting the culture medium by trypsin, sucking new culture medium for blowing the culture medium uniformly, sucking the culture medium along a glass bottom culture dish (with the diameter of 15 mm) for parallel operation after proper multiple dilution, adding the experimental group and the control group into A5 mu M probe solution for incubation for 30min after the culture medium is attached, flushing the culture medium for 2 to 3 times by PBS (small) after the incubation, adding the drug into the control group without adding the drug, adding the drug nystatin (with the final concentration of 10 mu M), monensin (with the final concentration of 10 mu M) and rapamycin (with the final concentration of 30 mu M), placing the culture medium into the carbon dioxide cell incubator for incubation for 30min, flushing the culture medium for 2 to 3 times by PBS carefully after the incubation, adding 1mL of PBS, and carrying out cell imaging experiments on a confocal instrument.
After a549 cells were treated with monensin, nystatin, and rapamycin, respectively, confocal laser fluorescence images of the probes were obtained, as shown in fig. 12 (a) and (B). The cell fluorescence intensity of the added nystatin, monensin and rapamycin groups is higher than that of the control group, and the result shows that the two ionophores cause obvious change of mitochondrial viscosity, and the probe DCN-C can sense the change of the mitochondrial viscosity of the cells caused by the ionophores. In addition, the observed mitochondrial viscosity increases with the presence of ionophores, as shown in fig. 12 (a), and changes in mitochondrial swelling or other ultrastructural structure are presumed to be related to changes in viscosity.
Example 12: viscosity imaging of fluorescent probe DCN-C in cell mitochondrial autophagy
Resuscitates A549 cells and cultures the cells to the bottom of a confluent cell culture bottle, digests the cells by trypsin digestion, sucks a new culture medium, blows the cells to be resuspended uniformly, dilutes the redispersed A549 cells by a proper multiple, sets a control group, a serum-free incubation and dosing CCCP experiment group, sucks the cell suspension, adds the experiment group and the control group along the parallel operation of a glass bottom culture dish (with the diameter of 15 mm), cultures the cell suspension to the wall (about 12 h), and sets an autophagy process monitoring lysosome experiment group and a mitochondria experiment group A, B, C, D, E group respectively.
Lysosomal group A added with probes DCN-C10. Mu.M and Lyo-Tracker 200nM, mitochondrial group A added with probes DCN-C10. Mu.M and Mito-Tracker 200nM, while lysosomal and mitochondrial serum-free incubation experiment group B used serum-free medium, lysosomal group B added with probes DCN-C10. Mu.M and Lyso-Tracker 200nM, mitochondrial group B added with probes DCN-C10. Mu.M and Mito-Tracker 200nM; and CCCP dosing groups are set as 30min C group, 60min D group and 120min E group, A, B, C, D, E group are added with probe DCN-C and incubated with Lyo-Tracker or Mito-Tracker for 30min, B group is incubated with serum-free culture medium for 60min, C group and D, E group are incubated with 20 mu M CCCP for 30min, 60min and 120min respectively, after incubation in a cell incubator is completed, after the glass culture dish is carefully washed 2-3 times by PBS, cell imaging experiments are carried out on a confocal instrument. Cell laser confocal imaging data are processed and exported in instrument software and Zenblue software.
Positioning of the probe on lysosomes and mitochondria in the a549 cell mitochondrial autophagy state, compared with the control group, the serum-free incubation group can generate mitochondrial autophagy by cells without adding fetal bovine serum in the culture medium, and as shown in A5 and b5 in fig. 13 and 14, the laser confocal imaging shows an ascending trend for lysosomes, in the serum-free incubation group, the positioning coefficient of the probe DCN-C on lysosomes is increased from 0.25 to 0.36 compared with the control group. The co-localization coefficient of the probe DCN-C to mitochondria was reduced from 0.83 to 0.37 compared to the control group, showing a decreasing trend. As shown in (a 4) and (b 4) of fig. 13 and 14, the degree of overlap of the fluorescent image of the probe DCN-C and the fluorescent image of Lyso-Tracker increases, and the degree of overlap of the fluorescent image of the probe DCN-C and the fluorescent image of Mito-Tracker decreases. The addition of CCCP stimulated autophagy in cells, and for lysosomes, as shown in fig. 13, C5-e5, co-localization coefficient of probe DCN-C to lysosomes increased with increasing incubation time from 0.25 to 0.46, and the degree of overlap of fluorescence images of probe DCN-C and Lyso-Tracker increased with increasing time, compared to the control group. For mitochondria, as shown in (C5) - (e 5) in fig. 14, the co-localization coefficient of mitochondria was decreased with the increase of incubation time compared to the control group from 0.83 to 0.28, and the degree of overlap of the fluorescence image of probe DCN-C and the fluorescence image of Mito-Tracker was gradually decreased with the increase of time. In the state of mitochondrial autophagy, the data show that the positioning property of the probe DCN-C on lysosomes is improved to a certain extent, and the co-localization coefficient is increased passively; the probe has good positioning property to mitochondria, so that the co-localization coefficient is low passively, and the probe can be used for monitoring the autophagy process of mitochondria for a long time.
The results of the above examples and the detection experiments thereof show that: the fluorescence emission of the small organic molecules DCN-SS-R and DCN-C is not influenced by the polarity of the solvent, is only positively correlated with the environmental viscosity, and has the characteristics of high sensitivity, wide linear range, strong pH stability, good biocompatibility and the like. At the same time, probe DCN-C showed significant tolerance to ROS and better targeting effect to wire granules. Therefore, the probes DCN-SS-R and DCN-C have important biomedical significance in the aspects of diagnosis of viscosity-related diseases and pathogenesis research.
In addition, compared with the currently commercialized mitochondrial imaging fluorescent probe, the probe DCN-C reported by the invention has remarkable advantages in terms of ROS stability, and has wide development prospect in the commercial field.
The foregoing description of the preferred embodiment of the invention is merely illustrative of the invention and is not intended to be limiting. It will be appreciated by persons skilled in the art that many variations, modifications, and even equivalents may be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. An active oxygen tolerant dicyanoisophorone fluorescent probe, wherein the fluorescent probe is (E) -2- ((2- ((2-hydroxyethyl) thio) propan-2-yl) thio) ethyl- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) ethyl)Alkenyl) phenyl) carbamate or (E) -N- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) carboxamide, said (E) -2- ((2- ((2-hydroxyethyl) thio) propan-2-yl) thio) ethyl- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) carbamate having the following structural formula:
the structural formula of the (E) -N- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) formamide is as follows:
2. a method for preparing an active oxygen tolerant dicyanoisophorone fluorescent probe according to claim 1, wherein the (E) -2- ((2- ((2-hydroxyethyl) thio) propan-2-yl) thio) ethyl- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) carbamate is synthesized as follows:
preparing a mixed system by using the compound dicyanoisophorone and 2,2'- (propane-2, 2-diylbis (sulfanediyl)) diethanol, preparing a mixed system by using the compound dicyanoisophorone and a condensing agent under the action of the condensing agent and the acid-binding agent, and reacting the mixed system with 2,2' - (propane-2, 2-diylbis (sulfanediyl)) diethanol in an organic solvent to obtain a target probe (E) -2- ((2- ((2-hydroxyethyl) thio) propane-2-yl) thio) ethyl- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) carbamate;
the condensing agent is di (trichloromethyl) carbonate.
3. The method for preparing an active oxygen-tolerant dicyanoisophorone fluorescent probe according to claim 2, wherein the acid-binding agent is selected from any one of N, N-diisopropylethylamine, triethylamine, ethylenediamine, anhydrous potassium carbonate, anhydrous sodium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, and 1, 8-diazabicyclo [5.4.0] undec-7-ene.
4. The method for preparing the active oxygen tolerant dicyanoisophorone probe according to claim 2, wherein the organic solvent is selected from any one of toluene, methanol, acetonitrile, chloroform, dichloromethane, tetrahydrofuran, DMF or DMSO.
5. The method for preparing the active oxygen tolerant dicyanoisophorone fluorescent probe according to claim 2, wherein the reaction temperature in the step of preparing a mixed system of dicyanoisophorone and a condensing agent is 0-200 ℃, the molar ratio of dicyanoisophorone to an acid-binding agent is 1:1-1:20, and the reaction temperature of the mixed system and 2,2' - (propane-2, 2-diylbis (sulfanediyl)) diethanol is 0-100 ℃.
6. A method for preparing an active oxygen tolerant dicyanoisophorone fluorescent probe according to claim 1, wherein the synthetic route of (E) -N- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) carboxamide is as follows:
the compound dicyanoisophorone and formic acid directly react under the heating condition to obtain the target probe (E) -N- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) formamide.
7. The method for preparing an active oxygen tolerant dicyanoisophorone probe according to claim 6, wherein the reaction temperature of dicyanoisophorone and formic acid is 0 to 200 ℃.
8. Use of an active oxygen tolerant dicyanoisophorone fluorescent probe according to claim 1 for monitoring viscosity of living cells when visualized under interference with active oxygen in the living cells.
9. Use of an active oxygen tolerant dicyanoisophorone fluorescent probe according to claim 1 for visual real-time monitoring of mitochondrial viscosity and/or physiological activity in living cells.
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