CN110590664A - Preparation method of fluorescent probe and application of fluorescent probe - Google Patents
Preparation method of fluorescent probe and application of fluorescent probe Download PDFInfo
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Abstract
The invention discloses a preparation method of a fluorescent probe and application of the fluorescent probe. The fluorescent probe has a structure shown in the following formula (I), and the applicant finds that the fluorescent probe can react with reduced glutathione in tumor cells in experiments and can well track the level of the reduced glutathione in the cells. The preparation method of the fluorescent probe mainly comprises the following steps: putting a compound shown as a formula (II) and N, N-dimethylethylenediamine into an organic solvent for reaction, and recovering the solvent after the reaction is finished to obtain a crude product of the target product;
Description
Technical Field
The invention relates to a preparation method of a fluorescent probe and application of the fluorescent probe, belonging to the technical field of medicines.
Background
Cancer is the first killer seriously threatening human health, and has high morbidity and high mortality. Chemotherapy is an important means for treating malignant tumor, but chemotherapy drugs damage normal cells by interfering cell metabolism to form oxygen free radicals, and reduced Glutathione (GSH) can scavenge oxygen free radicals formed in the chemotherapy process or form low-toxicity products with the chemotherapy drugs and metabolites thereof, so as to protect or recover the functions of damaged cells or damaged organs. However, for tumor cells, the presence of GSH makes them very susceptible to resistance to chemotherapeutic drugs. An increase in GSH levels over their drug-sensitive counterparts was observed in various multidrug-resistant cells. GSH may be involved in chemotherapy resistance by eliminating oxidative stimuli caused by oxidant therapy. Therefore, GSH is recommended as a potential biomarker for cancer diagnosis or prognosis.
The presence of free sulfhydryl groups is a typical structural feature of GSH, and most of the specific chemical properties of GSH, such as its reducibility, its ability to bind drugs or metals, etc., are determined by the free sulfhydryl groups. Virtually all reported fluorescent probes for GSH detection are designed based on the nucleophilicity of the sulfhydryl group. Most of the probes are michael type receptors at present, and they are reported to have potential side effects of inducing the aldehyde ketone reductase AKR1C 1. However, applicants contemplate that non-michael receptor-type probes may be more biocompatible.
The l, 8-naphthalimide derivative is a common optical material, generally has strong yellow-green fluorescence, higher fluorescence quantum efficiency and larger Stokes shift, and the compound also has good light stability and thermal stability. At present, no report related to the introduction of aryl sulfide at the electron-deficient 4-position of a 1, 8-naphthalimide fluorophore exists, and no report related to the use of the obtained compound as a fluorescent probe for tracking endogenous GSH fluctuation in living cells exists.
Disclosure of Invention
The invention aims to provide a method for preparing a fluorescent probe by introducing aryl sulfide to the electron-deficient 4-position of a 1, 8-naphthalimide fluorophore and application of the fluorescent probe.
The invention relates to a preparation method of a fluorescent probe with a structure shown in the following formula (I), which mainly comprises the following steps: putting a compound shown as a formula (II) and N, N-dimethylethylenediamine into an organic solvent for reaction, and recovering the solvent after the reaction is finished to obtain a crude product of the target product;
the chemical name of the compound shown in the formula (I) is N- (2-N, N-dimethylamino) ethylamino-6- (4-fluorobenzene sulfinyl) -1, 8-naphthalimide, and the molecular weight is as follows: 410.46.
in the preparation method of the present invention, the organic solvent may be one or a combination of two or more selected from ethanol, methanol, Dichloromethane (DCM), N-Dimethylformamide (DMF) and Petroleum Ether (PE). When the organic solvent is selected from the combination of two or more of the above substances, the ratio of the organic solvent to the organic solvent may be any ratio. The amount of the organic solvent to be used may be determined as required, and in general, all the starting materials to be reacted are dissolved in a total amount of 5 to 10mL of the organic solvent based on 1mmol of the compound represented by the formula (II).
In the preparation method of the present invention, the reaction may be carried out without heating or with heating, and preferably without heating. Whether the reaction was complete can be followed by TLC.
In the preparation method of the present invention, the amount ratio of the compound represented by the formula (II) to N, N-dimethylethylenediamine is a stoichiometric ratio, and in actual experimental operation, the amount ratio of the compound represented by the formula (II) to the amount of N, N-dimethylethylenediamine is generally: 1: 1.5-3.
The preparation method of the invention prepares the crude product of the compound shown in the formula (I), and the crude product can be purified by adopting the conventional purification method so as to improve the purity of the compound shown in the formula (I). Usually, silica gel column chromatography is adopted for purification, and specifically, the prepared crude target product is subjected to silica gel column chromatography, and the volume ratio of the crude target product to the silica gel column chromatography is 5-20: 1, eluting with an eluent consisting of Ethyl Acetate (EA) and methanol, and evaporating the eluent to remove the solvent to obtain the purified target substance.
The chemical name of the compound shown as the raw material formula (II) related in the preparation method is 6- (4-fluorobenzenesulfinyl) -1, 8-naphthalimide, and the compound can be synthesized by referring to the existing literature and also can be synthesized by a self-designed route. The preparation is preferably carried out as follows:
1) putting 4-chloro-1, 8-naphthalic anhydride and p-fluorobenzenethiol into a first solvent, reacting under a heating condition under an alkaline condition of a system, recovering the solvent, acidifying the obtained reactant, separating out a precipitate, and collecting the precipitate to obtain a compound 2;
2) and (2) putting the compound 2 and m-chloroperoxybenzoic acid (m-CBPA) into a second solvent, reacting without heating, washing a reactant with a saturated NaHCO3 solution, extracting with an extracting agent, collecting an organic phase, and recovering the solvent to obtain a compound 3, namely the compound shown in the formula (II).
In step 1) of the method for preparing a compound represented by the formula (II), the first solvent is preferably N, N-dimethylformamide; the alkalescence substance (such as sodium bicarbonate or sodium carbonate and the like) is added into the system to adjust the system to be alkaline so as to be beneficial to the reaction; the reaction is generally carried out at 40 ℃ to the reflux temperature of the first solvent, and preferably under stirring, and the completion of the reaction is checked by TLC tracking; after the reaction is completed, the alkalinity of each system in an acid solution (usually a dilute hydrochloric acid solution, such as a 5 v/v% hydrochloric acid solution) is adopted to maintain the system at a pH value of about 7. The product obtained in this step can be further purified (such as recrystallization, and the solvent for recrystallization can be common solvent such as ethanol) and then used for subsequent operation.
In step 2) of the method for preparing a compound represented by the above formula (II), the second solvent is preferably N, N-dimethylformamide; the reaction is usually carried out at normal temperature, and whether the reaction is complete or not is detected by TLC tracking; after the reaction is finished, the acid possibly existing in the system is removed by washing preferably by using saturated NaHCO3 solution; the extractant preferably adopts dichloromethane. The product obtained in this step is a crude product of the compound represented by formula (II), and is preferably purified by silica gel column chromatography (eluting with a mixed solvent composed of ethyl acetate and petroleum ether at a volume ratio of 1: 2-50, eluting with a mixed solvent composed of dichloromethane, ethyl acetate and methanol at a volume ratio of 9:3:1, and collecting the eluate eluted with the mixed solvent composed of dichloromethane, ethyl acetate and methanol) and then used in the subsequent operations.
The molar ratio of the starting materials in the reactions involved in the steps of the process for the preparation of the compound represented by the above formula (II) is the stoichiometric ratio.
In experiments, researchers of the application find that the fluorescent probe (also referred to as NA-21 or probe NA-21 or fluorescent probe NA-21) with the structure shown in the formula (I) can be combined with reduced glutathione under certain conditions to generate a fluorescent substance (III).
Therefore, the invention also comprises the application of the fluorescent probe with the structure shown in the formula (I) in the preparation of an indicator for indicating the level of reduced glutathione in cells;
in a specific experimental process, researchers of the application select a hepatoma cell strain as a test cell strain, and track the fluctuation of intracellular GSH through a fluorescence imaging method. To confirm whether probe NA-21 reacted with reduced glutathione, a reduced glutathione scavenger (N-ethylmaleimide) was chosen to indicate the dependence between probe NA-21 and glutathione dose.
The experimental result shows that when the probe NA-21 reacts with the reduced glutathione in the phosphate buffer solution at the temperature of 20-37 ℃, the fluorescence intensity after the reaction of the probe NA-21 and the reduced glutathione can be improved by 1500 times compared with the probe NA-21. In experiments, the researchers of the application also find that the probe NA-21 is time-dependent and dose-dependent relative to the reduced glutathione respectively.
Compared with the prior art, the invention provides a novel preparation method of the fluorescent probe for indicating the level of the intracellular reduced glutathione, and simultaneously discovers that the fluorescent probe can react with the reduced glutathione in the tumor cell, so that the level of the intracellular reduced glutathione can be well tracked.
Drawings
FIG. 1 is a fluorescence spectrum of probe NA-21 in Experimental example 1 of the present invention under the same conditions after incubation with GSH (reduced glutathione) and GSSG (oxidized glutathione) respectively at an excitation wavelength of 405nm and an emission wavelength of 498 nm; wherein (a) is a fluorescence spectrum of the probe NA-21, (b) is a fluorescence spectrum of the probe NA-21+ GSSG, and (c) is a fluorescence spectrum of the experiment 1 group (namely the probe NA-21+ GSH);
FIG. 2 is a graph showing the fluorometric reaction of probe NA-21 with GSH (1mM) for various periods in Experimental example 2 of the present invention; whereinIs the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 0min,is the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 5min,is the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 10min,is the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 15min,is the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 30min,is the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 45min,is the fluorescence spectrum of NA-21 incubated with GSH (1mM) for 60 min;
FIG. 3 is a graph showing the fluorometric reaction of probe NA-21 with GSH at various concentrations in Experimental example 3 of the present invention; whereinIs a fluorescence spectrum of NA-21 incubated with 0 mu M GSH for 1h,is the fluorescence spectrum of NA-21 when incubated with 200 mu M GSH for 1h,is the fluorescence spectrum of NA-21 when incubated with 400 mu M GSH for 1h,is a fluorescence spectrum of NA-21 incubated with 600 mu M GSH for 1h,is a fluorescence spectrum of NA-21 incubated with 800 mu M GSH for 1h,fluorescence spectra of NA-21 incubated with 1000. mu.M GSH for 1h,fluorescence spectra obtained by incubating NA-21 with 2000. mu.M GSH for 1 h;
FIG. 4 is a graph showing fluorescence images of probe NA-21 at different concentrations in experiment example 4 of the present invention in hepatoma cell line Hep G2; (a) the fluorescence imaging images of the blank group in the hepatoma cell line Hep G2, (b) the fluorescence imaging images of the experiment 1 group in the hepatoma cell line Hep G2, (c) the fluorescence imaging images of the experiment 2 group in the hepatoma cell line Hep G2, and (d) the fluorescence imaging images of the experiment 3 group in the hepatoma cell line Hep G2;
FIG. 5 is a photograph showing fluorescence images of fluorescent probe NA-21 in hepatoma cell line Hep G2 at different times in Experimental example 5; wherein (a) is a fluorescence imaging graph of a blank group in a hepatoma cell line Hep G2, (b) is a fluorescence imaging graph of an experiment 1 group in a hepatoma cell line Hep G2, (c) is a fluorescence imaging graph of an experiment 2 group in a hepatoma cell line Hep G2, and (d) is a fluorescence imaging graph of an experiment 3 group in a hepatoma cell line Hep G2;
FIG. 6 is a fluorescent image of probe NA-21 and exogenous GSH with different concentrations in hepatoma cell line Hep G2 in Experimental example 6 of the present invention; wherein (a) is a fluorescence imaging graph of a control group 1 in a liver cancer cell line Hep G2, (b) is a fluorescence imaging graph of an experiment group 1 in a liver cancer cell line Hep G2, (c) is a fluorescence imaging graph of an experiment group 2 in a liver cancer cell line Hep G2, and (d) is a fluorescence imaging graph of an experiment group 3 in a liver cancer cell line Hep G2;
FIG. 7 is a fluorescent image of probe NA-21 and exogenous GSH at different times in hepatoma cell line Hep G2 in Experimental example 7 of the present invention; wherein (a) is a fluorescence imaging graph of a control group 1 in a liver cancer cell line Hep G2, (b) is a fluorescence imaging graph of an experiment 1 group in a liver cancer cell line Hep G2, (c) is a fluorescence imaging graph of an experiment 2 group in a liver cancer cell line Hep G2, (d) is a fluorescence imaging graph of an experiment 3 group in a liver cancer cell line Hep G2, and (e) is a fluorescence imaging graph of an experiment 4 group in a liver cancer cell line Hep G2;
FIG. 8 is a photograph showing fluorescence images of probe NA-21 and NEM at different concentrations in hepatoma cell line Hep G2 in Experimental example 8 of the present invention; wherein (a) is a fluorescence imaging picture of a control group 1 in a liver cancer cell line Hep G2, (b) is a fluorescence imaging picture of an experiment group 1 in a liver cancer cell line Hep G2, and (c) is a fluorescence imaging picture of an experiment group 2 in a liver cancer cell line Hep G2;
FIG. 9 is a photograph showing fluorescence images of probe NA-21 and NEM at different times in hepatoma cell line Hep G2 in Experimental example 9 of the present invention; the fluorescence imaging picture of the control group in the hepatoma cell line Hep G2, (b) the fluorescence imaging picture of the experiment 1 group in the hepatoma cell line Hep G2, (c) the fluorescence imaging picture of the experiment 2 group in the hepatoma cell line Hep G2, and (d) the fluorescence imaging picture of the experiment 3 group in the hepatoma cell line Hep G2.
Detailed Description
The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.
Example 1: preparation of the Compound of formula (II)
The compound of formula (II) is prepared according to the following synthetic route:
the preparation method comprises the following steps:
1) to a 250mL round bottom flask were added 4-chloro-1, 8-naphthalic anhydride (2.32g,10mmol), DMF (30mL,28.35g), NaHCO in sequence with electromagnetic stirring3(0.84g) and p-fluorophenylthiophenol (1.59mL,15mmol), and the reaction was heated under reflux for 10 hours (reaction monitored by TLC, developer: V)EA:VPE1: 4). Cooling, removing solvent under reduced pressure, acidifying with 5% HCl solution to obtain yellow precipitate, filtering, and recrystallizing with ethanol to obtain 1.62g of compound 2 with yield of 69.8%;
2) to a round bottom flask containing DCM (10mL) solvent was added compound 2(0.30g,0.93mmol) sequentially with 1.5 equivalents of m-CBPA (85% pure, 0.28g,1.40mmol) under magnetic stirring for 5h at ambient temperature (TLC monitoring reaction, developing solvent: V)EA:VPE1:2), with saturated NaHCO3The solution was washed (4X 15mL), extracted with Dichloromethane (DCM), the organic phase was collected, dried, filtered, and the solvent was removed under reduced pressure, and purified by silica gel column chromatography (eluting with a mixed solvent composed of ethyl acetate and petroleum ether in a volume ratio of 1: 50, then with a mixed solvent composed of dichloromethane, ethyl acetate and methanol in a volume ratio of 9:3:1, and the eluate eluted with a mixed solvent composed of dichloromethane, ethyl acetate and methanol was collected) to obtain 0.81g of Compound 3 (pale yellow solid), i.e., the compound represented by formula (II).
The compound 3 obtained in this example was analyzed by nmr hydrogen spectroscopy and nmr carbon spectroscopy, and the data are shown below:
1H NMR(500MHz,CDCl3)δ8.80(d,J＝7.7Hz,1H),8.68(d,J＝7.3Hz,1H),8.59(dd,J＝11.4,8.5Hz,2H),7.89(t,J＝8.4Hz,1H),7.72(dd,J＝8.7,5.0Hz,2H),7.15(t,J＝8.4Hz,2H).
13C NMR(125MHz,CDCl3)δ164.8(d,J＝228.1Hz),159.6,159.5,149.7,139.2(d,J＝3.1Hz),133.4(d,J＝121.4Hz),130.5,129.5,128.7,128.1,128.0,127.7,124.1,120.6(d,J＝190.7Hz),117.4,117.3.
therefore, it was confirmed that the structure of the above pale yellow solid is represented by the following formula (II):
therefore, the compound is determined to be the compound shown in the formula (II), and the chemical name of the compound is 6- (4-fluorobenzenesulfinyl) -1, 8-naphthalimide.
Example 2: preparation of Compound represented by formula (I) (i.e., Probe NA-21)
To a round-bottomed flask containing ethanol (20mL) were added the compound represented by the formula (II) (0.27g,0.59mmol) and N, N-dimethylethylenediamine (0.12mL,0.71mmol) in this order under electromagnetic stirring, and reacted at ordinary temperature for 2 hours (reaction monitored by TLC, developing solvent: V)EA:VMeOH4:1), removing the solvent under reduced pressure, and performing silica gel column chromatography (eluent: V)EA:VMeOH10:1) to yield 0.21g of a pale yellow solid in 77.8% yield.
The light yellow solid obtained in this example was analyzed by nmr hydrogen spectroscopy, nmr carbon spectroscopy and electrospray mass spectroscopy, and the data are shown below:
1H NMR(500MHz,CDCl3)δ8.75(d,J＝7.7Hz,1H),8.62(d,J＝7.3Hz,1H),8.48(dd,J＝8.0,4.5Hz,2H),7.80(t,J＝7.6Hz,1H),7.69(dd,J＝8.7,5.0Hz,2H),7.12(t,J＝8.4Hz,2H),4.31(t,J＝6.8Hz,2H),2.64(t,J＝6.8Hz,2H),2.33(s,6H).
13C NMR(125MHz,CDCl3)δ:165.5,163.5,163.3,147.5,139.7(d,J＝3.2Hz),131.2(d,J＝125.0Hz),128.6,128.1(d,J＝17.3Hz),127.9(d,J＝9.1Hz),127.5,125.2,123.8,123.6,117.2,117.0,56.9,53.3,45.7,38.3.
electrospray mass spectrometry: ESI-MS M/z 411.1173[ M + H ] +.
Therefore, the light yellow solid can be determined to be the target product N- (2-N, N-dimethylamino) ethylamino-6- (4-fluorobenzene sulfinyl) -1, 8-naphthalimide, and the chemical structural formula of the light yellow solid is shown as the following formula (I):
example 3: preparation of Compounds of formula (I)
Example 2 was repeated except that:
replacing ethanol with N, N-dimethylformamide, changing reaction time to 1h, and changing eluent to VEA:VMeOHThe rest reaction conditions were unchanged at 6: 1. 0.08g of a pale yellow solid was obtained with a yield of about 29.6%.
The light yellow solid obtained in the embodiment is analyzed by nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum and electrospray mass spectrum, and is determined to be the target compound.
Example 4: preparation of Compounds of formula (I)
Example 2 was repeated except that:
using petroleum ether and dichloromethane in a ratio of 1: 1, changing the reaction time to 3h and the eluent to VEA:VMeOHThe rest reaction conditions were unchanged at 20: 1. 0.11g of a pale yellow solid was obtained with a yield of about 40.7%.
The light yellow solid obtained in the embodiment is analyzed by nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum and electrospray mass spectrum, and is determined to be the target compound.
Example 5: preparation of Compounds of formula (I)
Example 2 was repeated except that:
methanol is used to replace ethanol, and the rest reaction conditions are unchanged. 0.16g of a pale yellow solid was obtained in about 59.3% yield.
The light yellow solid obtained in the embodiment is analyzed by nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum and electrospray mass spectrum, and is determined to be the target compound.
Experimental example 1: fluorometric determination of Probe NA-21, Probe NA-21 reacting with GSH, GSSG, respectively
In the experiment, a fluorescence spectrophotometer is used for detecting the fluorescence intensity of the probe NA-21 at an excitation wavelength of 405nm and an emission wavelength of 498nm and the fluorescence intensity of the probe NA-21 incubated with GSH (reduced glutathione) and GSSG (oxidized glutathione) respectively under the same condition.
Experimental grouping conditions were:
experimental groups, including 2 subgroups, were as follows:
experiment 1 group: namely the probe NA-21+ GSH, the final concentration of the probe NA-21 is 5 mu mol/L and the final concentration is 10 mu mol/L, and the incubation is carried out for 1 h.
Experiment 2 group: namely the probe NA-21+ GSSG, and the final concentration of the probe NA-21 is 5 mu mol/L and the final concentration of the GSSG is 10 mu mol/L, and the incubation is carried out for 1 h.
Blank group: i.e., CON group, at a final concentration of 5. mu. mol/L of probe NA-21.
The measurement results of each group are shown in FIG. 1, in which (a) is the fluorescence spectrum of probe NA-21, (b) is the fluorescence spectrum of probe NA-21+ GSSG, and (c) is the fluorescence spectrum of experiment 1 group (i.e., probe NA-21+ GSH). As can be seen from FIG. 1, the probe NA-21 itself shows weak fluorescence, and the fluorescence intensity does not change significantly compared with NA-21 after incubation with GSSG; but after the culture medium is incubated with GSH, the fluorescence intensity can be increased by 40 percent, and the fluorescence intensity is obviously enhanced.
Experimental example 2: fluorescence measurement of probe NA-21 reacting with GSH for different time
Experimental example 1 was repeated except that the experimental 1 group and blank group in Experimental example 1 were retained. The incubation time of probe NA-21 with GSH (1mM) was varied on the basis of experiment 1 group. Sequentially comprises 0min,5min, 10min, 15min, 30min, 45min and 60 min. The results of the respective sets of measurements are shown in FIG. 2, whereinIs the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 0min,is the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 5min,is the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 10min,is the fluorescence spectrum of NA-21 incubated with GSH (1mM) for 15min，Is the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 30min,is the fluorescence spectrum of NA-21 when incubated with GSH (1mM) for 45min,is the fluorescence spectrum of NA-21 incubated with GSH (1mM) for 60 min.
As can be seen from FIG. 2, the fluorescence intensity of the co-incubation mother solution was increased with the increase of the incubation time of NA-21 and GSH, and was increased by nearly 1400 times at 60min of co-incubation.
Experimental example 3: fluorescence measurement of reaction of probe NA-21 with GSH of different concentrations
Experimental example 1 was repeated except that the experimental 1 group and blank group in Experimental example 1 were retained. The concentration of GSH was varied on the basis of experiment 1 group. The concentration of the active component is 0. mu.M, 200. mu.M, 400. mu.M, 600. mu.M, 800. mu.M, 1000. mu.M and 2000. mu.M in this order. The results of the respective sets of measurements are shown in FIG. 3, whereinIs a fluorescence spectrum of NA-21 incubated with 0 mu M GSH for 1h,is the fluorescence spectrum of NA-21 when incubated with 200 mu M GSH for 1h,is the fluorescence spectrum of NA-21 when incubated with 400 mu M GSH for 1h,is a fluorescence spectrum of NA-21 incubated with 600 mu M GSH for 1h,is a fluorescence spectrum of NA-21 incubated with 800 mu M GSH for 1h,fluorescence spectra of NA-21 incubated with 1000. mu.M GSH for 1h,fluorescence spectra for incubation of NA-21 with 2000. mu.M GSH for 1 h.
As can be seen from FIG. 3, when probe NA-21 was incubated with different concentrations of GSH, the fluorescence intensity was stronger as the concentration of GSH was higher.
Experimental example 4: fluorescent imaging of probe NA-21 with different concentrations in hepatoma cell line Hep G2
The experiment selects a human liver cancer Hep G2 cell line. The cell strain is cultured in DMEM culture solution containing 10 wt% calf serum, 100U/mL penicillin and 100U/mL streptomycin, and the DMEM culture solution is placed at 37 ℃ and contains 5% CO by volume concentration2Culturing in an incubator. The fluorescent probe NA-21 used was prepared as described in example 4 above, with a purity of 95% or more. In the experiment, DMSO is used as a solvent to be matched with 2mM of fluorescent probe NA-21 technical, the technical is diluted to a certain concentration by a culture solution and then added into a 6-hole plate,
experimental grouping conditions were:
experimental groups, including 3 subgroups, were as follows:
experiment 1 group: i.e., the NA-21 (5. mu.M) group, treated with probe NA-21 for 1 h;
experiment 2 group: i.e., the NA-21 (10. mu.M) group, treated with probe NA-21 for 1 h;
experiment 3 groups: i.e., the NA-21 (15. mu.M) group, treated with probe NA-21 for 1 h;
blank group: i.e., CON group, was not treated with probe probes, but otherwise the conditions were the same as those in the experimental group.
The measurement results of the respective groups are shown in fig. 4, in which (a) is a fluorescence image of the blank group in the hepatoma cell line Hep G2, (b) is a fluorescence image of the experiment 1 group in the hepatoma cell line Hep G2, (c) is a fluorescence image of the experiment 2 group in the hepatoma cell line HepG2, and (d) is a fluorescence image of the experiment 3 group in the hepatoma cell line Hep G2. As can be seen from FIG. 4, the fluorescence intensity of Hep G2 cells was increased in a manner that the concentration of probe NA-21 was dependent upon the incubation time of probe NA-21 with Hep G2 cells for 1 hour.
Experimental example 5: fluorescence imaging of probe NA-21 in hepatoma cell line Hep G2 at different times
Experimental example 4 was repeated, except that NA-21 (10. mu.M) was used in each experimental group, and the treatment time was 30min, 1h, and 5h in this order.
The measurement results of the respective groups are shown in fig. 5, in which (a) is a fluorescence image of the blank group in the hepatoma cell line Hep G2, (b) is a fluorescence image of the experiment 1 group in the hepatoma cell line Hep G2, (c) is a fluorescence image of the experiment 2 group in the hepatoma cell line HepG2, and (d) is a fluorescence image of the experiment 3 group in the hepatoma cell line Hep G2. As can be seen from FIG. 5, the fluorescence intensity of Hep G2 cells was increased in a time-dependent manner with probe NA-21 by incubating probe NA-21 with Hep G2 cells for 1 h.
Experimental example 6: fluorescent imaging of probe NA-21 and exogenous GSH with different concentrations in hepatoma cell line Hep G2
The experiment selects a human liver cancer Hep G2 cell line. The cell strain is cultured in DMEM culture solution containing 10 wt% calf serum, 100U/mL penicillin and 100U/mL streptomycin and cultured in an incubator containing CO2 with the volume concentration of 5% at 37 ℃. The fluorescent probe NA-21 used was prepared as described in example 1 above, with a purity of 95% or more. The GSH used was purchased from alatin. In the experiment, DMSO is used as a solvent to be matched with 2mM fluorescent probe NA-21 technical, PBS is used as a solvent to be matched with 10mM GSH, the technical solution is diluted to a certain concentration by a culture solution and then added into a 6-hole plate,
experimental grouping conditions were:
experimental groups, including 3 subgroups, were as follows:
experiment 1 group: GSH (0.20mM) + NA-21(10 μ M), treating with GSH for 1h, replacing with new culture solution, and treating with probe NA-21 for 1 h;
experiment 2 group: GSH (0.60mM) + NA-21(10 μ M), treating with GSH for 1h, replacing with new culture solution, and treating with probe NA-21 for 1 h;
experiment 3 groups: GSH (1mM) + NA-21(10 μ M), treating with GSH for 1h, replacing with new culture solution, and treating with probe NA-21 for 1 h;
blank group: namely CON group, the conditions are the same as those of the experimental group except that the probes NA-21 and GSH are not added;
control 1 group: i.e., the NA-21 (10. mu.M) group, was treated with probe NA-21 alone for 1h under the same experimental conditions without GSH treatment.
The measurement results of the respective groups are shown in fig. 6, in which (a) is a fluorescence image of the control 1 group in the hepatoma cell line Hep G2, (b) is a fluorescence image of the experiment 1 group in the hepatoma cell line Hep G2, (c) is a fluorescence image of the experiment 2 group in the hepatoma cell line HepG2, and (d) is a fluorescence image of the experiment 3 group in the hepatoma cell line Hep G2. As can be seen from fig. 6, the fluorescence intensity can be further enhanced by supplementing exogenous GSH, but the fluorescence imaging enhancement effect is not significant as the GSH concentration is excessively saturated.
Experimental example 7: fluorescent imaging of probe NA-21 and exogenous GSH at different time in hepatoma cell line Hep G2
Experimental example 6 was repeated except that the experimental group was treated with GSH (1mM) for 15min, 30min, 45min and 1h in this order, then replaced with a fresh culture solution, and then treated with probe NA-21 (10. mu.M) for 1h in this order. 4 groups in total
The measurement results of the groups are shown in fig. 7, wherein (a) is a fluorescence imaging graph of the control group 1 in the hepatoma cell line Hep G2, (b) is a fluorescence imaging graph of the experiment group 1 in the hepatoma cell line Hep G2, (c) is a fluorescence imaging graph of the experiment group 2 in the hepatoma cell line Hep G2, (d) is a fluorescence imaging graph of the experiment group 3 in the hepatoma cell line Hep G2, and (e) is a fluorescence imaging graph of the experiment group 4 in the hepatoma cell line Hep G2. As can be seen from FIG. 7, the exogenous GSH was supplemented to show strong fluorescence.
Experimental example 8: fluorescent probe NA-21 and different concentrations of NEM in hepatoma cell line Hep G2 for fluorescence imaging
Experimental example 6 was repeated except that the cells were treated with NEM (GSH chelator) for 1 hour at 25. mu.M and 50. mu.M, respectively, and the culture medium was replaced with a fresh one, and then treated with probe NA-21 (10. mu.M) for 1 hour in this order. The total number of the groups is 2.
The measurement results of the respective groups are shown in fig. 8, in which (a) is a fluorescence image of the control 1 group in the hepatoma cell line Hep G2, (b) is a fluorescence image of the experiment 1 group in the hepatoma cell line Hep G2, and (c) is a fluorescence image of the experiment 2 group in the hepatoma cell line HepG 2. As shown in FIG. 8, NEM was selected to clear GSH, since it was demonstrated that NA-21 shows that fluorescence in cells is indeed related to GSH. Cells were treated with NEM at 25. mu.M, 50. mu.M, respectively, and incubated with NA-21, showing that the fluorescence intensity gradually decreased as the concentration of NEM increased.
Experimental example 9: fluorescent imaging of probe NA-21 and NEM at different times in hepatoma cell line Hep G2
Experimental example 6 was repeated except that the cells were first treated with NEM (GSH chelator) (50. mu.M) for 30min, 1h, 5h, respectively.
The measurement results of the respective groups are shown in fig. 9, in which (a) is a fluorescence image of the control group in the hepatoma cell line Hep G2, (b) is a fluorescence image of the experiment 1 group in the hepatoma cell line Hep G2, (c) is a fluorescence image of the experiment 2 group in the hepatoma cell line HepG2, and (d) is a fluorescence image of the experiment 3 group in the hepatoma cell line Hep G2. As can be seen from FIG. 9, when the cells were treated with NEM (50. mu.M) for various periods of time and incubated with NA-21, the results showed that the fluorescence intensity gradually decreased as the NEM incubation period increased.
Claims (10)
1. A method for preparing a fluorescent probe having a structure represented by the following formula (I), characterized in that: the method mainly comprises the following steps: putting a compound shown as a formula (II) and N, N-dimethylethylenediamine into an organic solvent for reaction, and recovering the solvent after the reaction is finished to obtain a crude product of the target product;
2. a process for the preparation of a compound according to claim 1, characterized in that: the organic solvent is one or the combination of more than two of ethanol, methanol, dichloromethane, N-dimethylformamide and petroleum ether.
3. A process for the preparation of a compound according to claim 1, characterized in that: the reaction is carried out without heating or with heating.
4. A process for the preparation of a compound according to claim 1, characterized in that: further comprises a step of purifying the obtained crude target product.
5. The process for the preparation of a compound according to claim 4, wherein: and the purification step is to perform silica gel column chromatography on the prepared crude target product to obtain a purified target product.
6. A process for the preparation of a compound according to any one of claims 1 to 5, characterized in that: the compound shown in the formula (II) is prepared by the following method:
1) putting 4-chloro-1, 8-naphthalic anhydride and p-fluorobenzenethiol into a first solvent, reacting under a heating condition under an alkaline condition of a system, recovering the solvent, acidifying the obtained reactant, separating out a precipitate, and collecting the precipitate to obtain a compound 2;
2) and (3) putting the compound 2 and m-chloroperoxybenzoic acid into a second solvent, reacting without heating, washing a reactant with a saturated NaHCO3 solution, extracting with an extracting agent, collecting an organic phase, and recovering the solvent to obtain a compound 3, namely the compound shown in the formula (II).
7. The method of claim 6, wherein: in the step 1), the first solvent is N, N-dimethylformamide.
8. The method of claim 6, wherein: in the step 2), the second solvent is N, N-dimethylformamide.
9. The method of claim 6, wherein: in the step 2), the extracting agent is dichloromethane.
10. The application of the fluorescent probe with the structure shown in the formula (I) in the preparation of an indicator for indicating the level of reduced glutathione in cells;
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