CN111747918B - Biflavone derivative fluorescent probe, preparation method thereof and application thereof in brain glioma imaging - Google Patents
Biflavone derivative fluorescent probe, preparation method thereof and application thereof in brain glioma imaging Download PDFInfo
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- CN111747918B CN111747918B CN202010690780.2A CN202010690780A CN111747918B CN 111747918 B CN111747918 B CN 111747918B CN 202010690780 A CN202010690780 A CN 202010690780A CN 111747918 B CN111747918 B CN 111747918B
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
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- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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Abstract
The invention discloses a biflavone derivative fluorescent probe, a preparation method thereof and application thereof in brain glioma imaging, wherein the biflavone derivative fluorescent probe has a chemical name of 1, 4-phenyl bis (4-oxo-4H-benzopyran-2, 3-diyl) diacrylate (PPDC) and a molecular formula of C30H22O8. The probe PPDC is obtained by two-step synthesis, shows great Stokes shift (261 nm), and can effectively solve the defect of biological application resistance caused by fluorescence self-absorption. Fluorescence analysis research shows that the compound can be used for high-sensitivity detection of cysteine, the detection limit of the compound is 0.01 mu M, and in addition, the probe can effectively distinguish glutathione and homocysteine which are interferents of cysteine in organisms. Accurate detection of the trace cysteine in the brain glial cells is beneficial to early diagnosis of the brain glioma. In view of the defects of early detection and accurate excision of the brain glioma at present, the method is expected to realize accurate surgical navigation in clinical surgical excision of the brain glioma, and has good application prospect.
Description
Technical Field
The invention particularly relates to a biflavone derivative fluorescent probe, a preparation method thereof and application thereof in brain glioma imaging, belonging to the field of organic small-molecule biological fluorescent probes.
Background
Brain gliomas originate from brain and spinal glioblastomas and belong to the most common primary craniocerebral tumors, wherein malignant gliomas account for 80% of the total gliomas, and the 5-year mortality rate is listed as the third place in systemic tumors. Research shows that glioma grows in an infiltrative way, has no clear limit with normal brain tissue, and can be treated by surgical operation by completely cutting off the glioma on the premise of not damaging the normal brain tissue. In order to solve the problem, advanced detection methods and intraoperative navigation means such as intraoperative neuronavigation, intraoperative magnetic resonance imaging, intraoperative B-mode ultrasound imaging, intraoperative neuroelectrophysiological monitoring technology and the like are used for operation-assisted treatment, but have the defects of brain displacement, insufficient visualization, long anesthesia time and the like. Therefore, the intraoperative real-time, dynamic and visual detection of the brain glioma is an important exploration direction of the surgical treatment.
In recent years, fluorescence imaging technology has rapidly developed as an emerging molecular imaging technology. The method utilizes a specific fluorescent probe to mark specific molecules or cells, performs visual description on normal or abnormal physiological processes in space and time from the level of the molecules and the cells, is a non-invasive imaging mode, and has the advantages of high sensitivity, strong specificity, real-time detection and the like. The fluorescent probe serving as an imaging contrast agent can remarkably improve the imaging contrast between a target tumor signal and a background signal, realizes the target recognition of tumor cells, is further applied to the imaging of tumor tissues, initiates a new field of noninvasive, real-time and targeted tumor focus detection on the living body level, and is widely applied to the research of various tumors including breast cancer, lung cancer, colon cancer, cervical cancer, glioma and the like. Fluorescent probes based on small organic molecules have been developed and widely used in biomedical research to date, providing important information for diagnosis of related diseases, but their drawbacks of toxicity, poor biocompatibility and optical properties have led to their limited use in biological systems. The development of fluorescent probes applicable in vivo remains challenging.
Cysteine plays an important role in protein synthesis, detoxification, metabolism, post-translational modification and the like as an important biological thiol, and the abnormal content of Cys in a living body can cause various diseases, such as slow growth and development, skin injury, weakness, liver injury, lethargy, hair fading, cancer and the like. Therefore, the real-time, efficient and accurate quantitative detection of Cys under physiological conditions is of great significance in early diagnosis and biochemical research of certain diseases. In recent years, researches show that cysteine is an important marker of brain glioma, and attracts extensive attention of researchers. The design and preparation of the fluorescent probe for detecting cysteine in a living body have important significance for early diagnosis and treatment of brain glioma. However, the fluorescent probes available for the study of brain glioma are still very lacking, so that the development of such fluorescent probes is necessary and urgent. The flavonoid compounds generated from natural products are ideal low-toxicity fluorescent parent bodies and are widely used for construction of fluorescent probes, but the application of the flavonoid compounds in brain glioma detection is not found, so that the flavonoid compounds serving as the fluorescent parent bodies to construct novel fluorescent probes for brain glioma detection and treatment have important significance.
Disclosure of Invention
The technical problem to be solved is as follows: in order to solve the problem of lack of fluorescent probes for glioma, the invention provides a biflavone derivative fluorescent probe, a preparation method thereof and application thereof in glioma detection.
The technical scheme is as follows: the invention provides a biflavone derivative fluorescent probe, the chemical name of the fluorescent probe is 1, 4-phenyl bis (4-oxygen-4H-benzopyran-2, 3-diyl) diacrylate, the molecular formula is C30H22O8The structural formula is shown as the formula (I):
formula (I).
The invention also provides a preparation method of the biflavone derivative fluorescent probe, which comprises the following steps: weighing a certain amount of 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-ketone) to be dissolved in an organic solvent, adding alkali in an ice water bath, reacting for 30-50 min, then dropwise adding acryloyl chloride, continuing to react for 20-40 min in the ice water bath after dropwise adding, then heating to room temperature for reaction, tracking by using a thin-layer chromatography dot plate until the reaction solution is changed from red to orange yellow, spin-drying the filtrate after the reaction is finished, and separating to obtain the target product 1, 4-phenylbis (4-oxo-4H-benzopyran-2, 3-diyl) diacrylate.
The molar relation ratio of the 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-one), the alkali and the acryloyl chloride is 1: 2-4.
The alkali is at least one of triethylamine, piperidine, sodium ethoxide and piperidine.
The organic solvent is at least one of toluene, acetonitrile, dichloroethane, dichloromethane, chloroform, n-hexane, tetrahydrofuran, methanol and ethanol.
The separation mode is chromatographic separation, the chromatographic separation selects a mixed solvent of petroleum ether and ethyl acetate as a column chromatography eluent, and the volume ratio of the ethyl acetate to the petroleum ether in the eluent is 1: 3.
The 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-ketone) is prepared by the following steps:
s1, dissolving 2-hydroxyacetophenone and terephthalaldehyde in methanol, adding NaOH, heating and refluxing for 6-12 h, cooling to room temperature, and stirring the mixture at room temperature for 8-14 h;
s2, followed by addition of NaOH solution and H2O2And (3) continuously stirring the solution and the reaction solution at room temperature for 6 hours, pouring the reaction solution into ice water after the reaction is finished, placing the mixture into a refrigerator for standing for 4 to 8 hours, and filtering to obtain the 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-ketone).
The 2-hydroxyacetophenone, terephthalaldehyde, NaOH solution and H2O2The molar ratio of the solution is 1: 1: 0.0075: 0.5:9.9.
The invention also provides application of the biflavone derivative fluorescent probe in cysteine detection.
The invention also provides application of the biflavone derivative fluorescent probe in cysteine detection in brain glioma.
Has the advantages that:
1. the invention provides a novel biflavonoid compound fluorescent probe for the first time, enriches the types of cysteine fluorescent molecular probes, provides novel probe molecules for organic analysis and photochemistry, and can be widely applied to the field of fluorescence analysis or detection.
2. The fluorescent molecular probe realizes high-sensitivity detection of cysteine, and the detection limit of the fluorescent molecular probe is 0.01 mu M.
3. The fluorescent probe molecule can be applied to the detection of the content of cysteine in brain glioma and imaging research.
Drawings
FIG. 1 is a diagram showing the structural formula of a probe molecule PPDC (1, 4-phenylbis (4-oxo-4H-benzopyran-2, 3-diyl) diacrylate) described in example 1.
FIG. 2 shows the UV and fluorescence patterns and Stokes shift of the probe molecule PPDC described in example 1 in DMSO solution.
FIG. 3 is a fluorescence image of DMSO solutions of PPDC as probe molecules described in example 1 for detection of cysteine, homocysteine and glutathione.
FIG. 4 is a fluorescence plot of DMSO solutions of the probe molecule PPDC described in example 1 against other different types of interferents.
FIG. 5 is a fluorescent plot of DMSO solutions of the probe molecule PPDC described in example 1 against different concentrations of cysteine.
FIG. 6 is a graph of kinetics of cysteine detection by DMSO solutions of the probe molecule PPDC described in example 1.
FIG. 7 is a graph of the imaging of cysteine in brain glioma by the probe molecule PPDC described in example 1.
Specific experimental mode
The method of post-treatment after completion of the reaction in the following examples is not particularly limited, and those skilled in the art can separate the target product by a conventional organic separation method in combination with a separation method of common knowledge according to the physicochemical properties of the material. A preferred mode of separation is chromatographic separation. The chromatographic separation further preferably uses a mixed solvent of petroleum ether and ethyl acetate as a column chromatography eluent, and further preferably, the volume ratio of ethyl acetate to petroleum ether in the eluent is 1: 3.
In the following examples, the base is at least one of triethylamine, piperidine, sodium ethoxide, and piperidine. In the following examples, the organic solvent is at least one of toluene, acetonitrile, dichloroethane, dichloromethane, chloroform, n-hexane, tetrahydrofuran, methanol, and ethanol. The organic solvent is not particularly limited, but dichloromethane is preferred.
In the following examples, the end point of the reaction was monitored by TLC (thin layer chromatography) plate, and the reaction time was not particularly limited.
Example 1
Step 1, compound (ii): preparation of 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-one)
2-hydroxyacetophenone (0.68 g, 5 mmol) and terephthalaldehyde (0.67 g, 5 mmol) were dissolved in 40 ml of methanol and NaOH (1.5 g, 0.0375 mmol) was added, and after heating under reflux for 6h, cooling to room temperature and the mixture stirred at room temperature for 12 h. Then NaOH solution (0.5 mol/L, 5mL) and 30% H were added2O2Solution (9.9mol/L, 5mL), continuously stirring the reaction solution at room temperature for 6H, pouring the reaction solution into ice water (200 mL) after the reaction is finished, placing the ice water into a refrigerator for standing for 6H, and filtering to obtain an intermediate compound (II), namely 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-ketone).1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 4H), 8.05 (d, J = 8.0 Hz, 2H), 7.58 (dd, J = 20.2, 7.9 Hz, 4H), 7.24 (t, J = 7.6 Hz, 2H).。
Weighing intermediate compound (II) 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-one) (0.3986 g, 1 mmol), adding CH2Cl240 ml of the suspension was added Et in an ice-water bath3N (600 muL, 4 mmol), after reacting for 40 min, dropwise adding acryloyl chloride (360 muL, 4 mmol), continuing to react for about 30 min under an ice water bath, then raising the temperature to room temperature for reaction, monitoring the reaction end point through a point plate by Thin Layer Chromatography (TLC), and after about 10 h of reaction (the reaction solution is changed from red to orange yellow). After completion of the reaction the filtrate was spin dried by rotary evaporator and washed with ethyl acetate: petroleum ether =1:3, and the target product 1, 4-phenyl bis (4-oxo-4H-benzopyran-2, 3-diyl) diacrylate (PPDC) is obtained.1H NMR (400 MHz, Chloroform-d) δ 8.31 (dd, J = 8.0, 1.7 Hz, 2H), 8.06 (s, 4H), 7.78 (ddd, J = 8.7, 7.1, 1.7 Hz, 2H), 7.62 (dd, J = 8.6, 1.0 Hz, 2H), 7.49 (ddd, J = 8.1, 7.1, 1.1 Hz, 2H), 6.69 (dd, J = 17.4, 1.2 Hz, 2H), 6.46 – 6.38 (m, 2H), 6.12 (dt, J = 10.4, 1.3 Hz, 2H).13C NMR (101 MHz, Chloroform-d) δ 172.00, 163.05, 155.65, 154.86, 134.34, 134.23, 132.53, 128.59, 126.71, 126.31, 125.54, 123.65, 118.15 MS: m/z calcd for C30H19O8 + (M+), 507.1074; found, 507.1075.
Example 2
Step 1, compound (ii): the preparation of 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-one) was the same as in example 1.
Weighing 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-one) (0.3986 g, 1 mmol) as an intermediate compound (II), adding 40 ml of n-hexane, adding 300 muL, 2 mmol of hexahydropyridine in an ice-water bath, reacting for 40 min, dropwise adding acryloyl chloride (180 muL, 2 mmol), continuing to react for about 30 min in the ice-water bath, then heating to room temperature for reaction, monitoring the reaction end point through a Thin Layer Chromatography (TLC) dot plate, and after about 10H of reaction, changing the reaction liquid from red to orange yellow. After completion of the reaction the filtrate was spin dried by rotary evaporator and washed with ethyl acetate: petroleum ether =1:3, and the target product 1, 4-phenyl bis (4-oxo-4H-benzopyran-2, 3-diyl) diacrylate (PPDC) is obtained.1H NMR (400 MHz, Chloroform-d) δ 8.31 (dd, J = 8.0, 1.7 Hz, 2H), 8.06 (s, 4H), 7.78 (ddd, J = 8.7, 7.1, 1.7 Hz, 2H), 7.62 (dd, J = 8.6, 1.0 Hz, 2H), 7.49 (ddd, J = 8.1, 7.1, 1.1 Hz, 2H), 6.69 (dd, J = 17.4, 1.2 Hz, 2H), 6.46 – 6.38 (m, 2H), 6.12 (dt, J = 10.4, 1.3 Hz, 2H).13C NMR (101 MHz, Chloroform-d) δ 172.00, 163.05, 155.65, 154.86, 134.34, 134.23, 132.53, 128.59, 126.71, 126.31, 125.54, 123.65, 118.15 MS: m/z calcd for C30H19O8 + (M+), 507.1074; found, 507.1075.
Example 3
Step 1, compound (ii): the preparation of 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-one) was the same as in example 1.
WeighingThe intermediate compound (II), 2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-one) (0.3986 g, 1 mmol), 40 ml of ethanol was added, sodium ethoxide (450 muL, 3 mmol) was added in an ice-water bath, after 40 min of reaction, acryloyl chloride (240 muL, 3 mmol) was added dropwise, reaction was continued for about 30 min in an ice-water bath, then the reaction temperature was raised to room temperature for reaction, the reaction endpoint was monitored by Thin Layer Chromatography (TLC) dot plate, and after about 10H of reaction, the reaction solution turned from red to orange yellow. After completion of the reaction the filtrate was spin dried by rotary evaporator and washed with ethyl acetate: petroleum ether =1:3, and the target product 1, 4-phenyl bis (4-oxo-4H-benzopyran-2, 3-diyl) diacrylate (PPDC) is obtained.1H NMR (400 MHz, Chloroform-d) δ 8.31 (dd, J = 8.0, 1.7 Hz, 2H), 8.06 (s, 4H), 7.78 (ddd, J = 8.7, 7.1, 1.7 Hz, 2H), 7.62 (dd, J = 8.6, 1.0 Hz, 2H), 7.49 (ddd, J = 8.1, 7.1, 1.1 Hz, 2H), 6.69 (dd, J = 17.4, 1.2 Hz, 2H), 6.46 – 6.38 (m, 2H), 6.12 (dt, J = 10.4, 1.3 Hz, 2H).13C NMR (101 MHz, Chloroform-d) δ 172.00, 163.05, 155.65, 154.86, 134.34, 134.23, 132.53, 128.59, 126.71, 126.31, 125.54, 123.65, 118.15 MS: m/z calcd for C30H19O8 + (M+), 507.1074; found, 507.1075.
Example 4
Ultraviolet absorption spectrum and fluorescence spectrum property test of probe molecule PPDC
Testing an instrument: PE 950s type ultraviolet spectrometer, Hitachi F7100 type molecular fluorescence spectrometer. The experimental method comprises the following steps: the probe molecule PPDC prepared in example 1 was dissolved in DMSO solution to obtain 1 mM of a probe stock solution, which was stored at room temperature. The test was carried out by diluting the solution to a standard solution of 0.01 mM in an experimental assay.
During measurement, 3 ml of the DMSO solution of the probe was transferred to a 1 cm cuvette for measurement of the ultraviolet absorption spectrum and the fluorescence spectrum, respectively, as shown in FIG. 2. The results show that: the strongest ultraviolet absorption peak of the PPDC probe appears at about 315 nm, the fluorescence emission peak appears at about 576 nm, and the Stokes shift reaches 261 nm. The great Stokes displacement can effectively overcome the defect of difficult application in the living body due to fluorescence self-absorption, and realize the application of the probe in the living body.
Example 3
And (3) a fluorescence diagram for detecting cysteine, homocysteine and glutathione by using the probe molecule PPDC.
Testing an instrument: hitachi F7100 type molecular fluorescence spectrometer; the experimental method comprises the following steps: the probe molecule PPDC prepared in example 1 was dissolved in DMSO to obtain a 1 mM probe stock solution, which was stored at room temperature. Cysteine, homocysteine and glutathione are prepared into 1 mM mother liquor by secondary water. The test was carried out by diluting the solution to a standard solution of 0.01 mM in an experimental assay.
During measurement, three parts of DMSO solution of 3 ml probes are transferred to a 1 cm cuvette for fluorescence spectrum test, and 2 mM cysteine, homocysteine and glutathione solutions are respectively dripped. The results are shown in FIG. 3. The results show that: the probe PPDC shows different detection effects on cysteine, homocysteine and glutathione, and particularly shows an obvious fluorescence enhancement phenomenon on cysteine, which shows that the probe shows excellent selectivity on cysteine when applied in a life system and can effectively avoid the interference of the two biological thiols.
Example 4
Fluorescence of the probe molecule PPDC to other interfering ions.
Testing an instrument: hitachi F7100 type molecular fluorescence spectrometer; the experimental method comprises the following steps: the probe molecule PPDC prepared in example 1 was dissolved in DMSO to obtain a 1 mM probe stock solution, which was stored at room temperature. Cysteine, ZnSO4、AgNO3、Co(NO3)2、 CuSO4、Fe2(SO4)3、PbNO3、NaNO2、NaNO3、KH2PO4、NaHSO3、NaHSO41 mM stock solution was prepared with secondary water. The test was carried out by diluting the solution to a standard solution of 0.01 mM in an experimental assay. When in measurement, 3 ml of DMSO solution of the probe is transferred to a 1 cm cuvette, and 2 mM of cysteine and ZnSO are respectively dripped into the cuvette4、AgNO3、Co(NO3)2、 CuSO4、Fe2(SO4)3、PbNO3、NaNO2、NaNO3、KH2PO4、NaHSO3、 NaHSO4A fluorescence test was performed. The results are shown in FIG. 4. The results show that: the probe PPDC shows obvious fluorescence enhancement phenomenon on cysteine, but has little influence on metal cations and acid anions commonly seen in some organisms, and further shows that the probe PPDC has excellent selectivity and can be applied to the organisms.
Example 5
Quantitative analysis of cysteine in DMSO solution of PPDC.
Testing an instrument: hitachi F7100 type molecular fluorescence spectrometer; the experimental method comprises the following steps: the probe molecule PPDC prepared in example 1 was dissolved in DMSO to obtain a 1 mM probe stock solution, which was stored at room temperature. Cysteine was made up to 1 mM stock solution in secondary water. The test was carried out by diluting the solution to a standard solution of 0.01 mM in an experimental assay.
The fluorescence response of the probe molecules to the cysteine is tested by adopting a standard addition method, 3 mL of probe mother liquor (0.01 mM) is transferred into a cuvette, 2 mu L of cysteine is added each time to detect the change of fluorescence intensity, and the fluorescence intensity is measured every 5 mu L or 10 mu L after the fluorescence intensity is gradually enhanced until the fluorescence intensity reaches the maximum value. As shown in FIG. 5, the peak intensity of fluorescence at 576 nm increased with the increase in cysteine content, and the fluorescence was strongest when the cysteine content reached 100. mu.M. Therefore, the probe has higher sensitivity to cysteine and can be used for detecting trace cysteine in organisms.
Example 6
And (3) a kinetic experiment chart of the probe molecule PPDC in the presence of cysteine, homocysteine and glutathione.
Testing an instrument: hitachi F7100 type molecular fluorescence spectrometer; the experimental method comprises the following steps: the probe molecule PPDC prepared in example 1 was dissolved in DMSO to obtain a 1 mM probe stock solution, which was stored at room temperature. Cysteine, homocysteine and glutathione are prepared into 1 mM mother liquor by secondary water. The test was carried out by diluting the solution to a standard solution of 0.01 mM in an experimental assay.
3 mL of the probe stock solution (0.01 mM) was transferred to a cuvette and the fluorescence excitation wavelength was set at 380 nm, and the change in fluorescence intensity at different times (0.5 min, 1 min, 5 min, 10 min, 15 min, 20 min, 40 min) was measured for each of the probe, probe + cysteine, probe + homocysteine, probe + glutathione solution, as shown in FIG. 6. Experimental results show that the fluorescence intensity of the initial probe solution is enhanced along with the increase of time, the fluorescence intensity of the probe and cysteine reaches the maximum within 1 min, and the later fluorescence intensity tends to be stable, which indicates that the probe is rapid in response and good in stability.
Example 7
The probe molecule PPDC of the invention is used for imaging research on cysteine in brain glioma.
The experimental method comprises the following steps: the probe molecule PPDC prepared in example 1 was dissolved in DMSO to obtain a 1 mM probe stock solution, which was stored at room temperature. The test was carried out by diluting the solution to a standard solution of 0.01 mM in an experimental assay.
In order to prove the practical application of the probe in a biological system, the biological fluorescence imaging experiment of the cells under different pH values is carried out under a confocal fluorescence microscope. HeLa cells were plated on a petri dish and incubated at 37 ℃ for 24 h, then PPDC standard solution (10 mM) was added to the petri dish, and fluorescence imaging was performed after adding cysteine (5, 20, 50. mu.M) at various concentrations for further incubation for 2h, as shown in FIG. 7. The experimental result shows that the fluorescence of the probe molecule PPDC is continuously enhanced along with the increase of the concentration of cysteine. These results indicate that the probe PPDC can enter cells as a fluorescent label for detecting intracellular cysteine, and thus can be used for detecting cysteine in brain glioma cells.
Claims (10)
1. The biflavone derivative fluorescent probe is characterized in that the chemical name of the fluorescent probe is 1, 4-phenyl bis (4-oxo-4H-benzopyran-2, 3-diyl) diacrylate, and the molecular formula of the fluorescent probe is C30H22O8The structural formula is shown as the formula (I):
formula (I).
2. The preparation method of the biflavone derivative fluorescent probe is characterized by comprising the following steps of: weighing a certain amount of 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-ketone) to be dissolved in an organic solvent, adding alkali in an ice water bath, reacting for 30-50 min, then dropwise adding acryloyl chloride, continuing to react for 20-40 min in the ice water bath after dropwise adding, then heating to room temperature for reaction, tracking by using a thin-layer chromatography dot plate until the reaction solution is changed from red to orange yellow, spin-drying the filtrate after the reaction is finished, and separating to obtain the target product 1, 4-phenylbis (4-oxo-4H-benzopyran-2, 3-diyl) diacrylate.
3. The method for preparing the biflavone derivative fluorescent probe according to claim 2, wherein the molar relationship ratio of the 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-one), the alkali and the acryloyl chloride is 1: 2-4.
4. The method for preparing biflavone derivative fluorescent probe according to claim 2, wherein the base is at least one of triethylamine, piperidine, sodium ethoxide and piperidine.
5. The method of claim 2, wherein the organic solvent is at least one of toluene, acetonitrile, dichloroethane, dichloromethane, chloroform, n-hexane, tetrahydrofuran, methanol, and ethanol.
6. The method for preparing the biflavone derivative fluorescent probe according to claim 2, wherein the separation mode is chromatographic separation, a mixed solvent of petroleum ether and ethyl acetate is selected as a column chromatography eluent, and the volume ratio of ethyl acetate to petroleum ether in the eluent is 1: 3.
7. The method for preparing biflavone derivative fluorescent probe according to claim 2, wherein the 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-one) is prepared by the following steps:
s1, dissolving 2-hydroxyacetophenone and terephthalaldehyde in methanol, adding NaOH, heating and refluxing for 6-12 h, cooling to room temperature, and stirring the mixture at room temperature for 8-14 h;
s2, followed by addition of NaOH solution and H2O2And (3) continuously stirring the solution and the reaction solution at room temperature for 6 hours, pouring the reaction solution into ice water after the reaction is finished, placing the mixture into a refrigerator for standing for 4 to 8 hours, and filtering to obtain the 2,2' - (1, 4-phenylene) bis (3-hydroxy-4 a, 8 a-dihydro-4H-chromium-4-ketone).
8. The method of claim 7, wherein the 2-hydroxyacetophenone, terephthalaldehyde, NaOH solution and H are added2O2The molar ratio of the solution is 1: 1: 0.0075: 0.5:9.9.
9. Use of a biflavone derivative fluorescent probe as claimed in claim 1 in cysteine detection.
10. Use of a biflavone derivative fluorescent probe as claimed in claim 1 for the detection of cysteine in brain glioma.
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| CN110172337A (en) * | 2019-05-30 | 2019-08-27 | 南京师范大学 | A kind of benzothiazole derivant fluorescence probe and its preparation method and application |
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