CN110963995A - Double-color fluorescent probe and synthetic method and application thereof - Google Patents
Double-color fluorescent probe and synthetic method and application thereof Download PDFInfo
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- CN110963995A CN110963995A CN201911320431.5A CN201911320431A CN110963995A CN 110963995 A CN110963995 A CN 110963995A CN 201911320431 A CN201911320431 A CN 201911320431A CN 110963995 A CN110963995 A CN 110963995A
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- 238000010189 synthetic method Methods 0.000 title abstract description 5
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- -1 dipyridyl methyl amine Chemical compound 0.000 claims abstract 2
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- 239000011261 inert gas Substances 0.000 claims description 10
- 238000004809 thin layer chromatography Methods 0.000 claims description 10
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- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 5
- 150000008064 anhydrides Chemical class 0.000 claims description 5
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- 239000012467 final product Substances 0.000 claims description 4
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- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 3
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- ISWQCIVKKSOKNN-UHFFFAOYSA-L Tiron Chemical compound [Na+].[Na+].OC1=CC(S([O-])(=O)=O)=CC(S([O-])(=O)=O)=C1O ISWQCIVKKSOKNN-UHFFFAOYSA-L 0.000 description 2
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- GRSMWKLPSNHDHA-UHFFFAOYSA-N Naphthalic anhydride Chemical compound C1=CC(C(=O)OC2=O)=C3C2=CC=CC3=C1 GRSMWKLPSNHDHA-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- Engineering & Computer Science (AREA)
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- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Materials Engineering (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a bicolor fluorescent probe, a synthetic method and application thereof, which are particularly suitable for simultaneously detecting O in a living body2 ·‑And Zn2+The bicolor fluorescent probe is composed of dipyridyl methyl amine (DPA), caffeic acid and 4-bromo-1, 8-naphthalic anhydride, and the structural formula is as follows:
the double-color fluorescent probe has the advantages of simple preparation, sensitive detection, good selectivity and the like. The invention successfully realizes the imaging analysis of O in the brain of the tristimania mouse on the living body level for the first time2 ·‑And Zn2+The change of the pressure sensor has good practical application value.
Description
Technical Field
The invention belongs to the technical field of biological detection and fluorescent probe preparation, and particularly relates to a bicolor fluorescent probe and a synthetic method and application thereof.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Depression, as a group of mental diseases, has extremely high morbidity and mortality, and brings serious harm to human health. However, the pathophysiology of depression is still poorly understood, and in order to effectively prevent and treat depression, we must have a thorough understanding of the development and progression of depression. The fluorescence imaging technology has the obvious advantages of high sensitivity, non-invasiveness, high space-time resolution capability and the like, so that the application of fluorescence microscopic imaging for visually tracing bioactive molecules in cells is one of the hot problems of research in the fields of chemistry, biology and medicine at present.
Superoxide anion radical (O)2 ·-) Is the first ROS produced in the organism and can be converted into other ROS (H)2O2ONOO-, etc.), O produced in excess2 ·-Are closely related to cell damage, disease and aging; as the second most abundant transition metal ion, Zn, in the human body2+Is important for maintaining biological functions, including regulation of biological redox systems, cell signaling, and the like. Many assays for O have been developed in recent years2 ·-Or Zn2+But all of them are used for monocomponent detection and imaging at the cellular level, and due to the complex biological background of the brain and the presence of the blood-brain barrier, for simultaneous detection of O in vivo2 ·-And Zn2+The probe (2) has not been reported.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a bicolor fluorescent probe, a synthetic method and application thereof, which are particularly suitable for simultaneously detecting O in a living body2 ·-And Zn2+The double-color fluorescent probe has the advantages of simple preparation, sensitive detection, good selectivity and the like. The invention successfully realizes the imaging analysis of O in the brain of the tristimania mouse on the living body level for the first time based on the prepared bicolor fluorescent probe2 ·-And Zn2+Has good practical applicationAnd (4) value.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, a two-color fluorescent probe is provided, which is composed of Dipyridylmethylamine (DPA), caffeic acid, and 4-bromo-1, 8-naphthalic anhydride, and has a structural formula as follows:
in the present invention, DPA is taken as Zn2+Recognition group caffeic acid as O2 ·-A recognition group and a fluorophore, 4-bromo-1, 8-naphthalic anhydride as fluorophore. Proved by research, the double-color fluorescent probe prepared by the invention can be not limited by the complex biological background of the brain and the existence of blood brain barrier, and can directly detect O in the brain of a living body (such as a mouse) at the same time2 ·-And Zn2+. The two-color fluorescent probe prepared by the invention has the fluorescence emission intensity of the probe at 450nm (caffeic acid part) along with O under the excitation of 800nm two-photon2 ·-Increases with increasing concentration of (B), the fluorescence intensity at 540nm (the anhydride moiety of naphthalene) increases with Zn2+The concentration is increased, the two fluorescence parts do not interfere with each other, and the probe can well reflect O in cells and living bodies2 ·-And Zn2+The concentration of (c) is varied.
In a second aspect of the present invention, a method for preparing the above-mentioned two-color fluorescent probe is provided, the method comprising the following steps:
in a third aspect of the invention, there is provided the use of the above-described dual color fluorescent probe, said use comprising detecting, identifying O in an environment or in a biological sample2 ·-And Zn2+。
The biological sample includes a biological organ, a biological tissue, and a biological cell.
Wherein the biological organ is a brain of a living organism (e.g., mouse brain).
The invention has the beneficial technical effects that:
1. the synthesis is simple, the detection speed is high, and long-time incubation is not needed in the detection process.
2. The detection process does not need to add other chemical substances, has simple operation and small cytotoxicity, and is beneficial to endogenous O at living cell level, tissue level and organ level2 ·-And Zn2+The detection, in particular the invention successfully realizes the imaging analysis of O in the brain of the tristimania mouse on the living body level for the first time2 ·-And Zn2+While the probe prepared by the invention has wide pH adaptability and O resistance2 ·-And Zn2+Has high selectivity, thereby having good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph of the optical properties of a probe according to example 1 of the present invention;
FIG. 2 is a photograph showing an image of a cell of a probe according to example 1 of the present invention;
FIG. 3 is an image of a living body of the probe of example 1 of the present invention;
FIG. 4A is a mass spectrum of an intermediate product A in the preparation of a probe according to example 1 of the present invention;
FIG. 4B is a mass spectrum of an intermediate product B in the preparation of the probe of example 1 according to the present invention;
FIG. 4C is a mass spectrum of an intermediate product C in the preparation of the probe of example 1 according to the present invention;
FIG. 4D is a mass spectrum of the final product of the probe preparation process of example 1.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
As previously mentioned, although many tests for O have been developed in recent years2 ·-Or Zn2+But all of them are used for monocomponent detection and imaging at the cellular level, and due to the complex biological background of the brain and the presence of the blood-brain barrier, for simultaneous detection of O in vivo2 ·-And Zn2+The probe (2) has not been reported.
In view of the above, in one embodiment of the present invention, a two-color fluorescent probe is provided, which is composed of Dipyridylmethylamine (DPA), caffeic acid and 4-bromo-1, 8-naphthalic anhydride, and has the following structural formula:
in the present invention, DPA is taken as Zn2+Recognition group caffeic acid as O2 ·-A recognition group and a fluorophore, 4-bromo-1, 8-naphthalic anhydride as fluorophore. Proved by research, the double-color fluorescent probe prepared by the invention can be not limited by the complex biological background of the brain and the existence of the blood brain barrier, and can be directly used in living bodies(e.g. mouse) brain Simultaneous detection of O2 ·-And Zn2+. The two-color fluorescent probe prepared by the invention has the fluorescence emission intensity of the probe at 450nm (caffeic acid part) along with O under the excitation of 800nm two-photon2 ·-Increases with increasing concentration of (B), the fluorescence intensity at 540nm (the anhydride moiety of naphthalene) increases with Zn2+The concentration is increased, the two fluorescence parts do not interfere with each other, and the probe can well reflect O in cells and living bodies2 ·-And Zn2+The concentration of (c) is varied.
In another embodiment of the present invention, a method for preparing the above-mentioned dual-color fluorescent probe is provided, which comprises the following steps:
in another embodiment of the present invention, the step a specifically includes:
under the protection of inert gas, bromopropylamine, DPA and potassium carbonate are mixed and stirred, and light yellow solid A is obtained after purification. A.
In another embodiment of the present invention, the step b specifically includes:
under the protection of inert gas, caffeic acid, DMAP, DCC, triethylamine and DMF are mixed and stirred under ice bath condition to obtain activated caffeic acid solution;
under the protection of inert gas, mixing ethylenediamine and DMF, then dropwise adding activated caffeic acid solution, stirring at room temperature to obtain brown turbid liquid, and carrying out thin layer chromatography to obtain light yellow solid B.
Eluent is dichloromethane: methanol 10: 1.
in another embodiment of the present invention, the step c specifically includes:
and under the protection of inert gas, mixing the product B, the anhydride naphthalene, the ethanol and the DMF, stirring at a low temperature to obtain a brown solution, and carrying out thin-layer chromatography to obtain a light yellow solid C. The low temperature is 28-35 ℃, and the further optimization is 30 ℃.
Wherein, the eluent is dichloromethane: methanol 10: 1.
in another embodiment of the present invention, the step d specifically includes:
under the protection of inert gas, the product A, the product C and the product K2CO3Mixing, stirring at high temperature overnight, and performing thin layer chromatography to obtain yellow solid as final product. The high temperature is 75-85 ℃, and more preferably 80 ℃.
Wherein, the eluent is dichloromethane: methanol 10: 1.
in yet another embodiment of the present invention, there is provided the use of the above-described dual color fluorescent probe, which comprises detecting, identifying O in an environment or in a biological sample2 ·-And Zn2+。
The biological sample includes a biological organ, a biological tissue, and a biological cell.
Wherein the biological organ is a brain of a living organism, such as a brain of a mouse.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are indicated, and are generally carried out under conventional conditions.
Example 1
Detecting cell and in vivo O2 ·-And Zn2+The preparation method of the novel fluorescent probe comprises the following steps:
step a, under the protection of nitrogen, 2.18g of bromopropylamine, 1.99g of DPA and 0.50g of 0.50g K are added2CO3Adding 30ml of acetonitrile into a two-mouth bottle, stirring for 12 hours at room temperature, filtering, and performing rotary evaporation to obtain a light yellow solid A. The mass spectrum is shown in FIG. 4A.
Step b: under the protection of nitrogen, 1.8g of caffeic acid, 1.2g of DMAP, 2g of DCC, 1.4ml of triethylamine and 12ml of DMF were put into a two-necked bottle, and stirred for 30min under ice-bath conditions to obtain an activated caffeic acid solution. 670 microliter of ethylenediamine and 5ml of DMF were added to a three-necked flask under nitrogen protection, and then the activated caffeic acid solution was added dropwise to the three-necked flask and stirred at room temperature for 12 hours to obtain a brown turbid liquid. Thin layer chromatography (eluent dichloromethane: methanol 10: 1) afforded B as a pale yellow solid. The mass spectrum is shown in FIG. 4B.
Step c: under the protection of nitrogen, the product B in the step B, 2.7g of naphthalic anhydride, 10ml of ethanol and 10ml of DMF are added into a two-mouth bottle, stirred for 10 hours at 30 ℃ to obtain a brown solution, and then subjected to thin layer chromatography (eluent is dichloromethane: methanol 10: 1) to obtain a yellow solid C. The mass spectrum is shown in FIG. 4C.
Step d: under the protection of nitrogen, the product A of the step a, the product C of the step C and 0.5g K2CO3Put into a two-necked flask and stirred at 80 ℃ overnight. Thin layer chromatography (eluent dichloromethane: methanol 10: 1) gave the final product as a yellow solid. The mass spectrum is shown in FIG. 4D.
Optical properties of the invention. As shown in FIG. 1, the fluorescence emission intensity of the probe at 450nm (caffeic acid moiety) with O under 800nm two-photon excitation2 ·-Increases with increasing concentration of (B), the fluorescence intensity at 540nm (the anhydride moiety of naphthalene) increases with Zn2+The concentration is increased, the two fluorescence parts do not interfere with each other, and the probe can well reflect O in cells and living bodies2 ·-And Zn2+The concentration of (c) is varied. And O of the probe2 ·-And Zn2+The two-part recognition groups are hardly interfered by the change of pH value, and can be well used for cell and living body imaging. While the probe pair O2 ·-And Zn2+Has high selectivity and is not interfered by other ROS and metal ions.
Experiments of the invention in cells. Stimulation of PC12 cells with glutamate causes the cells to develop oxidative stress. Two-photon imaging with probe addition, we found that cells stimulated with glutamate exhibited intense blue and green fluorescence, as compared to the control, demonstrating O in the cells2 ·-And Zn2+The concentration all increased. To determine that the fluorescence change is caused by a change in oxidative stress, we next used Tiron (O)2 ·-Scavenger of (b) treatment of cells stimulated with glutamate, Tiron scavenges O2 ·-After that, the blue fluorescence intensity and the green fluorescence intensity were significantly reduced, indicating that O2 ·-And Zn2+The concentration of (c) is reduced. The imaging results show that the probe can observe O in living cells2 ·-And Zn2+Temporal variations of (2).
Experiments of the present invention in vivo. The mouse brain is imaged by injecting a probe into the abdominal cavity, and the blue fluorescence intensity of an experimental group is obviously enhanced and the green fluorescence intensity is obviously reduced compared with a control group. Explanation, Zn in the brain of depressed mice2+Significant reduction of O2 ·-Is significantly higher (fig. 3). Through the experiments, the Zn in the brain of the depressed mouse is analyzed through fluorescence imaging for the first time2+、O2 ·-The levels varied to different extents compared to normal mice. The result is O2 ·-、Zn2+A positive-negative correlation between levels and extent of depression provides direct evidence.
The invention is not the best known technology.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
1. A double-color fluorescent probe is characterized by comprising dipyridyl methylamine, caffeic acid and 4-bromo-1, 8-naphthalic anhydride, and the structural formula of the double-color fluorescent probe is as follows:
2. the dual color fluorescent probe of claim 1, wherein dipyridylmethylamine is used as Zn2+Recognition group caffeic acid as O2 ·-A recognition group and a fluorophore, 4-bromo-1, 8-naphthalic anhydride as fluorophore.
3. The method for preparing the two-color fluorescent probe according to claim 1 or 2, which is characterized by comprising the following reactions:
4. the preparation method according to claim 3, wherein the step a is specifically:
under the protection of inert gas, bromopropylamine, DPA and potassium carbonate are mixed and stirred, and light yellow solid A is obtained after purification.
5. The preparation method according to claim 3, wherein the step b is specifically:
under the protection of inert gas, caffeic acid, DMAP, DCC, triethylamine and DMF are mixed and stirred under ice bath condition to obtain activated caffeic acid solution; under the protection of inert gas, mixing ethylenediamine and DMF, then dropwise adding activated caffeic acid solution, stirring at room temperature to obtain brown turbid liquid, and carrying out thin layer chromatography to obtain light yellow solid B.
6. The preparation method according to claim 3, wherein the step c is specifically:
under the protection of inert gas, mixing the product B, anhydride naphthalene, ethanol and DMF, stirring at low temperature to obtain brown solution, and performing thin-layer chromatography to obtain a light yellow solid C; preferably, the low temperature is 28-35 ℃, and more preferably 30 ℃.
7. The preparation method according to claim 3, wherein the step d is specifically:
under the protection of inert gas, the product A, the product C and the product K2CO3Mixing, stirring at high temperature overnight, and performing thin layer chromatography to obtain yellow solid as final product; preferably, the high temperature is 75-85 ℃, and more preferably 80 ℃.
8. The method of any one of claims 5 to 7, wherein the eluent in thin layer chromatography is dichloromethane: methanol (v: v) ═ 10: 1.
9. use of the dual-color fluorescent probe of claim 1 or 2, characterized in that the use comprises detecting, identifying O in an environment or in a biological sample2 ·-And Zn2+。
10. The use of claim 9, wherein the biological sample comprises a biological organ, a biological tissue, and a biological cell; the biological organ is the brain of a living organism (mouse brain).
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| CN113072537A (en) * | 2021-03-25 | 2021-07-06 | 山东师范大学 | Fluorescent probe and preparation method and application thereof |
| CN114456079A (en) * | 2021-12-23 | 2022-05-10 | 山东师范大学 | Fluorescent probe compound, preparation method and application of fluorescent probe compound as superoxide anion indicator |
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| CN113072537B (en) * | 2021-03-25 | 2022-04-15 | 山东师范大学 | Fluorescent probe and preparation method and application thereof |
| CN114456079A (en) * | 2021-12-23 | 2022-05-10 | 山东师范大学 | Fluorescent probe compound, preparation method and application of fluorescent probe compound as superoxide anion indicator |
| CN114456079B (en) * | 2021-12-23 | 2023-06-23 | 山东师范大学 | Fluorescent probe compound, preparation method and application of fluorescent probe compound as superoxide anion indicator |
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